BIOCHEMISTRY FREE

FOR ALL

Kevin Ahern, Indira Rajagopal, & Taralyn

Tan
Oregon State University
 Oregon State University
Biochemistry Free For All

Kevin Ahern, Indira Rajagopal, & Taralyn Tan

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TABLE OF CONTENTS
Licensing

1: In The Beginning
1.1: Introduction - Basic Biology
1.2: Introduction - Basic Chemistry
1.3: Introduction - Water and Buffers

2: Structure and Function

2.1: Prelude to Structure and Function
2.4: Structure and Function- Proteins II
2.5: Structure and Function- Protein Function II
2.6: Structure and Function - Nucleic Acids
2.7: Structure and Function- Carbohydrates
2.8: Structure and Function - Lipids and Membranes
2.2: Structure & Function - Amino Acids
2.3: Structure & Function- Proteins I

3: Membranes
3.1: Basic Concepts in Membranes
3.2: Transport in Membranes
3.3: Other Considerations in Membranes

4: Catalysis
4.1: Basic Principles of Catalysis
4.2: Control of Enzymatic Activity
4.3: Mechanisms of Catalysis
4.4: Blood Clotting

5: Energy
5.1: Basics of Energy
5.3: Energy - Photophosphorylation
5.2: Electron Transport and Oxidative Phosphorylation

6: Metabolism
6.1: Metabolism - Sugars
6.2: Citric Acid Cycle & Related Pathways
6.3: Fats and Fatty Acids
6.4: Other Lipids
6.5: Amino Acids and the Urea Cycle
6.6: Nucleotides

7: Information Processing
7.1: Prelude to Information Processing
7.2: Genes and Genomes

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 7.3: DNA Replication
7.4: DNA Repair
7.5: Transcription
7.6: RNA Processing
7.7: Translation
7.8: Gene Expression
7.9: Signaling

8: Basic Techniques
8.1: Cell Lysis
8.2: Fractionation and Chromatography Techniques
8.3: Electrophoresis
8.4: Detection, identification and quantitation of specific nucleic acids and proteins
8.5: Transcriptomics
8.6: Isolating Genes
8.7: Polymerase Chain Reaction (PCR)
8.8: Reverse Transcription
8.9: FRET
8.10: Genome Editing (CRISPR)
8.11: Protein Cleavage
8.12: Membrane Dynamics (FRAP)

9: Chapter 10
9.1: Section 1-
9.2: Section 2-
9.3: Section 3-
9.4: Section 4-
9.5: Section 5-
9.6: Section 6-

10: Chapter 11
10.1: Section 1-
10.2: Section 2-
10.3: Section 3-
10.4: Section 4-
10.5: Section 5-
10.6: Section 6-

11: Point by Point

11.1: In the Beginning
11.2: Structure and Function
11.3: Membranes
11.4: Catalysis
11.5: Energy
11.6: Metabolism
11.7: Information Processing
11.8: Techniques

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Index
Glossary

Detailed Licensing

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Licensing
A detailed breakdown of this resource's licensing can be found in Back Matter/Detailed Licensing.

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 CHAPTER OVERVIEW

1: In The Beginning
1.1: Introduction - Basic Biology
1.2: Introduction - Basic Chemistry
1.3: Introduction - Water and Buffers

This page titled 1: In The Beginning is shared under a CC BY-NC-SA license and was authored, remixed, and/or curated by Kevin Ahern, Indira
Rajagopal, & Taralyn Tan.

1
1.1: Introduction - Basic Biology

Figure 1.1 - Tardigrade Wikipedia

Figure 1.2 Slices of cork as seen by Hooke

Figure 1.17 - Nerve cell anatomy Image by Pehr Jacobson

YouTube Lectures by Kevin HERE & HERE
Graphic images in this book were products of the work of several talented students. Links to their Web pages are below
Click HERE for Martha Baker’s Web Page
Click HERE for Pehr Jacobson’s Web
Page Click HERE for Aleia Kim’s Web Page
Click HERE for Penelope Irving’s Web Page
Problem set related to this section HERE
Point by Point summary of this section HERE
To get a certificate for mastering this section of the book, click HERE

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Kevin Ahern’s free iTunes U Courses - Basic / Med School / Advanced
Biochemistry Free & Easy (our other book) HERE / Facebook Page
Kevin and Indira’s Guide to Getting into Medical School - iTunes U Course / Book
To see Kevin Ahern’s OSU ecampus courses - BB 350 / BB 450 / BB 451
To register for Kevin Ahern’s OSU ecampus courses - BB 350 / BB 450 / BB 451
Biochemistry Free For All Facebook Page (please like us)
Kevin Ahern’s Web Page / Facebook Page / Taralyn Tan’s Web Page
Kevin Ahern’s free downloads HERE
OSU’s Biochemistry/Biophysics program HERE
OSU’s College of Science HERE
Oregon State University HERE
Email Kevin Ahern / Indira Rajagopal / Taralyn Tan

This page titled 1.1: Introduction - Basic Biology is shared under a CC BY-NC-SA license and was authored, remixed, and/or curated by Kevin
Ahern, Indira Rajagopal, & Taralyn Tan.

1.1.2 https://bio.libretexts.org/@go/page/7801
1.2: Introduction - Basic Chemistry
Source: BiochemFFA_1_2.pdf. The entire textbook is available for free from the authors at
http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy

“Organic chemistry is the chemistry of carbon compounds. Biochemistry is the chemistry

of carbon compounds that crawl” -Michael Adams.
To understand biochemistry, one must possess at least a basic understanding of organic and general chemistry. In this brief section,
we will provide a rapid review of the simple concepts necessary to understand cellular chemistry. Chemistry is chemistry, whether
in a cell or outside it, but biological chemistry is a particular subset of organic chemistry that often involves enormous
macromolecules, and that happens in the aqueous environment of the cell.

Figure 1.18 shows the various organic functional groups common in biochemistry. You will encounter these functional groups as
you study the biosynthetic and breakdown pathways that build and recycle the chemical compounds of which cells are made. In
addition to knowing the names and structures of these groups, students need a basic understanding of covalent and ionic bonds.

Figure 1.18 - Important functional groups in biochemistry Image by Aleia Kim

Covalent bonds, as you know, are the result of sharing of electrons between two atoms. Ionic bonds, by contrast, are formed when
one atom donates an electron to another, such as in the formation of sodium chloride. Single covalent bonds can rotate freely, but

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double bonds cannot. Single bonds around a carbon atom are arranged in a tetrahedron with bond angles of 109.5° relative to each
other, with the carbon at the center (Figure 1.19). Double bonded carbons create a planar structure with bond angles typically of
about 120°.

Electronegativity
Electronegativity is a measure of the affinity a nucleus has for outer shell electrons (Table 1.2). High electronegativity corresponds
to high affinity. Electrons in a covalent bond are held closer to the nucleus with a greater electronegativity compared to a nucleus
with lower electronegativity.

Table 1.2 Image by Aleia Kim

For example, in a molecule of water, with hydrogen covalently bonded to oxygen, the electrons are “pulled” toward the oxygen,
which is more electronegative. Because of this, there is a slightly greater negative charge near the oxygen atom of water, compared
to the hydrogen (which, correspondingly has a slightly higher positive charge). This unequal charge distribution sets up a dipole,
with one side being somewhat negative and the other somewhat positive. Because of this, the molecule is described as polar.

Figure 1.19 - Tetrahedral structure Wikipedia

Hydrogen bonds between water molecules are the result of the attraction of the partial positive and partial negative charges on
different water molecules (Figure 1.20). Hydrogen bonds can also form between hydrogens with a partial positive charge and other
strongly electronegative atoms, like nitrogen, with a partial negative charge. It is important to remember that hydrogen bonds are
interactions between molecules (or parts of molecules) and are not bonds between atoms, like covalent or ionic bonds. Bonds
between hydrogen and carbon do not form significant partial charges because the electronegativities of the two atoms are similar.
Consequently, molecules containing many carbon-hydrogen bonds will not form hydrogen bonds and therefore, do not mix well
with water. Such molecules are called hydrophobic. Other compounds with the ability to make hydrogen bonds are polar and can
dissolve in water. They are called hydrophilic. Molecules possessing both characteristics are called amphiphilic.

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 Figure 1.20 Hydrogen bonds (dotted lines) between water molecules Wikipedia

Weak interactions
Hydrogen bonds are one kind of electrostatic (i.e., based on charge) interaction between dipoles. Other forms of electrostatic
interactions that are important in biochemistry include weak interactions between a polar molecule and a transient dipole, or
between two temporary dipoles. These temporary dipoles result from the movement of electrons in a molecule. As electrons move
around, the place where they are, at a given time, becomes temporarily more negatively charged and could now attract a temporary
positive charge on another molecule. Since electrons don’t stay put, these dipoles are very short-lived. Thus, the attraction that
depends on these dipoles fluctuates and is very weak. Weak interactions like these are sometimes called van der Waals forces.
Many molecular interactions in cells depend on weak interactions. Although the individual hydrogen bonds or other dipole-dipole
interactions are weak, because of their large numbers, they can result in quite strong interactions between molecules.
Oxidation/reduction
Oxidation involves loss of electrons and reduction results in gain of electrons. For every biological oxidation, there is a
corresponding reduction - one molecule loses electrons to another molecule. Oxidation reactions tend to release energy and are a
source of bioenergy for chemotrophic cells.
Ionization
Ionization of biomolecules, by contrast does not involve oxidation/reduction. In ionization, a hydrogen ion (H+) leaves behind its
electron as it exits (leaving behind a negative charge) or joins a group (adding a positive charge). Biological ionizations typically
involve carboxyl groups or amines, though phosphates or sulfates can also be ionized. A carboxyl group can have two ionization
states - a charge of -1 corresponds to the carboxyl without its proton and a charge of zero corresponds to the charge of the carboxyl
with its proton on. An amine also has two ionization states. A charge of zero corresponds to a nitrogen with three covalent bonds
(usually in the form of C-NH2) and a charge of +1 corresponds to a nitrogen making four covalent bonds (usually X-NH3 +).
Stereochemistry
A carbon has the ability to make four single bonds (forming a tetrahedral structure) and if it bonds to four different chemical
groups, their atoms can be arranged around the carbon in two different ways, giving rise to stereochemical “handedness” (Figure
1.21). Each carbon with such a property is referred to as an asymmetric center. The property of handedness only occurs when a
carbon has four different groups bonded to it. Enzymes have very specific 3-D structures, so for biological molecules that can exist
in different stereoisomeric forms, an enzyme that synthesizes it would make only one of the possible isomers. By contrast, the same
molecules made chemically (not using enzymes) end up with equal amounts of both isomers, called a racemic mix.

Figure 1.21 - Mirror images of lactic acid

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Gibbs free energy
The Gibbs free energy calculation allows us to determine whether a reaction will be spontaneous, by taking into consideration two
factors, change in enthalpy (ΔH) and change in entropy (ΔS). The free energy content of a system is given by the Gibbs free energy
(G) and is equal to the enthalpy (H ) for a process minus the absolute temperature (T) times the entropy (S)
G = H = TS (1.2.1)

For a process, the change in the Gibbs free energy ΔG is given by

ΔG = ΔH − T ΔS (1.2.2)

A negative ΔG corresponds to release of free energy. Reactions that release energy are exergonic, whereas those that absorb
energy are called endergonic.
The biological standard Gibbs free energy change (ΔG°’) corresponds to the ΔG for a process under standard conditions of
temperature, pressure, and at pH = 7. For a reaction
aA + bB ⇌ cC + dD, (1.2.3)

the equilibrium constant, K eq is equal to

c d
[C ]eq [D]eq
Keq = (1.2.4)
a b
[A]eq [B]eq

where a , b , c , and d are integers in the balanced equation. Large values of K correspond to favorable reactions (more C and D
eq

produced than A and B) and small values of K mean the opposite. At equilibrium,
eq

o′
ΔG = −RT ln Keq (1.2.5)

If a process has a ΔG = Z and a second process has a ΔG = Y , then if the two processes are linked, ΔG and ΔG values for the
o′

overall reaction will be the sum of the individual ΔG and ΔG°’ values.
ΔGtotal = ΔG1 + ΔG2 = Z + Y (1.2.6)

o′ o′ o′
ΔG = ΔG + ΔG (1.2.7)
total 1 2

Catalysis
Catalysis is an increase in the rate of a reaction induced by a substance that is, itself, unchanged by the reaction. Because catalysts
remain unchanged at the end of a reaction, a single catalyst molecule can be reused for many reaction cycles. Proteins that catalyze
reactions in cells are called enzymes, while ribozymes are RNA molecules that act as catalysts.

Figure 1.22 - Glyceraldehyde-3-phosphate dehydrogenase in the midst of catalysis Wikipedia

Contributors
This page titled 1.2: Introduction - Basic Chemistry is shared under a CC BY-NC-SA license and was authored, remixed, and/or curated by Kevin
Ahern, Indira Rajagopal, & Taralyn Tan.

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1.3: Introduction - Water and Buffers
Source: BiochemFFA_1_3.pdf. The entire textbook is available for free from the authors at
http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy

When it comes to water, we’re literally drowning in it, as water is by far the most abundant component of every cell. To understand
life, we begin the discussion with the basics of water, because everything that happens in cells, even reactions buried deep inside
enzymes, away from water, is influenced by water’s chemistry.
The water molecule has wide ‘V’ shape (the HO-H angle is 104°) with uneven sharing of electrons between the oxygen and the
hydrogen atoms (Figure 1.23). Oxygen, with its higher electronegativity, holds electrons closer to itself than the hydrogens do. The
hydrogens, as a result, are described as having a partial positive charge (typically designated as δ+) and the oxygen has a partial
negative charge (written as δ- ). Thus, water is a polar molecule because charges are distributed around it unevenly, not
symmetrically.

Water as a solvent
Water (Figure 1.23) is described as a solvent because of its ability to solvate (dissolve) many, but not all, molecules. Molecules that
are ionic or polar dissolve readily in water, but non-polar substances dissolve poorly in water, if at all. Oil, for example, which is
non-polar, separates from water when mixed with it. On the other hand, sodium chloride, which ionizes, and ethanol, which is
polar, are able to form hydrogen bonds, so both dissolve in water. Ethanol’s solubility in water is crucial for brewers, winemakers,
and distillers – but for this property, there would be no wine, beer or spirits. As explained in an earlier section, we use the term
hydrophilic to describe substances that interact well with water and dissolve in it and the term hydrophobic to refer to materials that
are non-polar and do not dissolve in water. Table 1.3 illustrates some polar and non-polar substances. A third term, amphiphilic,
refers to compounds that have both properties. Soaps, for example are amphiphilic, containing a long, non-polar aliphatic tail and a
head that ionizes.

Table 1.3 Image by Aleia Kim

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Solubility

Figure 1.23 - Arrangement of atoms in water Image by Aleia Kim

The solubility of materials in water is based in free energy changes, as measured by ΔG. Remember, from chemistry, that H is the
enthalpy (heat at constant pressure) and S is entropy. Given this,
ΔG = ΔH − T ΔS (1.3.1)

where T is the temperature in Kelvin. For a process to be favorable, the ΔG for it must be less than zero.

Figure 1.24 - Structure of a Soap

From the equation, lowered ΔG values will be favored with decreases in enthalpy and/or increases in entropy. Let us first consider
why non-polar materials do not dissolve in water. We could imagine a situation where the process of dissolving involves the
“surrounding” of each molecule of the nonpolar solute in water, just like each sodium and each chloride ion gets surrounded by
water molecules as salt dissolves.

Water organization

Figure 1.25 - Structures formed by amphiphilic substances in water. Image by Aleia Kim
There is a significant difference, though between surrounding a non-polar molecule with water molecules and surrounding ions (or
polar compounds) with water molecules.
The difference is that since non-polar molecules don’t really interact with water, the water behaves very differently than it does
with ions or molecules that form hydrogen bonds. In fact, around each non-polar molecule, water gets very organized, aligning
itself regularly. As any freshman chemistry student probably remembers, entropy is a measure of disorder, so when something
becomes ordered, entropy decreases, meaning the ΔS is negative, so the TΔS term in the equation is positive (negative of a
negative).

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 Figure 1.26 - A phospholipid - an amphiphilic substance

Since mixing a non-polar substance with water doesn’t generally have any significant heat component, the ΔG is positive. This
means, then, that dissolving a non-polar compound in water is not favorable and does not occur to any significant extent. Further,
when the non-polar material associates with itself and not water, then the water molecules are free to mix, without being ordered,
resulting in an increase of entropy. Entropy therefore drives the separation of non-polar substances from aqueous solutions.

Figure 1.27 - Vinegar (black) and oil (yellow) A mix of polar and nonpolar compounds Wikipedia

Amphiphilic substances
Next, we consider mixing of an amphiphilic substance, such as a soap, with water (Figure 1.24). The sodium ions attached to the
fatty acids in soap readily come off in aqueous solution, leaving behind a negatively charged molecule at one end and a non-polar
region at the other end. The ionization of the soap causes in an increase in entropy - two particles instead of one. The non-polar
portion of the negatively charged soap ion is problematic - if exposed to water, it will cause water to organize and result in a
decrease of entropy and a positive ΔG.

Figure 1.28 - Environment of a lipid bilayer. Water is concentrated away from the hydrophobic center, being saturated on the
outside, semi-saturated near the head-tail junction and fully dehydrated in the middle. Image by Aleia Kim
Since we know fatty acids dissolve in water, there must be something else at play. There is. Just like the non-polar molecules in the
first example associated with each other and not water, so too do the non-polar portions of the soap ions associate with each other

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and exclude water. The result is that the soap ions arrange themselves as micelles (Figure 1.25) with the non-polar portions on the
interior of the structure away from water and the polar portions on the outside interacting with water.

Figure 1.29 - Protein folding arranges hydrophobic amino acids (black dots) inside the protein
The interaction of the polar heads with water returns the water to its more disordered state. This increase in disorder, or entropy,
drives the formation of micelles. As will be seen in the discussion of the lipid bilayer, the same forces drive glycerophospholipids
and sphingolipids to spontaneously form bilayers where the non-polar portions of the molecules interact with each other to exclude
water and the polar portions arrange themselves on the outsides of the bilayer (Figure 1.28).

Figure 1.30 - Common hydrogen bonds in biochemistry Image by Aleia Kim

Yet another example is seen in the folding of globular proteins in the cytoplasm. Nonpolar amino acids are found in the interior
portion of the protein (water excluded). Interaction of the non-polar amino acids turns out to be a driving force for the folding of
proteins as they are being made in an aqueous solution.

Hydrogen bonds
The importance of hydrogen bonds in biochemistry (Figure 1.30) is hard to overstate. Linus Pauling himself said,
“ . . . . I believe that as the methods of structural chemistry are further applied to physiological problems it will be found that the
significance of the hydrogen bond for physiology is greater than that of any other single structural feature.”

Figure 1.31 - Hydrogen bonds between water molecules Image by Pehr Jacobson

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In 2011, an IUPAC task group gave an evidence-based definition of hydrogen bonding that states,
“The hydrogen bond is an attractive interaction between a hydrogen atom from a molecule or a molecular fragment X–H in which
X is more electronegative than H, and an atom or a group of atoms in the same or a different molecule, in which there is evidence
of bond formation.”

Partial Charges
The difference in electronegativity between hydrogen and the molecule to which it is covalently bound give rise to partial charges
as described above. These tiny charges (δ+ and δ- ) result in formation of hydrogen bonds, which occur when the partial positive
charge of a hydrogen atom is attracted to the partial negative of another molecule. In water, that means the hydrogen of one water
molecule is attracted to the oxygen of another (Figure 1.31). Since water is an asymmetrical molecule, it means also that the
charges are asymmetrical. Such an uneven distribution is what makes a dipole. Dipolar molecules are important for interactions
with other dipolar molecules and for dissolving ionic substances (Figure 1.32).
Hydrogen bonds are not exclusive to water. In fact, they are important forces holding together macromolecules that include proteins
and nucleic acids. Hydrogen bonds occur within and between macromolecules.

Figure 1.32 - Example dipole interactions in biochemistry Image by Aleia Kim

The complementary pairing that occurs between bases in opposite strands of DNA, for example, is based on hydrogen bonds. Each
hydrogen bond is relatively weak (compared to a covalent bond, for example - Table 1.4), but collectively they can be quite strong.

Table 1.4 Image by Aleia Kim

Benefits of weak interactions

Their weakness, however, is actually quite beneficial for cells, particularly as regards nucleic acids (Figure 1.33). The strands of
DNA, for example, must be separated over short stretches in the processes of replication and the synthesis of RNA. Since only a
few base pairs at a time need to be separated, the energy required to do this is small and the enzymes involved in the processes can
readily take them apart, as needed. Hydrogen bonds also play roles in binding of substrates to enzymes, catalysis, and protein-
protein interaction, as well as other kinds of binding, such as protein-DNA, or antibody-antigen.

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 Figure 1.33 - Hydrogen bonds in a base pair of DNA Image by Aleia Kim

As noted, hydrogen bonds are weaker than covalent bonds (Table 1.4) and their strength varies form very weak (1-2 kJ/mol) to
fairly strong (29 kJ/mol). Hydrogen bonds only occur over relatively short distances (2.2 to 4.0 Å). The farther apart the hydrogen
bond distance is, the weaker the bond is.
The strength of the bond in kJ/mol represents the amount of heat that must be put into the system to break the bond - the larger the
number, the greater the strength of the bond. Hydrogen bonds are readily broken using heat. The boiling of water, for example,
requires breaking of H-bonds. When a biological structure, such as a protein or a DNA molecule, is stabilized by hydrogen bonds,
breaking those bonds destabilizes the structure and can result in denaturation of the substance - loss of structure. It is partly for this
reason that most proteins and all DNAs lose their native, or folded, structures when heated to boiling.

Image by Aleia Kim Table 1.5

For DNA molecules, denaturation results in complete separation of the strands from each other. For most proteins, this means loss
of their characteristic three-dimensional structure and with it, loss of the function they performed. Though a few proteins can
readily reassume their original structure when the solution they are in is cooled, most can’t. This is one of the reasons that we cook
our food. Proteins are essential for life, so denaturation of bacterial proteins results in death of any microorganisms contaminating
the food.

The importance of buffers

Water can ionize to a slight extent (10-7 M) to form H+ (proton) and OH- (hydroxide). We measure the proton concentration of a
solution with pH, which is the negative log of the proton concentration.
pH = -Log[H+]

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If the proton concentration, [H+]= 10-7 M, then the pH is 7. We could just as easily measure the hydroxide concentration with the
pOH by the parallel equation,
pOH = -Log[OH- ]
In pure water, dissociation of a proton simultaneously creates a hydroxide, so the pOH of pure water is 7, as well. This also means
that
pH + pOH = 14
Now, because protons and hydroxides can combine to form water, a large amount of one will cause there to be a small amount of
the other. Why is this the case? In simple terms, if I dump 0.1 moles of H+ into a pure water solution, the high proton concentration
will react with the relatively small amount of hydroxides to create water, thus reducing hydroxide concentration. Similarly, if I
dump excess hydroxide (as NaOH, for example) into pure water, the proton concentration falls for the same reason.

Acids vs bases
Chemists use the term “acid” to refer to a substance which has protons that can dissociate (come off) when dissolved in water. They
use the term “base” to refer to a substance that can absorb protons when dissolved in water. Both acids and bases come in strong
and weak forms. (Examples of weak acids are shown in Table 1.5.) Strong acids, such as HCl, dissociate completely in water. If we
add 0.1 moles (6.02x1022 molecules) of HCl to a solution to make a liter, it will have 0.1 moles of H+ and 0.1 moles of Cl- or
6.02x1022 molecules of each . There will be no remaining HCl when this happens. A strong base like NaOH also dissociates
completely into Na+ and OH- .

Weak Acids
Weak acids and bases differ from their strong counterparts. When you put one mole of acetic acid (HAc) into pure water, only a
tiny percentage of the HAc molecules dissociate into H+ and Ac- . Clearly, weak acids are very different from strong acids. Weak
bases behave similarly, except that they accept protons, rather than donate them. Since we can view everything as a form of a weak
acid, we will not use the term weak base here.

Figure 1.34 - Dissociation of a weak acid Image by Aleia Kim

Students are often puzzled and expect that [H+] = [A- ] because the dissociation equation shows one of each from HA. This is, in
fact, true ONLY when HA is allowed to dissociate in pure water. Usually the HA is placed into solution that has protons and
hydroxides to affect things. Those protons and /or hydroxides change the H+ and Aconcentration unequally, since A- can absorb
some of the protons and/or HA can release H+ when influenced by the OH- in the solution. Therefore, one must calculate the
proton concentration from the pH using the Henderson Hasselbalch equation.
\[pH = pKa + log ([Ac- ]/[HAc])\]

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 Image by Aleia Kim Table 1.6
You may wonder why we care about weak acids. You may never have thought much of weak acids when you were in General
Chemistry. Your instructor described them as buffers and you probably dutifully memorized the fact that “buffers are substances
that resist change in pH” without really learning what Clearing Confusion - this meant. Buffers are much too important to be
thought of in this way.
UPS
Weak acids are critical for life because their affinity for protons causes them to behave like a UPS. We’re not referring to the UPS
that is the United Parcel Service, but instead, to the encased battery backup systems for computers called Uninterruptible Power
Supplies that kick on to keep a computer running during a power failure. The battery in a laptop computer is a UPS, for example.
We can think of weak acids as Uninterruptible Proton Suppliers within certain pH ranges, providing (or absorbing) protons as
needed. Weak acids thus help to keep the H+ concentration (and thus the pH) of the solution they are in relatively constant.
Consider the bicarbonate/carbonic acid system. Figure 1.35 shows what happens when H2CO3dissociates. Adding hydroxide ions
(by adding a strong base like NaOH) to the solution causes the H+ ions to react with OH- ions to make water. Consequently, the
concentration of H+ ions would go down and the pH would go up.

Figure 1.35 - Titration curve for carbonic acid Image by Aleia Kim
However, in contrast to the situation with a solution of pure water, there is a backup source of H+ available in the form of H2CO3.
Here is where the UPS function kicks in. As protons are taken away by the added hydroxyl ions (making water), they are partly
replaced by protons from the H2CO3. This is why a weak acid is a buffer. It resists changes in pH by releasing protons to
compensate for those “used up” in reacting with the hydroxyl ions.

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Henderson-Hasselbalch
It is useful to be able to predict the response of the H2CO3 system to changes in H+ concentration. The Henderson-Hasselbalch
equation defines the relationship between pH and the ratio of HCO3 - and H2CO3. It is
pH = pKa + log ([HCO3- ]/ [H2CO3])
This simple equation defines the relationship between the pH of a solution and the ratio of HCO3- and H2CO3 in it. The new term,
called the pKa, is defined as
pKa = -Log Ka,
just as
pH = -Log [H+].
The Ka is the acid dissociation constant and is a measure of the strength of an acid. For a general acid, HA, which dissociates as
HA ⇄ H+ + A -, Ka = [H+][A- ]/[HA]
Thus, the stronger the acid, the more protons that will dissociate from it when added to water and the larger the value its Ka will
have. Large values of Ka translate to lower values of pKa. As a result, the lower the pKa value is for a given acid, the stronger the
weak acid is.

Constant pKa
Please note that pKa is a constant for a given acid. The pKa for carbonic acid is 6.37. By comparison, the pKa for formic acid is
3.75. Formic acid is therefore a stronger acid than acetic acid. A stronger acid will have more protons dissociated at a given pH
than a weaker acid.
Now, how does this translate into stabilizing pH? Figure 1.35 shows a titration curve. In this curve, the titration begins with the
conditions at the lower left (very low pH). At this pH, the H2CO3 form predominates, but as more and more OH- is added (moving
to the 45 Why do we care about pH? Because biological molecules can, in some cases, be exquisitely sensitive to changes in it. As
the pH of a solution changes, the charges of molecules in the solution can change, as you will see. Changing charges on biological
molecules, especially proteins, can drastically affect how they work and even whether they work at all right), the pH goes up, the
amount of HCO3- goes up and (correspondingly), the amount of H2CO3 goes down. Notice that the curve “flattens” near the pKa
(6.37).

Buffering region
Flattening of the curve tells us is that the pH is not changing much (not going up as fast) as it did earlier when the same amount of
hydroxide was added. The system is resisting a change in pH (not stopping the change, but slowing it) in the region of about one
pH unit above and one pH unit below the pKa. Thus, the buffering region of the carbonic acid/ bicarbonate buffer is from about
5.37 to 7.37. It is maximally strong at a pH of 6.37.
Now it starts to become apparent how the buffer works. HA can donate protons when extras are needed (such as when OH- is
added to the solution by the addition of NaOH). Similarly, A- can accept protons when extra H+ are added to the solution (adding
HCl, for example). The maximum ability to donate or accept protons comes when
[A- ] = [HA]
This is consistent with the Henderson Hasselbalch equation and the titration curve. When [A- ] = [HA], pH = 6.37 + Log(1). Since
Log(1) = 0, pH = 6.37 = pKa for carbonic acid. Thus for any buffer, the buffer will have maximum strength and display flattening

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of its titration curve when [A- ] = [HA] and when pH = pKa. If a buffer has more than one pKa (Figure 1.36), then each pKa region
will display the behavior.
Buffered vs non-buffered
To understand how well a buffer protects against changes in pH, consider the effect of adding .01 moles of HCl to 1.0 liter of pure
water (no volume change) at pH 7, compared to adding it to 1.0 liter of a 1M acetate buffer at pH 4.76. Since HCl completely
dissociates, in 0.01M (10-2 M) HCl you will have 0.01M H+. For the pure water, the pH drops from 7.0 down to 2.0 (pH = -
log(0.01M)).
By contrast, the acetate buffer’s pH after adding the same amount of HCl is 4.74. Thus, the pure water solution sees its pH fall from
7 to 2 (5 pH units), whereas the buffered solution saw its pH drop from 4.76 to 4.74 (0.02 pH units). Clearly, the buffer minimizes
the impact of the added protons compared to the pure water.
Buffer capacity
It is important to note that buffers have capacities limited by their concentration. Let’s imagine that in the previous paragraph, we
had added the 0.01 moles HCl to an acetate buffer that had a concentration of 0.01M and equal amounts of Ac- and HAc. When we
try to do the math in parallel to the previous calculation, we see that there are 0.01M protons, but only 0.005M A- to absorb them.
We could imagine that 0.005M of the protons would be absorbed, but that would still leave 0.005M of protons unbuffered. Thus,
the pH of this solution would be approximately
pH = -log(0.005M) = 2.30
Exceeding buffer capacity dropped the pH significantly compared to adding the same amount of protons to a 1M acetate buffer.
Consequently, when considering buffers, it is important to recognize that their concentration sets their limits. Another limit is the
pH range in which one hopes to control proton concentration.
Multiple ionizable groups
Now, what happens if a molecule has two (or more) ionizable groups? It turns out, not surprisingly, that each group will have its
own pKa and, as a consequence, will have multiple regions of buffering.

Figure 1.36 - Titration of an acidic amino acid Image by Aleia Kim

Figure 1.36 shows the titration curve for the amino acid aspartic acid. Note that in- stead of a single flattening of the curve, as was
seen for acetic acid, aspartic acid’s titration curve displays three such regions. These are individual buffering regions, each centered
on the respective pKa values for the carboxyl group and the amine group.
Aspartic acid has four possible charges: +1 (α-carboxyl group, α-amino group, and Rgroup carboxyl each has a proton), 0 (α-
carboxyl group missing proton, α- amino group has a proton, R-group carboxyl has a proton), -1 (α-carboxyl group and R-group

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carboxyl each lack a proton, α-amino group retains a proton), -2 (α-carboxyl, R-group carboxyl, and α-amino groups all lack extra
proton).
Prediction
How does one predict the charge for an amino acid at a given pH? A good rule of thumb for estimating charge is that if the pH is
more than one unit below the pKa for a group (carboxyl or amino), the proton is on. If the pH is more than one unit above the pKa
for the group, the proton is off. If the pH is NOT more than one or less than one pH unit from the pKa, this simple assumption will
not work.
Further, it is important to recognize that these rules of thumb are estimates only. The pI (pH at which the charge of a molecule is
zero) is an exact value calculated as the average of the two pKa values on either side of the zero region. It is calculated at the
average of the two pKa values around the point where the charge of the molecule is zero. For aspartic acid, this corresponds to
pKa1and pKa2.

References
1. http://www.lpi.usra.edu/lunar/missions/apollo/ apollo_12/experiments/surveyor/
2. Arunan, Elangannan; Desiraju, Gautam R.; Klein, Roger A.; Sadlej, Joanna; Scheiner, Steve; Alkorta, Ibon; Clary, David C.;
Crabtree, Robert H.; Dannenberg, Joseph J.; Hobza, Pavel; Kjaergaard, Henrik G.; Legon, Anthony C.; Mennucci, Benedetta;
Nesbitt, David J. (2011). "Definition of the hydrogen bond". Pure Appl. Chem. 83 (8): 1637–1641. doi:10.1351/PAC-REC-10-
01-02

This page titled 1.3: Introduction - Water and Buffers is shared under a CC BY-NC-SA license and was authored, remixed, and/or curated by
Kevin Ahern, Indira Rajagopal, & Taralyn Tan.

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 CHAPTER OVERVIEW

2: Structure and Function

Topic hierarchy
2.1: Prelude to Structure and Function
2.4: Structure and Function- Proteins II
2.5: Structure and Function- Protein Function II
2.6: Structure and Function - Nucleic Acids
2.7: Structure and Function- Carbohydrates
2.8: Structure and Function - Lipids and Membranes
2.2: Structure & Function - Amino Acids
2.3: Structure & Function- Proteins I

Thumbanil: Structure of human hemoglobin. The proteins α and βsubunits are in red and blue, and the iron-containing hemegroups
in green. Image used with permission (CC BY-SA 3.0; Richard Wheeler).

This page titled 2: Structure and Function is shared under a CC BY-NC-SA license and was authored, remixed, and/or curated by Kevin Ahern,
Indira Rajagopal, & Taralyn Tan.

1
2.1: Prelude to Structure and Function
Source: BiochemFFA_2_1.pdf. The entire textbook is available for free from the authors at
http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy
"The man who does not read good books has no advantage over the man who cannot read them." Mark Twain

In this chapter, we will examine the structures of the major classes of biomolecules, with an eye to understanding how these
structures relate to function.
As noted earlier, water is the most abundant molecule in cells, and provides the aqueous environment in which cellular chemistry
happens. Dissolved in this water are inorganic ions like sodium, potassium and calcium. But the distinctiveness of biochemistry
derives from the vast numbers of complex, large, carbon compounds, that are made by living cells. You have probably learned that
the major classes of biological molecules are proteins, nucleic acids, carbohydrates and lipids. The first three of these major groups
are macromolecules that are built as long polymers made up of smaller subunits or monomers, like strings of beads. The lipids,
while not chains of monomers, also have smaller subunits that are assembled in various ways to make the lipid components of
cells, including membranes. The chemical properties and three dimensional conformations of these molecules determine all the
molecular interactions upon which life depends. Whether building structures within cells, transferring information, or catalyzing
reactions, the activities of biomolecules are governed by their structures. The properties and shapes of macromolecules, in turn,
depend on the subunits of which they are built.

Interactive 2.1: The enzyme Hexokinase: as for all enzymes, the activity of hexokinase depends on its structure. Protein Database
(PDB)
We will next examine the major groups of biological macromolecules: proteins, polysaccharides, nucleic acids, and lipids. The
building blocks of the first three, respectively, are amino acids, monosaccharides (sugars), and nucleotides. Acetyl-CoA is the most

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common building block of lipids.

This page titled 2.1: Prelude to Structure and Function is shared under a CC BY-NC-SA license and was authored, remixed, and/or curated by
Kevin Ahern, Indira Rajagopal, & Taralyn Tan.

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2.4: Structure and Function- Proteins II
Source: BiochemFFA_2_3.pdf. The entire textbook is available for free from the authors at
http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy
In this section, we hope to bring to life the connection between structure and function of proteins. So far, we have described notable
features of the four elements (primary, secondary, tertiary, and quaternary) of protein structure and discussed example
proteins/motifs exhibiting them. In this section, we will examine from a functional perspective a few proteins/domains whose
function relies on secondary, tertiary, or quaternary structure. It is, of course a bit of a narrow focus to ascribe protein function to
any one component of structure, but our hope is by presenting these examples, we can bring to life the way in which a protein’s
secondary, tertiary, and quarternary structure lead to the functions it has.

Hemoglobin Wikipedia

Fibrous proteins - secondary structure

Proteins whose cellular or extracellular roles have a strong structural component are composed primarily of primary and second
structure, with little folding of the chains. Thus, they have very little tertiary structure and are fibrous in nature. Proteins exhibiting
these traits are commonly insoluble in water and are referred to as fibrous proteins (also called scleroproteins). The examples
described in this category are found exclusively in animals where they serve roles in flesh, connective tissues and hardened external
structures, such as hair. They also contain the three common fibrous protein structures α -helices (keratins), β-strands/sheets
(fibroin & elastin) and triple helices (collagen). The fibrous proteins have some commonality of amino acid sequence. Each
possesses an abundance of repeating sequences of amino acids with small, non-reactive side groups. Many contain short repeats of
sequences, often with glycine.
Keratins

Figure 2.56 - The horns of an impala are composed of keratin Wikipedia

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The keratins are a family of related animal proteins that take numerous forms. α-keratins are structural components of the outer
layer of human skin and are integral to hair, nails, claws, feathers, beaks, scales, and hooves. Keratins provide strength to tissues,
such as the tongue, and over 50 different keratins are encoded in the human genome. At a cellular level, keratins comprise the
intermediate filaments of the cytoskeleton. α- keratins primarily contain α-helices, but can also have β-strand/sheet structures.
Individual α-helices are often intertwined to form coils of coiled structures and these strands can also be further joined together by
disulfide bonds, increasing structural strength considerably. This is particularly relevant for α-keratin in hair, which contains about
14% cysteine. The odor of burned hair and that of the chemicals used to curl/uncurl hair (breaking/re-making disulfide bonds) arise
from their sulfurous components. β-keratins are comprised of β-sheets, as their name implies.

Figure 2.57 - The repeating amino acid sequence of fibroin

Fibroin
An insoluble fibrous protein that is a component of the silk of spiders and the larvae of moths and other insects, fibroin is
comprised of antiparallel β-strands tightly packed together to form β- sheets. The primary structure of fibroin is a short repeating
sequence with glycine at every other residue (Figure 2.57). The small R-groups of the glycine and alanine in the repeating sequence
allows for the tight packing characteristic of the fibers of silk. Wikipedia link HERE Elastin As suggested by its name, elastin is a
protein with elastic characteristics that functions in many tissues of the body to allow them to resume their shapes after expanding
or contracting. The protein is rich in glycine and proline and can comprise over 50% of the weight of dry, defatted arteries.

Figure 2.58 - Weaving of a silk sari Wikipedia

Elastin

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 Figure 2.59 - Desmosine Wikipedia
is made by linking tropoelastin proteins together through lysine residues to make a durable complex crosslinked by desmosine. In
arteries, elastin helps with pressure wave propagation for facilitating blood flow.
Collagen

Figure 2.60 - Collagen’s triple helix Wikipedia

Collagen is the most abundant protein in mammals, occupying up to a third of the total mass. There are at least 16 types of
collagen. Its fibers are a major component of tendons and they are also found abundantly in skin. Collagen is also prominent in
cornea, cartilage, bone, blood vessels and the gut.
Collagen’s structure is an example of a helix of helices, being composed of three lefthanded helical chains that each are coiled
together in a right-handed fashion to make the collagen fiber (Figure 2.60). Each helix is stretched out more than an α-helix, giving
it an extended appearance. On the inside of the triple helical structure, only residues of glycine are found, since the side chains of
other amino acids are too bulky. Collagen chains have the repeating structure glycinem-n where m is often proline and n is often
hydroxyproline (Figure 2.61).

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 Figure 2.61 - Repeating sequences in collagen
Collagen is synthesized in a pre-procollagen form. Processing of the pre-procollagen in the endoplasmic reticulum results in
glycosylation, removal of the ‘pre’ sequence, and hydroxylation of lysine and proline residues (see below). The hydroxides can
form covalent cross-links with each other, strengthening the collagen fibers. As pro-collagen is exported out of the cell, proteases
trim it, resulting in a final form of collagen called tropocollagen.
Hydroxylation
Hydroxylation of proline and lysine side chains occurs post-translationally in a reaction catalyzed by prolyl-4-hydroxylase and
lysyl-hydroxylase (lysyl oxidase), respectively. The reaction requires vitamin C. Since hydroxylation of these residues is essential
for formation of stable triple helices at body temperature, vitamin C deficiency results in weak, unstable collagen and,
consequently, weakened connective tissues. It is the cause of the disease known as scurvy. Hydrolyzed collagen is used to make
gelatin, which is important in the food industry. collagens. Wikipedia link HERE

Figure 2.62 - Oxidation and cross-linking of lysine residues in tropocollagen. Only two strands of the triple helix are shown for
simplicity Image by Aleia Kim
Lamins
Lamins are fibrous proteins that provide structure in the cell nucleus and play a role in transcription regulation. They are similar to
proteins making up the intermediate filaments, but have extra amino acids in one coil of the protein. Lamins help to form the
nuclear lamin in the interior of the nuclear envelope and play important roles in assembling and disassembling the latter in the
process of mitosis. They also help to position nuclear pores. In the process of mitosis, disassembly of the nuclear envelope is
promoted by phosphorylation of lamins by a protein called mitosis promoting factor and assembly is favored by reversing the
reaction (dephosphorylation).
Structural domains - tertiary structure
Every globular protein relies on its tertiary structure to perform its function, so rather than trying to find representative proteins for
tertiary structure (an almost impossible task!), we focus here on a few elements of tertiary structure that are common to many
proteins. These are the structural domains and they differ from the structural motifs of supersecondary structure by being larger
(25-500 amino acids), having a conserved amino acid sequence, and a history of evolving and functioning independently of the

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protein chains they are found in. Structural domains are fundamental units of tertiary structure and are found in more than one
protein. A structural domain is selfstabilizing and often folds independently of the rest of the protein chain.
Leucine zipper

Figure 2.63 - Leucine zipper bound to DNA Wikipedia

A common feature of many eukaryotic DNA binding proteins, leucine zippers are characterized by a repeating set of leucine
residues in a protein that interact like a zipper to favor dimerization. Another part of the domain has amino acids (commonly
arginine and lysine) that allow it to interact with the DNA double helix (Figure 2.63). Transcription factors that contain leucine
zippers include Jun-B, CREB, and AP-1 fos/ jun.

Figure 2.64 - Leucine zipper structure. Leucines are indicated by orange and purple balls. Image by Penelope Irving
Zinc fingers
The shortest structural domains are the zinc fingers, which get their name from the fact that one or more coordinated zinc ions
stabilize their finger-like structure. Despite their name, some zinc fingers do not bind zinc. There are many structural domains
classified as zinc fingers and these are grouped into different families. Zinc fingers were first identified as components of DNA
binding transcription factors, but others are now known to bind RNA, protein, and even lipid structures. Cysteine and histidine side
chains commonly play roles in coordinating the zinc.
Src SH2 domain

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 Figure 2.65 - SH2 Domain Wikipedia
The Src oncoprotein contains a conserved SH2structural domain that recognizes and binds phosphorylated tyrosine side chains in
other proteins (Figure 2.65). Phosphorylation is a fundamental activity in signaling and phosphorylation of tyrosine and interaction
between proteins carrying signals is critically needed for cellular communication. The SH2domain is found in over 100 human
proteins.
Helix-turn-helix domain

Figure 2.66 - Helix-Turn-Helix Domain of a Protein Bound to DNA Wikipedia

Helix-turn-helix is a common domain found in DNA binding proteins, consisting of two α-helices separated by a small number of
amino acids. As seen in Figure 2.66, the helix parts of the structural domain interact with the bases in the major groove of DNA.
Individual α-helices in a protein are part of a helix-turn-helix structure, where the turn separates the individual helices.
Pleckstrin homology domain
Pleckstrin Homology (PH) domains are protein domains with important functions in the process of signaling. This arises partly
from the affinity for binding phosphorylated inositides, such as PIP2 and PIP3, found in Figure 2.66 - Helix-Turn-Helix Domain of
a Protein Bound to DNA Wikipedia Figure 2.65 - SH2 Domain Wikipedia biological membranes. PH domains can also bind to G-
proteins and protein kinase C. The domain spans about amino acids and is found in numerous signaling proteins. These include
Akt/Rac Serine/ Threonine Protein Kinases, Btk/ltk/Tec tyrosine protein kinases, insulin receptor substrate (IRS-1),
Phosphatidylinositolspecific phospholipase C, and several yeast proteins involved in cell cycle regulation.

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 Figure 2.67 - Pleckstrin homology domain of Btk tyrosine protein kinase. The protein is embedded in a membrane (above blue line)
Wikipedia

Structural globular proteins

Enzymes catalyze reactions and proteins such as hemoglobin perform important specialized functions. Evolutionary selection has
reduced and eliminated waste so that we can be sure every protein in a cell has a function, even though in some cases we may not
know what it is. Sometimes the structure of the proFigure 2.68 - Relationship of basement membrane to epithelium, endothelium,
and connective tissue tein is its primary function because the structure provides stability, organization, connections other important
properties. It is with this in mind that we present the following proteins.
Basement membrane
The basement membrane is a layered extracellular matrix of tissue comprised of protein fibers (type IV collagen) and
glycosaminoglycans that separates the epithelium from other tissues (Figure 2.68). More importantly, the basement membrane acts
like a glue to hold tissues together. The skin, for example, is anchored to the rest of the body by the basement membrane. Basement
membranes provide an interface of interaction between cells and the environment around them, thus facilitating signaling
processes. They play roles in differentiation during embryogenesis and also in maintenance of function in adult organisms.

Figure 2.68 - Relationship of basement membrane to epithelium, endothelium, and connective tissue
Actin
Actin is the most abundant globular protein found in most types of eukaryotic cells, comprising as much as 20% of the weight of
muscle cells. Similar proteins have been identified in bacteria (MreB) and archaeons (Ta0583). Actin is a monomeric subunit able
to polymerize readily into two different types of filaments. Microfilaments are major component of the cytoskeleton and are acted
on by myosin in the contraction of muscle cells (See HERE). Actin will be discussed in more detail in the next section HERE.
Intermediate Filaments

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 Figure 2.69 - Assembly of intermediate filaments
Intermediate filaments are a part of the cytoskeleton in many animal cells and are comprised of over 70 different proteins. They are
called intermediate because their size (average diameter = 10 nm) is between that of the microfilaments (7 nm) and the
microtubules (25 nm).
The intermediate filament components include fibrous proteins, such as the keratins and the lamins, which are nuclear, as well as
cytoplasmic forms. Intermediate filaments give flexibility to cells because of their own physical properties. They can, for example,
be stretched to several times of their original length.
Six types
There are six different types of intermediate filaments. Type I and II are acidic or basic and attract each other to make larger
filaments. They include epithelial keratins and trichocytic keratins (hair components). Type III proteins include four structural
proteins - desmin, GFAP (glial fibrillary acidic protein), peripherin, and vimentin. Type IV also is a grouping of three proteins and
one multiprotein structure (neurofilaments). The three proteins are α-internexin, synemin, and syncoilin. Type V intermediate
filaments encompass the lamins, which give structure to the nucleus. Phosphorylation of lamins leads to their disassembly and this
is important in the process of mitosis. The Type VI category includes only a single protein known as nestin.
Tubulin
A third type of filament found in cells is that of the microbutules. Comprised of a polymer of two units of a globular protein called
tubulin, microtubules provide “rails” for motor proteins to move organelles and other “cargo” from one part of a cell to another.
Microtubules and tubulin are discussed in more detail HERE.
Vimentin
Vimentin (Figure 2.70) is the most widely distributed protein of the intermediate filaments. It is expressed in fibroblasts,
leukocytes, and blood vessel endothelial cells. The protein has a significant role maintaining the position of organelles in the
cytoplasm, with attachments to the nucleus, mitochondria, and endoplasmic reticulum (Figure 2.70). Vimentin provides elasticity to
cells and resilience that does not arise from the microtubules or microfilaments. Wounded mice that lack the vimentin gene survive,
but take longer to heal wounds than wild type mice. Vimentin also controls the movement of cholesterol from lysosomes to the site
of esterification. The result is a reduction in the amount of cholesterol stored inside of cells and has implications for adrenal cells,
which must have esters of cholesterol.

Figure 2.70 - Vimentin in cells Wikipedia

Mucin

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Mucins are a group of proteins found in animal epithelial tissue that have many glycosyl residues on them and typically are of high
molecular weight (1 to 10 million Da). They are gel-like in their character and are often used for lubrication. Mucus is comprised of
mucins. In addition to lubrication, mucins also help to control mineralization, such as bone formation in vertebrate organisms and
calcification in echinoderms. They also play roles in the immune system by helping to bind pathogens. Mucins are commonly
secreted onto mucosal surfaces (nostrils, eyes, mouth, ears, stomach, genitals, anus) or into fluids, such as saliva. Because of their
extensive mucosylation, mucins hold a considerable amount of water (giving them the “slimy” feel) and are resistant to proteolysis.
Vinculin

Figure 2.71 - Actin filaments (green) attached to vinculin in focal adhesion (red) Wikipedia
Vinculin (Figure 2.72) is a membrane cytoskeletal protein found in the focal adhesion structures of mammalian cells. It is found at
cell-cell and cell-matrix junctions and interacts with integrins, talin, paxillins and F-actin. Vinculin is thought to assist (along with
other proteins) in anchoring actin microfilaments to the membrane (Figure 2.71). Binding of vinculin to actin and to talin is
regulating by polyphosphoinositides and can be inhibited by acidic phospholipids.

Figure 2.72 - Vinculin Wikipedia

Syndecans
Syndecans are transmembrane proteins that make a single pass with a long amino acid chain (24-25 residues) through plasma
membranes and facilitate G proteincoupled receptors’ interaction with Figure 2.71 - Actin filaments (green) attached to vinculin in
focal adhesion (red) Wikipedia ligands, such as growth factors, fibronectin, collagens (I, III, and IV) and antithrombin-1.
Syndecans typically have 3-5 heparan sulfate and chondroitin sulfate chains attached to them.
Heparan sulfate can be cleaved at the site of a wound and stimulate action of fibroblast growth factor in the healing process. The
role of syndecans in cell-cell adhesion is shown in mutant cells lacking syndecan I that do not adhere well to each other. Syndecan
4 is also known to adhere to integrin. Syndecans can also inhibit the spread of tumors by the ability of the syndecan 1 ectodomain
to suppress growth of tumor cells without affecting normal epithelial cells.
Defensin

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 Figure 2.73 - Defensin monomer (top) and dimer (bottom) - Cationic residues in blue, hydrophobic residues in orange, and anionic
residues in red Wikipedia
Defensins (Figure 2.73) are a group of small cationic proteins (rich in cysteine residues) that serve as host defense peptides in
vertebrate and invertebrate organisms. They protect against infection by various bacteria, fungi, and viruses. Defensins contain
between 18 and 45 amino acids with (typically) about 6- 8 cysteine residues. In the immune system, defensins help to kill bacteria
engulfed by phagocytosis by epithelial cells and neutrophils. They kill 120 Figure 2.72 - Vinculin Wikipedia bacteria by acting like
ionophores - binding the membrane and opening pore-like structures to release ions and nutrients from the cells.
Focal adhesions
In the cell, focal adhesions are structures containing multiple proteins that mechanically link cytoskeletal structures (actin bundles)
with the extracellular matrix. They are dynamic, with proteins bringing and leaving with signals regarding the cell cycle, cell
motility, and more almost constantly. Focal adhesions serve as anchors and as a signaling hub at cellular locations where integrins
bind molecules and where membrane clustering events occur. Over 100 different proteins are found in focal adhesions.
Focal adhesions communicate important messages to cells, acting as sensors to update information about the status of the
extracellular matrix, which, in turn, adjusts/ affects their actions. In sedentary cells, they are stabler than in cells in motion because
when cells move, focal adhesion contacts are established at the “front” and removed at the rear as motion progresses. This can be
very important in white blood cells’ ability to find tissue damage.
Ankyrin

Figure 2.74 - Ankyrin’s membrane-binding domain

Ankyrins (Figure 2.74) are a family of membrane adaptor proteins serving as “anchors” to interconnect integral membrane proteins
to the spectrin-actin membrane cytoskeleton. Ankyrins are anchored to the plasma membrane by covalently linked palmitoyl-CoA.

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They bind to the β subunit of spectrin and at least a dozen groups of integral membrane proteins. The ankyrin proteins contain four
functional domains: an N-terminal region with 24 tandem ankyrin repeats, a central spectrin-binding domain, a “death domain”
interacting with apoptotic proteins, and a C-terminal regulatory domain that is highly varied significantly among different ankyrins.
Spectrin

Figure 2.75 - Spectrin and other proteins in the cytoskeleton Wikipedia

Spectrin (Figures 2.75 & 2.76) is a protein of the cellular cytoskeleton that plays an important role in maintaining its structure and
the integrity of the plasma membrane. In animals, spectrin gives red blood cells their shape. Spectrin is located inside the inner
layer of the eukaryotic plasma membrane where it forms a network of pentagonal or hexagonal arrangements.
Spectrin fibers collect together at junctional complexes of actin and is also attached to ankyrin for stability, as well as numerous
integral membrane proteins, such as glycophorin.

Figure 2.76 - Spectrin (green) and nuclei (Blue) Wikipedia

Integrins

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 Figure 2.77 - Integrin and its binding site (on top left) Wikipedia
In multicellular organisms, cells need connections, both to each other and to the extracellular matrix. Facilitating these attachments
at the cellular end are transmembrane proteins known as integrins (Figure 2.77). Integrins are found in all metazoan cells. Ligands
for the integrins include collagen, fibronectin, laminin, and vitronectin. Integrins function not only in attachment, but also in
communication, cell migration, virus linkages (adenovirus, for example), and blood clotting. Integrins are able to sense chemical
and mechanical signals about the extracellular matrix and move that information to intracellular domains as part of the process of
signal transduction. Inside the cells, responses to the signals affect cell shape, regulation of the cell cycle, movement, or changes in
other cell receptors in the membrane. The process is dynamic and allows for rapid responses as may be necessary, for example in
the process of blood clotting, where the integrin known as GPIbIIIa (on the surface of blood platelets) attaches to fibrin in a clot as
it develops.
Integrins work along with other receptors, including immunoglobulins, other cell adhesion molecules, cadherins, selectins, and
syndecans. In mammals the proteins have a large number of subunits - 18 α- and 8 β-chains. They are a bridge between its links
outside the cell to the extracellular matrix (ECM) and its links inside the cell to the cytoskeleton. Integrins play central role in
formation and stability of focal adhesions. These are large molecular complexes arising from clustering of integrin-ECM
connections. In the process of cellular movement, integrins at the “front” of the cell (in the direction of the movement), make new
attachments to substrate and release connections to substrate in the back of the cell. These latter integrins are then endocytosed and
reused.
Integrins also help to modulate signal transduction through tyrosine kinase receptors in the cell membrane by regulating movement
of adapters to the plasma membrane. β1c integrin, for example, recruits the Shp2 phosphatase to the insulin growth factor receptor
to cause it to become dephosphorylated, thus turning off the signal it communicates. Integrins can also help to recruit signaling
molecules inside of the cell to activated tyrosine kinases to help them to communicate their signals.
Cadherins

Figure 2.78 - Extracellular ectodomain of a cadherin

Cadherins (Figure 2.78) constitute a type-1 class of transmembrane proteins playing important roles in cell adhesion. They require
calcium ions to function, forming adherens junctions that hold tissues together (See Figure 2.69). Cells of a specific cadherin type
will preferentially cluster with each other in preference to associating with cells containing a different cadherin type. Caderins are

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both receptors and places for ligands to attach. They assist in the proper positioning of cells in development, separation of different
tissue layers, and cell migration.
Selectins

Figure 2.79 - Selectin bound to a sugar Wikipedia

Selectins (Figure 2.79) are cell adhesion glycoproteins that bind to sugar molecules. As such, they are a type of lectin - proteins that
bind sugar polymers (see HERE also). All selectins have an N-terminal calcium-dependent lectin domain, a single transmembrane
domain, and an intracellular cytoplasmic tail.
There are three different types of selectins, 1) E-selectin (endothelial); 2) L (lymphocytic; and 3) P (platelets and endothelial cells.
Selectins function in lymphocyte homing (adhesion of blood lymphocytes to cells in lymphoid organs), in inflammation processes,
and in cancer metastasis. Near the site of inflammation, P-selectin on the surface of blood capillary cells interacts with
glycoproteins on leukocyte cell surfaces. This has the effect of slowing the movement of the leukocyte. At the target site of
inflammation, E- selectin on the endothelial cells of the blood vessel and L-selectin on the surface of the leukocyte bind to their
respective carbohydrates, stopping the leukocyte movement. The leukocyte then crosses the wall of the capillary and begins the
immune response. Selectins are involved in the inflammatory processes of asthma, psoriasis, multiple scleroris, and rheumatoid
arthritis.
Laminins
Laminins are extracellular matrix glycoproteins that a major components of the basal lamina and affect cell differentiation,
migration, and adhesion. They are secreted into the extracellular matrix where they are incorporated and are essential for tissue
maintenance and survival. When laminins are defective, muscles may not form properly and give rise to muscular dystrophy.
Laminins are associated with fibronectin, entactin, and perlecan proteins in type IV collagen networks and bind to integrin
receptors in the plasma membrane. As a consequence, laminins contribute to cellular attachment, differentiation, shape, and
movement. The proteins are trimeric in structure, having one α-chain, a β-chain, and a γ-chain. Fifteen combinations of different
chains are known.
Vitronectin
Vitronectin is a glycoprotein (75kDa) found in blood serum (platelets), the extracellular matrix, and in bone. It promotes the
process of cell adhesion and spreading and binds to several protease inhibitors (serpins). It is secreted from cells and is believed to
play roles in blood clotting and the malignancy of tumors. One domain of vitronectin binds to plasminogen activator inhibitor and
acts to stabilize it. Another domain of the protein binds to cellular integrin proteins, such as the vitronectin receptor that anchors
cells to the extracellular matrix.
Catenins

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Catenins are a family of proteins interacting with cadherin proteins in cell adhesion (Figure 2.69). Four main types of catenins are
known, α-, β-, γ-, and δ-catenin. Catenins play roles in cellular organization before development occurs and help to regulate cellular
growth. α-catenin and β-catenin are found at adherens junctions with cadherin and help cells to maintain epithelial layers.
Cadherins are connected to actin filaments of the cytoskeleton and catenins play the critical role. Catenins are important for the
process whereby cellular division is inhibited when cells come in contact with each other (contact inhibition).
When catenin genes are mutated, cadherin cell adhesions can disappear and tumorigenesis may result. Catenins have been found to
be associated with colorectal and numerous other forms of cancer.
Glycophorins

Figure 2.80 - Glycophorin a

All of the membrane proteins described so far are notable for the connections they make to other proteins and cellular structures.
Some membrane proteins, though, are designed to reduce cellular connections to proteins of other cells. This is particularly
important for blood cells where “stickiness” is undesirable except where clotting is concerned.
Glycophorins (Figure 2.80) are membrane-spanning sialoglycoproteins of red blood cells. They are heavily glycosylated (60%).and
rich in sialic acid, giving the cells a very hydrophilic (and negatively charged) coat, which enables them to circulate in the
bloodstream without adhering to other cells or the vessel walls.
Five glycophorins have been identified - four (A,B,C,and D) from isolated membranes and a fifth form (E) from coding in the
human genome. The proteins are abundant, forming about 2% of the total membrane proteins in these cells. Glycophorins have
important roles in regulating RBC membrane mechanical properties and shape. Because some glycophorins can be expressed in
various nonerythroid tissues (particularly Glycophorin C), the importance of their interactions with the membrane skeleton may
have a considerable biological significance.
Cooperativity and allosterism - quaternary structure

Figure 2.81 - Two polypeptide units of a protein interact in quaternary structure Wikipedia

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Quaternary structure, of course describes the interactions of individual subunits of a multi-subunit protein (Figure 2.81). The result
of these interactions can give rise to important biological phenomena, such as cooperative binding of substrates to a protein and
allosteric effects on the action of an enzyme.
Allosteric effects can occur by a series of mechanisms, but a common feature is that binding of an effector to an enzyme subunit
causes (or locks) the enzyme in either a Tstate (less activity) or an R-state (more activity). Effectors can be enzyme substrates
(homotropic effectors) or non-substrates (heterotropic effectors). Allosterism will be covered in more depth in the Catalysis chapter
HERE.
We begin our consideration of quaternary structure with a discussion of cooperativity, how it arises in the multi-subunit protein
hemoglobin and how its properties contrast with those of the related, single subunit protein myoglobin.
Cooperativity
Cooperativity is defined as the phenomenon where binding of one ligand molecule by a protein favors the binding of additional
molecules of the same type. Hemoglobin, for example, exhibits cooperativity when the binding of an oxygen molecule by the iron
of the heme group in one of the four subunits causes a slight conformation change in the subunit. This happens because the heme
iron is attached to a histidine side chain and binding of oxygen ‘lifts’ the iron along with the histidine ring (also known as the
imidazole ring).

Movie 2.3 - Hemoglobin’s structural changes on binding oxygen Wikipedia

Since each hemoglobin subunit interacts with and influences the other subunits, they too are induced to change shape slightly when
the first subunit binds to oxygen (a transition described as going from the T-state to the R-state). These shape changes favor each of
the remaining subunits binding oxygen, as well. This is very important in the lungs where oxygen is picked up by hemoglobin,
because the binding of the first oxygen molecule facilitates the rapid uptake of more oxygen molecules. In the tissues, where the
oxygen concentration is lower, the oxygen leaves hemoglobin and the proteins flips from the R-state back to the Tstate.
CO2 transport

Figure 2.82 - Heme structure within hemoglobin Image by Aleia Kim

Cooperativity is only one of many fascinating structural aspects of hemoglobin that help the body to receive oxygen where it is
needed and pick it up where it is abundant. Hemoglobin also assists in the transport of the product of cellular respiration (carbon
dioxide) from the tissues producing it to the lungs where it is exhaled. Like the binding of oxygen to hemoglobin, binding of other
molecules to hemoglobin affects its affinity for oxygen. The effect is particularly pronounced when comparing the oxygen binding

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characteristics of hemoglobin’s four subunits with the oxygen binding of the related protein myoglobin’s single subunit (Figure
2.83).
Different oxygen binding

Figure by Aleia Kim

Like hemoglobin, myoglobin contains an iron in a heme group that binds to oxygen. The structure of the globin protein in
myoglobin is very similar to the structure of the globins in hemoglobin and hemoglobin is thought to have evolved from myoglobin
in evolutionary history. As seen in Figure 2.83, the binding curve of hemoglobin for oxygen is S-shaped (sigmoidal), whereas the
binding curve for myoglobin is hyperbolic. What this tells us is that hemoglobin’s affinity for oxygen is low at a low concentration
oxygen, but increases as the oxygen concentration increases. Since myoglobin very quickly saturates with oxygen, even under low
oxygen concentrations, it says that its affinity for oxygen is high and doesn’t change.

Figure 2.84 - Binding of oxygen at the heme center of hemoglobin Image by Aleia Kim
Because myoglobin has only a single subunit, binding of oxygen by that subunit can’t affect any other subunits, since there are no
other subunits to affect. Consequently, cooperativity requires more than one subunit. Therefore, hemoglobin can exhibit
cooperativity, but myoglobin can’t. It is worth noting that simply having multiple subunits does not mean cooperativity will exist.
Hemoglobin is one protein that exhibits the characteristic, but many multisubunit proteins do not.

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 Interactive 2.2 - Hemoglobin in the presence (top) and absence (bottom) of oxygen
Storage vs. delivery
The lack of ability of myoglobin to adjust its affinity for oxygen according to the oxygen concentration (low affinity at low oxygen
concentration, such as in tissues and high affinity at high oxygen concentration, such as in the lungs) means it is better suited for
storing oxygen than for delivering it according to the varying oxygen needs of and animal body. As we shall see, besides
cooperativity, hemoglobin has other structural features that allow it to deliver oxygen precisely where it is needed most in the body.

Figure 2.85 - Sequential model of binding. The sequential model is one way to explain hemoglobin’s cooperativity. Squares
represent no oxygen bound. Circles represent subunits bound with oxygen and rounded subunits correspond to units whose affinity
for oxygen increases by interacting with a subunit that has bound oxygen. Image by Aleia Kim 131
Bohr effect

Figure 2.86 - The Bohr effect with respect to pH changes Image by Aleia Kim
The Bohr Effect was first described over 100 years ago by Christian Bohr, father of the famous physicist, Niels Bohr. Shown
graphically (Figures 2.86, 2.87, and 2.88), the observed effect is that hemoglobin’s affinity for oxygen decreases as the pH
decreases and as the concentration of carbon dioxide increases. Binding of the protons and carbon dioxide by amino Figure 2.85 -
Sequential model of binding. The sequential model is one way to explain hemoglobin’s cooperativity. Squares represent no oxygen
bound. Circles represent subunits bound with oxygen and rounded subunits correspond to units whose affinity for oxygen increases
by interacting with a subunit that has bound oxygen. Image by Aleia Kim acid side chains in the globin proteins helps to facilitate

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structural changes in them. Most commonly, the amino acid affected by protons is histidine #146 of the β strands. When this
happens, the ionized histidine can form an ionic bond with the side chain of aspartic acid #94, which has the effect of stabilizing
the T-state (reduced oxygen binding state) and releasing oxygen. Other histidines and the amine of the amino terminal amino acids
in the α-chains are also binding sites for protons.

Figure 2.87 - Binding affinity of hemoglobin for oxygen under different conditions Image by Aleia Kim
2,3-BPG

Figure 2.88 - The Bohr effect physiologically - oxygen binding curves for resting muscle (blue), active muscle (green) and
reference muscle (orange) with respect to pH, 2,3-BPG, and CO2 Image by Aleia Kim
Another molecule favoring the release of oxygen by hemoglobin is 2,3- bisphosphoglycerate (also called 2,3-BPG or just BPG -
Figure 2.89). Like protons and carbon dioxide, 2,3-BPG is produced by actively respiring tissues, as a byproduct of glucose
metabolism. The 2,3-BPG mole cule fits into the ‘hole of the donut’ of adult hemoglobin (Figure 2.89). Such binding of 2,3-BPG
favors the T-state (tight - low oxygen binding) of hemoglobin, which has a reduced affinity for oxygen. In the absence of 2,3-BPG,
hemoglobin can more easily exist in the R-state (relaxed - higher oxygen binding), which has a high affinity for oxygen.
Smokers

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 Figure 2.89 - The structure of 2,3 bisphosphoglycerate (2,3-BPG)
Notably, the blood of smokers is higher in the concentration of 2,3-BPG than non-smokers, so more of their hemoglobin remains in
the T-state and thus the oxygen carrying capacity of smokers is lower than non-smokers.Another reason why smokers’ oxygen
carrying capacity is lower than that of non-smokers is that cigarette smoke contains carbon monoxide and this molecule, which has
almost identical dimensions to molecular oxygen, effectively outcompetes with oxygen for binding to the iron atom of heme
(Figure 2.90). Part of carbon monoxide’s toxicity is due to its ability to bind hemoglobin and prevent oxygen from binding.

Figure 2.90 - Binding of oxygen (left) and carbon monoxide (right) by a heme group of hemoglobin Image by Aleia Kim
Carbon dioxide

Figure 2.91 - Hemoglobin’s hole of the donut for binding 2,3-BPG Wikipedia
Carbon dioxide binds to form a carbamate when binding the α-amine of each globin chain. The process of forming this structure
releases a proton, which helps to further enhance the Bohr effect. Physiologically, the binding of CO2 and H+ has significance
because actively respiring tissues (such as contracting muscles) require oxygen and release protons and carbon dioxide. The higher
the concentration of protons and carbon dioxide, the more oxygen is released to feed the tissues that need it most.
About 40% of the released protons and about 20% of the carbon dioxide are carried back to the lungs by hemoglobin. The
remainder travel as part of the bicarbonate buffering system or as dissolved CO2. In the lungs, the process reverses itself. The lungs
have a higher pH than respiring tissues, so protons are released from hemoglobin and CO2 too is freed to be exhaled.

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Fetal hemoglobin

Figure 2.92 - Formation of bicarbonate from CO2 in blood

Adult hemoglobin releases oxygen when it binds 2,3- BPG. This is in contrast to fetal hemoglobin, which has a slightly different
configuration (α2γ2) than adult hemoglobin (α2β2). Fetal hemoglobin has a greater affinity for oxygen than maternal hemoglobin,
allowing the fetus to obtain oxygen effectively from the mother’s blood. Part of the reason for fetal hemoglobin’s greater affinity
for oxygen is that it doesn’t bind 2,3-BPG. Consequently, fetal hemoglobin remains in the R-state much more than adult
hemoglobin and because of this, fetal hemoglobin has greater affinity for oxygen than adult hemoglobin and can take oxygen away
from adult hemoglobin. Thus, the fetus can get oxygen from the mother.

Figure 2.93 - Comparison of oxygen binding of myoglobin (blue), fetal hemoglobin (orange), and adult hemoglobin (green) Image
by Aleia Kim
Sickle cell disease

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 Figure 2.94 - Four normal red blood cells (right) and one sickled red blood cell (left) Wikipedia
Mutations to the globin genes coding for hemoglobin can sometimes have deleterious consequences. Sickle cell disease (also called
sickle cell anemia) is a genetically transmitted disease that arises from such mutations. There are different forms of the disease. It is
a recessive trait, meaning that to be afflicted with it, an individual must inherit two copies of the mutated gene.

Figure 2.95 - Movement of blood in capillaries. Top - normal red blood cells. Bottom - sickled red blood cells
The predominant form of hemoglobin in adults is hemoglobin A, designated HbA (two α chains and two β chains). The mutant
form is known as HbS. The most common mutation is an A to T mutation in the middle of the codon for the seventh amino acid
(some counting schemes call it the sixth amino acid) of the β-chain. This results in conversion of a GAG codon to GTG and thus
changes the amino acid specified at that position from a glutamic acid to a valine. This minor change places a small hydrophobic
patch of amino acids on the surface of the β-globin chains.
Polymerization

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 Figure 2.96 - Pattern of expression of six different globins of hemoglobin - α,β,γ,ε,δ, and ζ Image by Aleia Kim
Under conditions of low oxygen, these hydrophobic patches will associate with each other to make long polymers of hemoglobin
molecules. The result is that the red blood cells containing them will change shape from being rounded to forming the shape of a
sickle (Figure 2.94). Rounded red blood cells readily make it through tiny capillaries, but sickleshaped cells do not.
Worse, they block the flow of other blood cells. Tissues where these blockages occur are already low in oxygen, so stopping the
flow of blood through them causes them to go quickly anaerobic, causing pain and, in some cases, death of tissue. In severe
circumstances, sickled red blood cells death may result. The disease is referred to as an anemia because the sickling of the red
blood cells targets them for removal by the blood monitoring system of the body, so a person with the disease has chronically
reduced numbers of red blood cells.
Heterozygote advantage
Interestingly, there appears to be a selective advantage to people who are heterozygous for the disease in areas where malaria is
prominent. Heterozygotes do not suffer obvious ill effects of the disease, but their red blood cells appear to be more susceptible to
rupture when infected. As a consequence, the parasite gets less of a chance to reproduce and the infected person has a greater
chance of survival.
The protective effect of the mutant gene, though, does not extend to people who suffer the full blown disease (homozygotes for the
mutant gene). Treatments for the disease include transfusion, pain management, and avoidance of heavy exertion. The drug
hydroxyurea has been linked to reduction in number and severity of attacks, as well as an increase in survival time1,2. It appears to
work by reactivating expression of the fetal hemoglobin gene, which typically is not synthesized to any significant extent normally
after about 6 weeks of age.
Oxygen binding
Animals have needs for oxygen that differ from all other organisms. Oxygen, of course, is the terminal electron acceptor in animals
and is necessary for electron transport to work. When electron transport is functioning, ATP generation by cells is many times more
efficient than when it is absent. Since abundant ATP is essential for muscular contraction and animals move around a lot - to catch
prey, to exercise, to escape danger, etc., having an abundant supply of oxygen is important.
This is particularly a concern deep inside tissues where diffusion of oxygen alone (as occurs in insects) does not deliver sufficient
quantities necessary for long term survival. The issue is not a problem for plants since, for the most part, their motions are largely
related to growth and thus don’t have rapidly changing needs/demands for oxygen that animals have. Unicellular organisms have a
variety of mechanisms for obtaining oxygen and surviving without it. Two other important oxygen binding proteins besides
hemoglobin are myoglobin and hemocyanin.
Myoglobin

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 Figure 2.97 - Myoglobin bound to oxygen
Myoglobin is the primary oxygen-storage protein found in animal muscle tissues. In contrast to hemoglobin, which circulates
throughout the body, myoglobin protein is only found in muscle tissue and appears in the blood only after injury. Like hemoglobin,
myoglobin binds oxygen at a prosthetic heme group it contains.
The red color of meat arises from the heme of myoglobin and the browning of meat by cooking it comes from oxidation of the
ferrous (Fe++) ion of myoglobin’s heme to the ferric (Fe+++) ion via oxidation in the cooking process. As meat sits in our
atmosphere (an oxygen-rich environment), oxidation of Fe++ to Fe+++ occurs, leaving the brown color noted above. If meat is
stored in a carbon monoxide (CO) environment, CO binds to the heme group and reduces the amount of oxidation, keeping meat
looking red for a longer period of time.
High affinity
Myoglobin (Figure 2.97) displays higher affinity for oxygen at low oxygen concentrations than hemoglobin and is therefore able to
absorb oxygen delivered by hemoglobin under these conditions. Myoglobin’s high affinity for oxygen makes it better suited for
oxygen storage than delivery. The protein exists as a single subunit of globin (in contrast to hemoglobin, which contains four
subunits) and is related to the subunits found in hemoglobin. Mammals that dive deeply in the ocean, such as whales and seals,
have muscles with particularly high abundance of myoglobin. When oxygen concentration in muscles falls to low levels,
myoglobin releases its oxygen, thus functioning as an oxygen “battery” that delivers oxygen fuel when needed and holding onto it
under all other conditions. Myoglobin holds the distinction of being the first protein for which the 3D structure was determined by
X-ray crystallography by John Kendrew in 1958, an achievement for which he later won the Nobel Prize.

Figure 2.98 - Oxygen bound at heme of myoglobin Wikipedia

Hemocyanin

Figure 2.99 - Oxygen binding in hemocyanin Wikipedia

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Hemocyanin is the protein transporting oxygen in the bodies of molluscs and arthropods. It is a coppercontaining protein found not
within blood cells of these organisms, but rather is suspended in the circulating hemolymph they possess. The oxygen binding site
of hemocyanin contains a pair of copper(I) cations directly coordinated to the protein by the imidazole rings of six histidine side
chains.

Figure 2.100 - Hemocyanin (purple) in a red rock crab Wikipedia

Most, but not all hemocyanins bind oxygen non-cooperatively and are less efficient than hemoglobin at transporting oxygen.
Notably, the hemocyanins of horseshoe crabs and some other arthropods do, in fact, bind oxygen cooperatively. Hemocyanin
contains many subunit proteins, each with two copper atoms that can bind one oxygen molecule (O2). Subunit proteins have atomic
masses of about 75 kilodaltons (kDa). These may be arranged in dimers or hexamers depending on species. Superstructures
comprised of dimer or hexamer complexes are arranged in chains or clusters and have molecular weights of over 1500 kDa.

This page titled 2.4: Structure and Function- Proteins II is shared under a CC BY-NC-SA license and was authored, remixed, and/or curated by
Kevin Ahern, Indira Rajagopal, & Taralyn Tan.

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2.5: Structure and Function- Protein Function II
Source: BiochemFFA_2_4.pdf. The entire textbook is available for free from the authors at
http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy
To this point, the proteins we have discussed have not been catalysts (enzymes). The majority of proteins in cells, however,
catalyze reactions. In this section we begin our discussion of a subclass of proteins that catalyze reactions releasing energy and
convert it into mechanical force. These operate at the cellular and organismal level and are known as motor proteins. Motor
proteins rely on globular structural proteins, so it is important that we describe how these cellular “railways” are assembled before
discussing the motor proteins themselves. There are two relevant fibrous structures serving as rails for motor proteins. They are:
1. microfilaments (composed of an actin polymer) and
2. microtubules (composed of a polymer of tubulin.

Actin
The monomeric unit of actin is called G-actin (globular actin) and the polymer is known as F-actin (filamentous actin). Filaments
of Factin comprise the smallest filaments of cells known as microfilaments (Figure 2.101). Actin is essential for muscular
contraction and also has diverse roles in cellular signaling and maintenance of cell junctions. In conjunction with other proteins,
actin has numerous interactions with the cell membrane. The β- and γ-forms of actin are components of the cytoskeleton and
facilitate motility inside of cells. α-actin is important in muscle tissues, where it is used by myosin in the mechanical process of
contraction (See HERE).

Figure 2.101 - Model of actin filaments. Image used with permission (CC BY-SA 3.0; Thomas Splettstoesser).
Monomeric and polymeric forms of actin play roles in cellular activities relating to motion. Two parallel F-actin strands can pair
with each other and create a double helical structure with 2.17 subunits per turn of the helix. Helical F-actin in muscles contains
tropomyosin, which covers the actin binding sites for myosin in resting muscles to prevent contraction. Other proteins bound to
actin muscle filaments include the troponins (I, T, and C).

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Actin Cellular Action
Examples of actin action at the cellular level include cell motility, cytokinesis, intracellular transport of vesicles and organelles, and
cell shape. Each actin monomer is bound to a molecule of ATP or ADP and the presence of one of these is essential for proper G-
actin functioning.

Figure 2.102 - Attachment of actin at the cell membrane complex known as the adherens junction Wikipedia

The role of ATP

In the monomer, actin is more commonly bound to ATP, whereas in the filaments, it is typically bound to ADP. Actin is an
inefficient ATPase, breaking the molecule down slowly, but the catalysis speeds up as much as 40,000 fold when the monomer
begins to polymerize. Actin also has a binding site for divalent cations - either calcium or magnesium. F-
Actin binds to structural proteins at the adherens junction (Figure 2.102). These include α-actinin, vinculin (provides a membrane
connection and connections to the catenins and cadherin).
Polymerization

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 Figure 2.103 - Polymerization of G-actin monomers into Factin polymers
Polymerization of actin begins with a nucleating event (Figure 2.103). One factor known to affect the process is known as the Arp
2/3 complex. It does this by mimicking an actin dimer, starting an autocatalytic process of actin assembly. The Arp 2/3 complex
plays roles both in the initiation of polymerization of new actin filaments as well as the formation of branches in the filaments.
Two proteins play roles in modulating polymer growth. Thymosin functions on the end of actin filaments to control growth.
Profilin works on G-actin monomers exchanging ADP for ATP, promoting addition of monomers to a growing chain.
F-actin filaments are held together by relatively weak bonds compared to the covalent bonds of the monomers of nucleic acids, thus
allowing for easier disassembly when desired. Actin’s amino acid sequence is optimized, having diverged only a relatively small
amount (20%) between algae and humans. Mutations in the actin gene result in muscular diseases and/or deafness.
Tubulin

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 Figure 2.104 Microtubule structure Wikipedia
Tubulin proteins are the monomeric building blocks of eukaryotic microtubules (Figure 2.104 & 2.105). Bacterial (TubZ) and
archaeon (FtsZ) equivalents are known. The α-tubulin and β-tubulin proteins polymerize to make microtubule structures in the
cytoplasm of cells. Microtubules are major components of the cytoskeleton of eukaryotic cells, providing structural support,
transport within the cell, and functions necessary for segregation of DNAs during cell division.
Dimerization of the α-tubulin and β-tubulin proteins is necessary for polymerization and requires that the subunits bind to GTP.
Microtubules only grow in one direction. β- tubulin is found on the plus end of the tubule (growth end = plus end) and α-tubulin is
exposed on the other end (non-growth end = minus end). Dimers of α-tubulin/β-tubulin are incorporated into growing microtubules
in this orientation. If a dimer is bound to GDP instead of GTP, it tends to be unstable and fall apart, whereas those bound to GTP
stably assemble into microtubules.

Figure 2.105 - Microtubule anatomy Wikipedia

Microtubules
Microtubules, along with microfilaments and intermediate filaments (see HERE) constitute the cytoskeleton of cells. Found in the
cytoplasm, they are found in eukaryotic cells, as well as some bacteria. Microtubules help to give cells structure. They comprise the
inner structure of flagella and cilia and provide rail-like surfaces for the transport of materials within cells.

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 Figure 2.106 - Kinesin and dynein “walk” along microtubules, but move in opposite directions Image by Aleia Kim
Polymerization of α- tubulin and β-tubulin to form microtubules occurs after a nucleating event. Individual units get arranged in
microtubule organizing centers (MTOCs), an example of which is the centrosome. Centrosomes are focal points of connection of
microtubules. Basal bodies of cilia and flagella also help to organize microtubules.

Motor proteins
From the transport of materials within a cell to the process of cytokinesis where one cell splits into two in mitosis, a cell has needs
for motion at the molecular level. Secretory vesicles and organelles must be transported. Chromosomes must be separated in
mitosis and meiosis.
The proteins dynein and kinesin (Figure 2.106) are necessary for intracellular movement. These motor proteins facilitate the
movement of materials inside of cells along microtubule “rails”. These motor proteins are able to move along a portion of the
cytoskeleton by converting chemical energy into motion with the hydrolysis of ATP. An exception is flagellar rotation, which uses
energy provided from a gradient created by a proton pump.
Kinesins and dyneins
As noted, kinesins and dyneins navigate in cells on microtubule tracks (Figure 2.108 & Movie 2.4). Most kinesins move in the
direction of the synthesis of the microtubule (+ end movement), which is generally away from the cell center and the opposite
direction of movement of dyneins, which are said to do retrograde transport toward the cell center. Both proteins provide
movement functions necessary for the processes of mitosis and meiosis. These include spindle formation, chromosome separation,
and shuttling of organelles, such as the mitochondria, Golgi apparatuses, and vesicles.

Figure 2.107 - Kinesin. “Feet” are at the top.

Kinesins are comprised of two heavy chains and two light chains. The head motor domains of heavy chains (in the feet) use energy
of ATP hydrolysis to do mechanical work for the movement along the microtubules. There are at least fourteen distinct kinesin
families and probably many related ones in addition.

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 Figure 2.105 are the stalk and globular head of the structure here. Wikipedia
Dyneins are placed into two groups - cytoplasmic and axonemal (also called ciliary or flagellar dyneins - Figure 2.109). Dyneins
are more complex in structure than kinesins with many small polypeptide units. Notably, plants do not have dynein motor proteins,
but do contain kinesins.

Movie 2.4 The motor protein kinesin walking down a microtubule. Image used with permission (Public Domain; zp706).

Myosin
An important group of motor proteins in the cell is the myosins. Like kinesins and dyneins, myosins use energy from hydrolysis of
ATP for movement. In this case, the movement is mostly not along microtubules, but rather along microfilaments comprised of a
polymer of actin (F-actin). Movement of myosin on actin is best known as the driving force for muscular contraction. Myosins are
a huge family of proteins, all of which bind to actin and all of which involve motion. Eighteen different classes of myosin proteins
are known.

Figure 2.109 - Dynein in an axoneme Wikipedia

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Myosin II is the form responsible for generating muscle contraction. It is an elongated protein formed from two heavy chains with
motor heads and two light chains. Each myosin motor head binds actin and has an ATP binding site. The myosin heads bind and
hydrolyze ATP. This hydrolysis produces the energy necessary for myosin to walk toward the plus end of an actin filament.

Figure 2.110 - Actin filament anatomy Wikipedia

Non-muscle myosin IIs provide contraction needed to power the action of cytokinesis. Other myosin proteins are involved in
movement of non-muscle cells. Myosin I is involved in intracellular organization. Myosin V performs vesicle and organelle
transport. Myosin XI provides movement along cellular microfilament networks to facilitate organelle and cytoplasmic streaming
in a particular direction.
Structure
Myosins have six subunits, two heavy chains and four light chains. Myosin proteins have domains frequently described as a head
and a tail (Figure 2.111). Some also describe an intermediate hinge region as a neck. The head portion of myosin is the part that
binds to actin. It uses energy from ATP hydrolysis to move along the actin filaments. In muscles, myosin proteins form aggregated
structures referred to as thick filaments. Movements are directional.

Figure 2.111 - Myosin protein anatomy Wikipedia

Structural considerations of muscular contraction
Before we discuss the steps in the process of muscular contraction, it is important to describe anatomical aspects of muscles and
nomenclature.
There are three types of muscle tissue - skeletal (striated), smooth, and (in vertebrates) cardiac. We shall concern ourselves mostly
here with skeletal muscle tissue. Muscles may be activated by the central nervous system or, in the case of smooth and cardiac
muscles, may contract involuntarily. Skeletal muscles may be slow twitch or fast twitch.

Figure 2.112 - Structural components of muscle. Wikipedia

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Sarcomeres
Sarcomeres are described as the basic units comprising striated muscles and are comprised of thick (myosin) and thin (actin)
filaments and a protein called titin. The filaments slide past each other in muscular contraction and then backwards in muscular
relaxation. They are not found in smooth muscles.
Under the microscope, a sarcomere is the region between two Z-lines of striated muscle tissue (Figure 2.112). The Z-line is the
distinct, narrow, dark region in the middle of an I-band. Within the sarcomere is an entire Aband with its central H-zone. Within the
Hzone are located tails of myosin fibers, with the head pointed outwards from there projecting all the way to the I-band. The
outside of the Aband is the darkest and it gets lighter moving towards the center.
Within the Iband are located thin filaments that are not occupied with thick myosin filaments. The Aband contains intact thick
filaments overlaying thin filaments except in the central H zone, which contains only thick filaments. In the center of the H-zone is
a line, known as the M-line. It contains connecting elements of the cellular cytoskeleton. In muscular contraction, myosin heads
walk along pulling their tails over the actin thin filaments, using energy from hydrolysis of ATP and pulling them towards the
center of the sarcomere.

Figure 2.113 - Skeletal muscle longitudinal section. Wikipedia

Sarcolemma
The sarcolemma (also known as the myolemma) is to muscle cells what the plasma membrane is to other eukaryotic cells - a barrier
between inside and outside. It contains a lipid bilayer and a glycocalyx on the outside of it. The glyocalyx contains polysaccharides
and connects with the basement membrane. The basement membrane serves a s scaffolding to connect muscle fibers to. This
connection is made by transmembrane proteins bridging the actin cytoskeleton on the inside of the cell with the basement
membrane on the outside. On the ends of the muscle fibers, each sarcolemma fuses with a tendon fiber and these, in turn, adhere to
bones.
Sarcoplasmic reticulum
The sarcoplasmic reticulum (Figure 2.114) is a name for the structure found within muscle cells that is similar to the smooth
endoplasmic reticulum found in other cells. It contains a specialized set of proteins to meet needs unique to muscle cells. The
organelle largely serves as a calcium “battery,” releasing stored calcium to initiate muscular contraction when stimulated and taking
up calcium when signaled at the end of the contraction cycle. It accomplishes these tasks using calcium ion channels for release of
the ion and specific calcium ion pumps to take it up.
Movement direction
All myosins but myosin VI move towards the + end (the growing end) of the microfilament. The neck portion serves to link the
head and the tail. It also a binding site for myosin light chain proteins that form part of a macromolecular complex with regulatory
functions. The tail is the point of attachment of molecules or other “cargo” being moved. It can also connect with other myosin
subunits and may have a role to play in controlling movement.

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 Figure 2.114 - Anatomy of a muscle fiber Wikipedia
Muscular contraction
The sliding filament model has been proposed to describe the process of muscular tension/contraction. In this process a repeating
set of actions slide a thin actin filament over a thick myosin filament as a means of creating tension/ shortening of the muscle fiber.
Steps in the process occur as follows:
A. A signal from the central nervous system (action potential) arrives at a motor neuron, which it transmits towards the
neuromuscular junction (see more on the neurotransmission part of the process HERE)

Figure 2.115 - 1. Activation of a muscle cell by release of calcium (step H) Wikipedia

B. At the end of the axon, the nerve signal stimulates the opening of calcium channels at the axon terminus causing calcium to flow
into the terminal.

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C. Movement of calcium into the axon of the nerve causes acetylcholine (a neurotransmitter) in synaptic vesicles to fuse with the
plasma membrane. This causes the acetylcholine to be expelled into the synaptic cleft between the axon and the adjacent skeletal
muscle fiber.

Figure 2.116 - 2. Calcium binding by troponin allows myosin to access actin sites (I). Wikipedia
D. Acetylcholine diffuses across the synapse and then binds to nicotinic acetylcholine receptors on the neuromuscular junction,
activating them.
E. Activation of the receptor stimulates opening gates of sodium and potassium channels, allowing sodium to move into the cell
and potassium to exit. The polarity of the membrane of the muscle cell (called a sarcolemma - Figure 2.111) changes rapidly
(called the end plate potential).

Figure 2.117 - 3. ATP cleavage by myosin allows actin attachment (J) Wikipedia
F. Change in the end plate potential results in opening of voltage sensitive ion channels specific for sodium or potassium only to
Figure 2.117 - 3. ATP cleavage by myosin allows actin attachment (J) Wikipediaopen, creating an action potential (voltage change)
that spreads throughout the cell in all directions.
G. The spreading action potential depolarizes the inner muscle fiber and opens calcium channels on the sarcoplasmic reticulum
(Figure 2.115).
H. Calcium released from the sarcoplasmic reticulum binds to troponin on the actin filaments (Figure 2.115).
I. Troponin alters the structure of the tropomyosin to which is it bound. This causes tropomyosin to move slightly, allowing access
to myosin binding sites on the microfilament (also called thin filament) that it was covering (Figure 2.116).
J. Myosin (bound to ATP) cleaves the ATP to ADP and Pi, which it holds onto in its head region and then attaches itself to the
exposed binding sites on the thin filaments causing inorganic phosphate to be released from the myosin followed by ADP (Figure

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2.117).

Figure 2.118 - 4. Release of Pi causes myosin hinge to bend. Thin filament pulled left (K). Wikipedia
K. Release of ADP and Pi is tightly coupled to a bending of the myosin hinge, resulting in what is called the power stroke. This
causes the thin filament to move relative to the thick fibers of myosin (Figures 2.118 & 2.119).

Figure 2.119 - 5. Release of ADP favors further bending of hinge and movement of thin filament leftward (K). Wikipedia
L. Such movement of the thin filaments causes the Z lines to be pulled closer to each other. This results in shortening of the
sarcomere as a whole (Figure 2.122) and narrowing of the I band and the H zones (Figure 2.123). M. If ATP is available, it binds to
myosin, allowing it to let go of the actin (Figures 2.120 & 2.121). If ATP is not available, the muscle will remain locked in this
state. This is the cause of rigor mortis in death - contraction without release of muscles

.
Figure 2.120 - When ATP is present, it binds to myosin (M). Wikipedia

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N. After myosin has bound the ATP, it hydrolyzes it, producing ADP and Pi, which are held by the head. Hydrolysis of ATP resets
the hinge region to its original state, unbending it. This unbent state is also referred to as the cocked position.
O.If tropomyosin is still permitting access to binding sites on actin, the process repeats so long as ATP is available and calcium
remains at a high enough concentration to permit it to bond to troponin.

Figure 2.121 - Binding of ATP favors release of myosin from actin site (N) Wikipedia
Relaxation of the muscle tension occurs as the action potential in the muscle cell dissipates. This happens because all of the
following things happen 1) the nerve signal stops; 2) the neurotransmitter is degraded by the enzyme acetylcholinesterase; and 3)
the calcium concentration declines because it is taken up by the sarcoplasmic reticulum.

Figure 2.122 - Sarcomere Anatomy Wikipedia

It should be noted that the sarcoplasmic reticulum is always taking up calcium. Only when its calcium gates are opened by the
action potential is it unable to reduce cellular calcium concentration. As the action potential decreases, then the calcium gates close
and the sarcoplasmic reticulum “catches up” and cellular calcium concentrations fall. At that point troponin releases calcium,
tropomyosin goes back to covering myosin binding sites on actin, myosin loses its attachment to actin and the thin filaments slide
back to their original positions relative to the myosin thick filaments.

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 Figure 2.123 - The Sliding Filament Model of Muscular Contraction Wikipedia
Tropomyosin

Figure 2.124 - Tropomyosin and troponin in muscle anatomy Wikipedia

Tropomyosins are proteins that interact with actin thin filaments to help regulate their roles in movement, both in muscle cells and
non-muscle cells (Figure 2.124). Tropomyosins interact to form head-to-toe dimers and perch along the α-helical groove of an actin
filament. The isoforms of tropomyosin that are in muscle cells control interactions between myosin and the actin filament within
the sarcomere and help to regulate contraction of the muscle. In other cells, nonmuscle tropomyosins help to regulate the
cytoskeleton’s functions.
The interactions of tropomyosin with the cytoskeleton are considerably more complicated than what occurs in muscle cells. Muscle
cells have five tropomyosin isoforms, but in the cytoskeleton of non-muscle cells, there are over 40 tropomyosins.
Troponin

Figure 2.125 - Troponin complex of muscle. Blue = troponin C, magenta = troponin T , green = troponin I
The troponins involved in muscular contraction are actually a complex of three proteins known as troponin I, troponin C, and
troponin T (Figure 2.125). They associate with each other and with tropomyosin on actin filaments to help regulate the process of
muscular contraction. Troponin I prevents binding of myosin’s head to actin and thus prevents the most important step in
contraction.

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Troponin C is a unit that binds to calcium ions. Troponin T is responsible for binding all three proteins to tropomyosin. Troponins
in the bloodstream are indicative of heart disorders. Elevation of troponins in the blood occurs after a myocardial infarction and can
remain high for up to two weeks.
Actinin
Actinin is a skeletal muscle protein that attaches filaments of actin to Z-lines of skeletal muscle cells. In smooth muscle cells, it
also connects actin to dense bodies.
Titin
Titin (also known as connectin) is the molecular equivalent of a spring that provides striated muscle cells with elasticity. It is the
third most abundant protein in muscle cells. The protein is enormous, with 244 folded individual protein domains spread across 363
exons (largest known number), with the largest known exon (17,106 base pairs long), and it is the largest protein known (27,000 to
33,000 amino acids, depending on splicing).
Unstructured sequences
The folded protein domains are linked together by unstructured sequences. The unstructured regions of the protein allow for
unfolding when stretching occurs and refolding upon relaxation. Titin connects the M and Z lines in the sarcomere (Figure 2.123).
Tension created in titin serves to limit the range of motion of the sarcomere, giving rise to what is called passive stiffness.
Skeletal and cardiac muscles have slight amino acid sequence variations in their ti tin proteins and these appear to relate to
differences in the mechanical characteristics of each muscle.
Energy backup for muscle energy
Myoglobin was described as a molecular batter for oxygen. Muscle cells have a better of their own for ATP. The is important for
animals, but not for plants because a plant’s need for energy is different than an animal’s. Plants do not need to access energy
sources as rapidly as animals do, nor do they have to maintain a constant internal temperature. Plants can neither flee predators, nor
chase prey. These needs of animals are much more immediate and require that energy stores be accessible on demand. Muscles, of
course, enable the motion of animals and the energy required for muscle contraction is ATP. To have stores of energy readily
available, muscles have, in addition to ATP, creatine phosphate for energy and glycogen for quick release of glucose to make more
energy. The synthesis of creatine phosphate is a prime example of the effects of concentration on the synthesis of high energy
molecules. For example, creatine phosphate has an energy of hydrolysis of -43.1 kJ/mol whereas ATP has an energy of hydrolysis
of -30.5 kJ/mol. Creatine phosphate, however, is made from creatine and ATP in the reaction shown in Figure 2.126. How is this
possible?

Figure 2.126 - Phosphorylation of creatine (phosphocreatine) - making of a creatine phosphate battery Image by Aleia Kim
The ∆G°’ of this reaction is +12.6 kJ/mol, reflecting the energies noted above. In a resting muscle cell, ATP is abundant and ADP
is low, driving the reaction downward, creating creatine phosphate. When muscular contraction commences, ATP levels fall and
ADP levels climb. The above reaction then reverses and proceeds to synthesize ATP immediately. Thus, creatine phosphate acts
like a battery, storing energy when ATP levels are high and releasing it almost instantaneously to create ATP when its levels fall.

This page titled 2.5: Structure and Function- Protein Function II is shared under a CC BY-NC-SA license and was authored, remixed, and/or
curated by Kevin Ahern, Indira Rajagopal, & Taralyn Tan.

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2.6: Structure and Function - Nucleic Acids
Source: BiochemFFA_2_5.pdf. The entire textbook is available for free from the authors at
http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy
The nucleic acids, DNA and RNA, may be thought of as the information molecules of the cell. In this section, we will examine the
structures of DNA and RNA, and how these structures are related to the functions these molecules perform.

We will begin with DNA, which is the hereditary information in every cell, that is copied and passed on from generation to
generation. The race to elucidate the structure of DNA was one of the greatest stories of 20th century science. Discovered in 1869
by Friedrich Miescher, DNA was identified as the genetic material in experiments in the 1940s led by Oswald Avery, Colin
MacLeod, and Maclyn McCarty. X-ray diffraction work of Rosalind Franklin and the observations of Erwin Chargaff were
combined by James Watson and Francis Crick to form a model of DNA that we are familiar with today. Their famous paper, in the
April 25, 1953 issue of Nature, opened the modern era of molecular biology. Arguably, that one-page paper has had more scientific
impact per word than any other research article ever published. Today, every high school biology student is familiar with the double
helical structure of DNA and knows that G pairs with C and A with T.
The double helix, made up of a pair of DNA strands, has at its core, bases joined by hydrogen bonds to form base pairs - adenine
always paired with thymine, and guanine invariably paired with cytosine. Two hydrogen bonds are formed between adenine and
thymine, but three hydrogen bonds hold together guanine and cytosine (Figure 2.127).

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 Figure 2.127 - A DNA duplex with base pairs, a closeup of base pairing, and a closeup of a nucleotide Wikipedia
The complementary structure immediately suggested to Watson and Crick how DNA might be (and in fact, is) replicated and it
further explains how information is DNA is transmitted to RNA for the synthesis of proteins. In addition to the hydrogen bonds
between bases of each strand, the double helix is held together by hydrophobic interactions of the stacked, non-polar bases.
Crucially, the sequence of the bases in DNA carry the information for making proteins. Read in groups of three, the sequence of the
bases directly specifies the sequence of the amino acids in the encoded protein.

Structure
A DNA strand is a polymer of nucleoside monophosphates held together by phosphodiester bonds. Two such paired strands make
up the DNA molecule, which is then twisted into a helix. In the most common Bform, the DNA helix has a repeat of 10.5 base
pairs per turn, with sugars and phosphate forming the covalent phosphodiester “backbone” of the molecule and the adenine,
guanine, cytosine, and thymine bases oriented in the middle where they form the now familiar base pairs that look like the rungs of
a ladder.

Building blocks
The term nucleotide refers to the building blocks of both DNA (deoxyribonucleoside triphosphates, dNTPs) and RNA
(ribonucleoside triphosphates, NTPs). In order to discuss this important group of molecules, it is necessary to define some terms.
Nucleotides contain three primary structural components. These are a nitrogenous base, a pentose sugar, and at least one phosphate.
Molecules that contain only a sugar and a nitrogenous base (no phosphate) are called nucleosides. The nitrogenous bases found in
nucleic acids include adenine and guanine (called purines) and cytosine, uracil, or thymine (called pyrimidines). There are two
sugars found in nucleotides - deoxyribose and ribose (Figure 2.128). By convention, the carbons on these sugars are labeled 1’ to
5’. (This is to distinguish the carbons on the sugars from those on the bases, which have their carbons simply labeled as 1, 2, 3,
etc.) Deoxyribose differs from ribose at the 2’ position, with ribose having an OH group, where deoxyribose has H.

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 Figure 2.128 - Nucleotides, nucleosides, and bases
Nucleotides containing deoxyribose are called deoxyribonucleotides and are the forms found in DNA. Nucleotides containing
ribose are called ribonucleotides and are found in RNA. Both DNA and RNA contain nucleotides with adenine, guanine, and
cytosine, but with very minor exceptions, RNA contains uracil nucleotides, whereas DNA contains thymine nucleotides. When a
base is attached to a sugar, the product, a nucleoside, gains a new name.
uracil-containing = uridine (attached to ribose) / deoxyuridine (attached to deoxyribose)
thymine-containing = ribothymidine (attached to ribose) / thymidine (attached to deoxyribose)
cytosine-containing = cytidine (attached to ribose - Figure 2.129) / deoxycytidine (attached to deoxyribose)
guanine-containing = guanosine (attached to ribose) / deoxyguanosine (attached to deoxyribose)
adenine-containing = adenosine (attached to ribose) / deoxyadenosine (attached to deoxyribose)
Of these, deoxyuridine and ribothymidine are the least common. The addition of one or more phosphates to a nucleoside makes it a
nucleotide. Nucleotides are often referred to as nucleoside phosphates, for this reason. The number of phosphates in the nucleotide
is indicated by the appropriate prefixes (mono, di or tri).

Figure 2.129 Cytidine

Thus, cytidine, for example, refers to a nucleoside (no phosphate), but cytidine monophosphate refers to a nucleotide (with one
phosphate). Addition of second and third phosphates to a nucleoside monophosphate requires energy, due to the repulsion of
negatively charged phosphates and this chemical energy is the basis of the high energy triphosphate nucleotides (such as ATP) that
fuel cells.
Note: Ribonucleotides as Energy Sources
Though ATP is the most common and best known cellular energy source, each of the four ribonucleotides plays important roles
in providing energy. GTP, for example, is the energy source for protein synthesis (translation) as well as for a handful of
metabolic reactions. A bond between UDP and glucose makes UDP-glucose, the building block for making glycogen. CDP is
similarly linked to several different molecular building blocks important for glycerophospholipid synthesis (such as CDP-
diacylglycerol).
The bulk of ATP made in cells is not from directly coupled biochemical metabolism, but rather by the combined processes of
electron transport and oxidative phosphorylation in mitochondria and/or photophosphorylation that occurs in the chloroplasts of
photosynthetic organisms. Triphosphate energy in ATP is transferred to the other nucleosides/nucleotides by action of enzymes
called kinases. For example, nucleoside diphosphokinase (NDPK) catalyzes the following reaction
ATP + NDP ⟷ ADP + NTP (2.6.1)

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 where ‘N’ of “NDP” and “NTP corresponds to any base. Other kinases can put single phosphates onto nucleosides or onto
nucleoside monophosphates using energy from ATP.

Deoxyribonucleotides
Individual deoxyribonucleotides are derived from corresponding ribonucleoside diphosphates via catalysis by the enzyme known as
ribonucleotide reductase (RNR). The deoxyribonucleoside diphosphates are then converted to the corresponding triphosphates
(dNTPs) by the addition of a phosphate group. Synthesis of nucleotides containing thymine is distinct from synthesis of all of the
other nucleotides and will be discussed later.

Building DNA strands

Each DNA strand is built from dNTPs by the formation of a phosphodiester bond, catalyzed by DNA polymerase, between the
3’OH of one nucleotide and the 5’ phosphate of the next. The result of this directional growth of the strand is that the one end of
the strand has a free 5’ phosphate and the other a free 3’ hydroxyl group (Figure 2.130). These are designated as the 5’ and 3’ ends
of the strand.

Figure 2.130 - 5’-3’ Polarity of a DNA strand

Figure 2.131 shows two strands of DNA (left and right). The strand on the left, from 5’ to 3’ reads T-C-G-A, whereas the strand on
the right, reading from 5’ to 3’ is T-C-G-A. The strands in a double-stranded DNA are arranged in an anti-parallel fashion with the
5’ end of one strand across from the 3’ end of the other.

Hydrogen bonds
Hydrogen bonds between the base pairs hold a nucleic acid duplex together, with two hydrogen bonds per A-T pair (or per A-U
pair in RNA) and three hydrogen bonds per G-C pair. The B-form of DNA has a prominent major groove and a minor groove
tracing the path of the helix (Figure 2.132). Proteins, such as transcription factors bind in these grooves and access the hydrogen
bonds of the base pairs to “read” the sequence therein.

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 Figure 2.131 - Anti-parallel orientation of a DNA duplex, phosphodiester backbone, and base pairing Image by Aleia Kim
Other forms of DNA besides the B-form (Movie 2.5) are known (Figure 2.133). One of these, the A-form, was identified by
Rosalind Franklin in the same issue of Nature as Watson and Crick’s paper. Though the A-form structure is a relatively minor form
of DNA and resembles the B-form, it turns out to be important in the duplex form of RNA and in RNA-DNA hybrids. Both the A
form and the B-form of DNA have the helix oriented in what is termed the right-handed form.

Figure 2.132 - Major and minor grooves of DNA. The minor groove has been bound by a dye Wikipedia

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 Movie 2.5 - B-form DNA duplex rotating in space Wikipedia

Z-DNA
The A-form and the B-form stand in contrast to another form of DNA, known as the Z-form. ZDNA, as it is known, has the same
base-pairing rules as the B and A forms, but instead has the helices twisted in the opposite direction, making a left-handed helix
(Figure 2.133). The Z-form has a sort of zig-zag shape, giving rise to the name Z-DNA.
In addition, the helix is rather stretched out compared to the A- and B-forms. Why are there different topological forms of DNA?
The answer relates to both superhelical tension and sequence bias. Sequence bias means that certain sequences tend to favor the
“flipping” of Bform DNA into other forms. ZDNA forms are favored by long stretches of alternating Gs and Cs. Superhelical
tension is discussed below.

Superhelicity

Figure 2.133 - From left to right, the A-, B-, and Zforms of DNA
Short stretches of linear DNA duplexes exist in the B-form and have 10.5 base pairs per turn. Double helices of DNA in the cell
can vary in the number of base pairs per turn they contain. There are several reasons for this. For example, during DNA replication,
strands of DNA at the site of replication get unwound at the rate of 6000 rpm by an enzyme called helicase. The effect of such local
unwinding at one place in a DNA has the effect increasing the winding ahead of it. Unrelieved, such ‘tension’ in a DNA duplex can
result in structural obstacles to replication.

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 Figure 2.134 - Negatively supercoiled, relaxed circular, and positively supercoiled DNA interconverted by topoisomerase enzymes
Wikipedia
Such adjustments can occur in three ways. First, tension can provide the energy for ‘flipping’ DNA structure. Z-DNA can arise as a
means of relieving the tension. Second, DNA can ‘supercoil’ to relieve the tension (Figures 2.134 & 2.135). In this method, the
duplex crosses over itself repeatedly, much like a rubber band will coil up if one holds one section in place and twists another part
of it. Third, enzymes called topoisomerases can act to relieve or, in some cases, increase the tension by adding or removing twists
in the DNA.

Figure 2.135 - Three topological forms of DNA Wikipedia

Topological isomers
As noted, so-called “relaxed” DNA has 10.5 base pairs per turn. Each turn corresponds to one twist of the DNA. Using enzymes, it
is possible to change the number of base pairs per turn. In either the case of increasing or decreasing the twists per turn, tension is
introduced into the DNA structure. If the tension cannot be relieved, the DNA duplex will act to relieve the strain, as noted. This is
most easily visualized for circular DNA, though long linear DNA (such as found in eukaryotic chromosomes) or DNAs constrained
in other ways will exhibit the same behavior.

Parameters

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 Figure 2.136 - Twists and writhes - DNA duplex coiling omitted for simplicity Wikipedia
To understand topologies, we introduce the concepts of ‘writhe’ and ‘linking number’. First, imagine either opening a closed circle
of DNA and either removing one twist or adding one twist and then re-forming the circle. Since the strands have no free ends, they
cannot relieve the induced tension by re-adding or removing the twists at their ends, respectively. Instead, the tension is relieved by
“superhelices” that form with crossing of the double strands over each other (figure 8 structures in Figure 2.136). Though it is not
apparent to visualize, each crossing of the double strands in this way allows twists to be increased or decreased correspondingly.
Thus, superhelicity allows the double helix to reassume 10.5 base pairs per turn by adding or subtracting twists as necessary and
replacing them with writhes.
We write the equation L= T + W where T is the number of twists in a DNA, W is the number of writhes, and L is the linking
number. The linking number is therefore the sum of the twists and writhes. Interestingly, inside of cells, DNAs typically are in a
supercoiled form. Supercoiling affects the size of the DNA (compacts it) and also the expression of genes within the DNA, some
having enhanced expression and some having reduced expression when supercoiling is present. Enzymes called topoisomerases
alter the superhelical density of DNAs and play roles in DNA replication, transcription, and control of gene expression. They work
by making cuts in one strand (Type I topoisomerases) or both strands (Type II topoisomerases) and then add or subtract twists as
appropriate to the target DNA. After that process is complete, the topoisomerase re-ligates the nick/cut it had made in the DNA in
the first step.
Topoisomerases may be the targets of antibiotics. The family of antibiotics known as fluoroquinolones work by interfering with the
action of bacterial type II topoisomerases. Ciprofloxacin also preferentially targets bacterial type II topoisomerases. Other
topoisomerase inhibitors target eukaryotic topoisomerases and are used in anti-cancer treatments.

RNA
The structure of RNA (Figure 2.137) is very similar to that of a single strand of DNA. Built of ribonucleotides, joined together by
the same sort of phosphodiester bonds as in DNA, RNA uses uracil in place of thymine. In cells, RNA is assembled by RNA
polymerases, which copy a DNA template in the much same way that DNA polymerases replicate a parental strand. During the
synthesis of RNA, uracil is used across from an adenine in the DNA template. The building of messenger RNAs by copying a
DNA template is a crucial step in the transfer of the information in DNA to a form that directs the synthesis of protein.
Additionally, ribosomal and transfer RNAs serve important roles in “reading” the information in the mRNA codons and in
polypeptide synthesis. RNAs are also known to play important roles in the regulation of gene expression.

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 Figure 2.137 A section of an RNA molecule Wikipedia
RNA world
The discovery, in 1990, that RNAs could play a role in catalysis, a function once thought to be solely the domain of proteins, was
followed by the discovery of many more so-called ribozymes- RNAs that functioned as enzymes. This suggested the answer to a
long-standing chicken or egg puzzle - if DNA encodes proteins, but the replication of DNA requires proteins, how did a replicating
system come into being? This problem could be solved if the first replicator was RNA, a molecule that can both encode
information and carry out catalysis. This idea, called the “RNA world” hypothesis, suggests that DNA as genetic material and
proteins as catalysts arose later, and eventually prevailed because of the advantages they offer. The lack of a 2’OH on deoxyribose
makes DNA more stable than RNA. The double-stranded structure of DNA also provides an elegant way to easily replicate it. RNA
catalysts, however, remain, as remnants of that early world. In fact, the formation of peptide bonds, essential for the synthesis of
proteins, is catalyzed by RNA.
Secondary structure

Figure 2.138 - tRNA Images - 3D projection (left) and 2D projection (inset) Wikipedia
With respect to structure, RNAs are more varied than their DNA cousin. Created by copying regions of DNA, cellular RNAs are
synthesized as single strands, but they often have self-complementary regions leading to “foldbacks” containing duplex regions.
These are most easily visualized in the ribosomal RNAs (rRNAs) and transfer RNAs (tRNAs) (Figure 2.138), though other RNAs,
including messenger RNAs (mRNAs), small nuclear RNAs (snRNAs), microRNAs (Figure 2.139), and small interfering RNAs
(siRNAs) may each have double helical regions as well.

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 Figure 2.139 - MicroRNA stem loops. Wikipedia
Base pairing
Base pairing in RNA is slightly different than DNA. This is due to the presence of the base uracil in RNA in place of thymine in
DNA. Like thymine, uracil forms base pairs with adenine, but unlike thymine, uracil can, to a limited extent, also base pair with
guanine, giving rise to many more possibilities for pairing within a single strand of RNA.
These additional base pairing possibilities mean that RNA has many ways it can fold upon itself that single-stranded DNA cannot.
Folding, of course, is critical for protein function, and we now know that, like proteins, some RNAs in their folded form can
catalyze reactions just like enzymes. Such RNAs are referred to as ribozymes. It is for this reason scientists think that RNA was the
first genetic material, because it could not only carry information, but also catalyze reactions. Such a scheme might allow certain
RNAs to make copies of themselves, which would, in turn, make more copies of themselves, providing a positive selection.
Stability
RNA is less chemically stable than DNA. The presence of the 2’ hydroxyl on ribose makes RNA much more prone to hydrolysis
than DNA, which has a hydrogen instead of a hydroxyl. Further, RNA has uracil instead of thymine. It turns out that cytosine is the
least chemically stable base in nucleic acids. It can spontaneously deaminate and in turn is converted to a uracil. This reaction
occurs in both DNA and RNA, but since DNA normally has thymine instead of uracil, the presence of uracil in DNA indicates that
deamination of cytosine has occurred and that the uracil needs to be replaced with a cytosine. Such an event occurring in RNA
would be essentially undetectable, since uracil is a normal component of RNA. Mutations in RNA have much fewer consequences
than mutations in DNA because they are not passed between cells in division.
Catalysis
RNA structure, like protein structure, has importance, in some cases, for catalytic function. Like random coils in proteins that give
rise to tertiary structure, single-stranded regions of RNA that link duplex regions give these molecules a tertiary structure, as well.
Catalytic RNAs, called ribozymes, catalyze important cellular reactions, including the formation of peptide bonds in ribosomes
(Figure 2.114). DNA, which is usually present in cells in strictly duplex forms (no tertiary structure, per se), is not known to be
involved in catalysis.
RNA structures are important for reasons other than catalysis. The 3D arrangement of tRNAs is necessary for enzymes that attach
amino acids to them to do so properly. Further, small RNAs called siRNAs found in the nucleus of cells appear to play roles in both
gene regulation and in cellular defenses against viruses. The key to the mechanisms of these actions is the formation of short
foldback RNA structures that are recognized by cellular proteins and then chopped into smaller units. One strand is copied and
used to base pair with specific mRNAs to prevent the synthesis of proteins from them.

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 Figure 2.140 - Structure of the 50S ribosomal subunit. rRNA shown in brown. Active site in red Wikipedia
Denaturing nucleic acids

Figure 2.141 - The hyperchromic effect Wikipedia

Like proteins, nucleic acids can be denatured. Forces holding duplexes together include hydrogen bonds between the bases of each
strand that, like the hydrogen bonds in proteins, can be broken with heat or urea. (Another important stabilizing force for DNA
arises from the stacking interactions between the bases in a strand.) Single strands absorb light at 260 nm more strongly than
double strands. This is known as the hyperchromic effect (Figure 2.141)and is a consequence of the disruption of interactions
among the stacked bases. The changes in absorbance allow one to easily follow the course of DNA denaturation. Denatured
duplexes can readily renature when the temperature is lowered below the “melting temperature” or Tm, the temperature at which
half of the DNA strands are in duplex form. Under such conditions, the two strands can re-form hydrogen bonds between the
complementary sequences, returning the duplex to its original state. For DNA, strand separation and rehybridization are important
for the technique known as the polymerase chain reaction (PCR). Strand separation of DNA duplexes is accomplished in the
method by heating them to boiling. Hybridization is an important aspect of the method that requires single stranded primers to
“find” matching sequences on the template DNA and form a duplex. Considerations for efficient hybridization (also called
annealing) include temperature, salt concentration, strand concentration, and magnesium ion levels (for more on PCR, see HERE).
DNA packaging
DNA is easily the largest macromolecule in a cell. The single chromosome in small bacterial cells, for example, can have a
molecular weight of over 1 billion Daltons. If one were to take all of the DNA of human chromosomes from a single cell and lay
them end to end, they would be over 7 feet long. Such an enormous molecule demands careful packaging to fit within the confines
of a nucleus (eukaryotes) or a tiny cell (bacteria). The chromatin system of eukaryotes is the best known, but bacteria, too, have a
system for compacting DNA.

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DNA in Bacteria
In bacteria, there is no nucleus for the DNA. Instead, DNA is contained in a structure called a nucleoid (Figure 2.142). It contains
about 60% DNA with much of the remainder comprised of RNAs and transcription factors. Bacteria do not have histone proteins
that DNA wrap around, but they do have proteins that help organize the DNA in the cell - mostly by making looping structures.

Figure 2.142 - Anatomy of a bacterial cell.

These proteins are known as Nucleoid Associated Proteins and include ones named HU, H-NS, Fis, CbpA, and Dps. Of these, HU
most resembles eukaryotic histone H2B and binds to DNA non-specifically. The proteins associate with the DNA and can also
cluster, which may be the origin of the loops. It is likely these proteins play a role in helping to regulate transcription and respond
to DNA damage. They may also be involved in recombination.
Eukaryotes

Figure 2.143 - Structure of a nucleosome Wikipedia

The method eukaryotes use for compacting DNA in the nucleus is considerably different, and with good reason - eukaryotic DNAs
are typically much larger than prokaryotic DNAs, but must fit into a nucleus that is not much bigger than a prokaryotic cell. Human
DNA, for example, is about 1000 times longer than c DNA. The strategy employed in eukaryotic cells is that of spooling - DNA is
coiled around positively charged proteins called histones. These proteins, whose sequence is very similar in cells as diverse as yeast
and humans, come in four types, dubbed H1, H2a, H2b, H3, and H4. A sixth type, referred to as H5 is actually an isoform of H1
and is rare. Two each of H2a, H2b, H3, and H4 are found in the core structure of what is called the fundamental unit of chromatin -
the nucleosome (Figure 2.143).
Octamer

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 Figure 2.144 - Detailed core particle structure - DNA in gray, histones in red, yellow, green, blue Wikipedia
The core of 8 proteins is called an octamer. The stretch of DNA wrapped around the octamer totals about 147 base pairs and makes
1 2/3 turns around it. This complex is referred to as a core particle (Figure 2.144). A linker region of about 50-80 base pairs
separate core particles. The term nucleosome then refers to a a core particle plus a linker region (Figure 2.143). Histone H1 sits
near the junction of the incoming DNA and the histone core. It is often referred to as the linker histone. In the absence of H1, non-
condensed nucleosomes resemble “beads on a string” when viewed in an electron microscope.
Histones

Figure 2.145 - Amino acid sequence (1- letter code) of histone H3 of S. cerevisiae. Arginines (R) and lysines (K) shown in red.
Histone proteins are similar in structure and are rich in basic amino acids, such as lysine and arginine (Figure 2.145). These amino
acids are positively charged at physiological pH, with enables them to form tight ionic bonds with the negatively charged
phosphate backbone of DNA.
For DNA, compression comes at different levels (Figure 2.146). The first level is at the nucleosomal level. Nucleosomes are
stacked and coiled into higher order structures. 10 nm fibers are the simplest higher order structure (beads on a string) and they
grow in complexity. 30 nm fibers consist of stacked nucleosomes and they are packed tightly. Higher level packing produces the
metaphase chromosome found in meiosis and mitosis.

Figure 2.146 Packaging of DNA into a chromosome

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The chromatin complex is a logistical concern for the processes of DNA replication and (particularly) gene expression where
specific regions of DNA must be transcribed. Altering chromatin structure is therefore an essential function for transcriptional
activation in eukaryotes. One strategy involves adding acetyl groups to the positively charged lysine side chains to “loosen their
grip” on the negatively charged DNA, thus allowing greater access of proteins involved in activating transcription to gain access to
the DNA. The mechanisms involved in eukaryotic gene expression are
Ames test
The Ames test (Figure 2.147) is an analytical method that allows one to determine whether a compound causes mutations in DNA
(is mutagenic) or not. The test is named for Dr. Bruce Ames, a UC Berkeley emeritus professor who was instrumental in creating it.
In the procedure, a single base pair of a selectable marker of an organism is mutated in a plasmid to render it nonfunctional. In the
example, a strain of Salmonella is created that lacks the ability to grow in the absence of histidine. Without histidine, the organism
will not grow, but if that one base in the plasmid’s histidine gene gets changed back to its original base, a functional gene will be
made and the organism will be able to grow without histidine.
A culture of the bacterium lacking the functional gene is grown with the supply of histidine it requires. It is split into two vials. To
one of the vials, a compound that one wants to test the mutagenicity of is added. To the other vial, nothing is added. The bacteria in
each vial are spread onto plates lacking histidine. In the absence of mutation, no bacteria will grow. The more colonies of bacteria
that grow, the more mutation happened. Note that even the vial without the possible mutagenic compound will have a few colonies
grow, as a result of mutations unlinked to the potential mutagen.

Figure 2.147 - The Ames test Wikipedia

Mutation happens in all cells at a low level. If the plate with the cells from the vial with the compound has more colonies than the
cells from the control vial (no compound), then that would be evidence that the compound causes more mutations than would
normally occur and it is therefore a mutagen. On the other hand, if there was no significant difference in the number of colonies on
each plate, then that would suggest it is not mutagenic. The test is not perfect - it identifies about 90% of known mutagens - but its
simplicity and inexpensive design make it an excellent choice for an initial screen of a compound.

This page titled 2.6: Structure and Function - Nucleic Acids is shared under a CC BY-NC-SA license and was authored, remixed, and/or curated
by Kevin Ahern, Indira Rajagopal, & Taralyn Tan.

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2.7: Structure and Function- Carbohydrates

Figure 2.171 - Repeating unit of amylose

Endogenous glycation, on the other hand, arises with a frequency that is proportional to the concentration of free sugar in the body.
These occur most frequently with fructose, galactose, and glucose in that decreasing order and are detected in the bloodstream.
Both proteins and lipids can be glycated and the accumulation of endogenous advanced glycation endproducts (AGEs) is associated
with Type 2 diabetes, as well as in increases in cardiovascular disease (damage to endothelium, cartilage, and fibrinogen),
peripheral neuropathy (attack of myelin sheath), and deafness (loss of myelin sheath).
The formation of AGEs increases oxidative stress, but is also thought to be exacerbated by it. Increased oxidative stress, in turn
causes additional harm. Damage to collagen in blood cells causes them to stiffen and weaken and is a factor in hardening of the
arteries and formation of aneurysms, respectively. One indicator of diabetes is increased glycation of hemoglobin in red blood cells,
since circulating sugar concentration are high in the blood of diabetics. Hemoglobin glycation is measured in testing for blood
glucose control in diabetic patients.

Homopolymer Monomeric Unit

Glycogen Glucose

Cellulose Glucose

Amylose Glucose

Callose Glucose

Chitin N-acetylglucosamine

Xylan Xylose

Mannan Mannose

Chrysolaminarin Glucose

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 Figure 2.189). Along with the proteoglycan called lubricin, hyaluronic acid turns water into lubricating material. Hyaluronic acid is
present as a coat around each cell of articular cartilage and forms complexes with proteoglycans that absorb water, giving resilience
(resistance to compression) to cartilage. Aging causes a decrease in size of hyaluronans, but an increase in concentration.
Function in skin
Hyaluronic acid is a major component of skin and has functions in tissue repair. With exposure to excess UVB radiation, cells in
the dermis produce less hyaluronan and increase its degradation.
For some cancers the plasma level of hyaluronic acid correlates with malignancy. Hyaluronic acid levels have been used as a
marker for prostate and breast cancer and to follow disease progression. The compound can to used to induce healing after cataract
surgery. Hyaluronic acid is also abundant in the granulation tissue matrix that replaces a fibrin clot during the healing of wounds. In
wound healing, it is thought that large polymers of hyaluronic acid appear early and they physically make room for white blood
cells to mediate an immune response.
Breakdown
Breakdown of hyaluronic acid is catalyzed by enzymes known as hyaluronidases. Humans have seven types of such enzymes, some
of which act as tumor suppressors. Smaller hyaluronan fragments can induce inflammatory response in macrophages and dendritic
cells after tissue damage. They can also perform proangiogenic functions.
Proteoglycans
Glycosaminoglycans are commonly found attached to proteins and these are referred to as proteoglycans. Linkage between the
protein and the glycosaminoglycan is made through a serine side-chain. Proteoglycans are made by glycosylation of target proteins
in the Golgi apparatus.

This page titled 2.7: Structure and Function- Carbohydrates is shared under a CC BY-NC-SA license and was authored, remixed, and/or curated
by Kevin Ahern, Indira Rajagopal, & Taralyn Tan.

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2.8: Structure and Function - Lipids and Membranes
Source: BiochemFFA_2_7.pdf. The entire textbook is available for free from the authors at
http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy

Lipids are a diverse group of molecules that all share the characteristic that at least a portion of them is hydrophobic. Lipids play
many roles in cells, including serving as energy storage (fats/oils), constituents of membranes (glycerophospholipids,
sphingolipids, cholesterol), hormones (steroids), vitamins (fat soluble), oxygen/ electron carriers (heme), among others. For lipids
that are very hydrophobic, such as fats/ oils, movement and storage in the aqueous environment of the body requires special
structures. Other, amphipathic lipids, such as glycerophospholipids and sphingolipids spontaneously organize themselves into lipid
bilayers when placed in water. Interestingly, major parts of many lipids can be derived from acetyl-CoA.

Fatty acids

Figure 2.190 - Saturated fatty acid (stearic acid) and unsaturated fatty acid (oleic acid)
The most ubiquitous lipids in cells are the fatty acids. Found in fats, glycerophospholipids, sphingolipids and serving as as
membrane anchors for proteins and other biomolecules, fatty acids are important for energy storage, membrane structure, and as
precursors of most classes of lipids. Fatty acids, as can be seen from Figure 2.190 are characterized by a polar head group and a
long hydrocarbon tail. Fatty acids with hydrocarbon tails that lack any double bonds are described as saturated, while those with
one or more double bonds in their tails are known as unsaturated fatty acids. The effect of double bonds on the fatty acid tail is to
introduce a kink, or bend, in the tail, as shown for oleic acid.

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 Figure 2.191 - Arachidonic acid - A polyunsaturated fatty acid Wikipedia
Stearic acid, a saturated fatty acid, by contrast has a straight hydrocarbon tail. Figures 2.190-2.194 show the most common
saturated and unsaturated fatty acids. Fatty acids with unsaturated tails have a lower melting temperature than those with saturated
tails of the same length. Shorter tails also decrease melting temperature. These properties carry over to the fats/oils containing
them.

Figure 2.192 - Saturated fatty acids. Number of carbons in right column Wikipedia
Fatty acids with more than one double bond are called polyunsaturated. Plants are excellent sources of unsaturated and
polyunsaturated fatty acids. The position of the double bond(s) in fatty acids has important considerations both for their synthesis
and for their actions in the body. Biochemically, the double bonds found in fatty acids are predominantly in the cis configuration.
So-called trans fats arise as a chemical by-product of partial hydrogenation of vegetable oil.

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 Figure 2.193 - Unsaturated fatty acids. Right column Indicates number of carbons and double bonds Wikipedia
In humans, consumption of trans fat raises low density lipoprotein (LDL) levels and lowers high density lipoprotein (HDL) levels.
Each is thought to contribute to the risk of developing coronary artery disease. The most Figure 2.194 - Fatty acid models.
Carboxyl end labeled in red Wikipedia common fatty acids in our body include palmitate, stearate, oleate, linolenate, linoleate, and
arachidonate. Two notable shorter fatty acids are nonanoic (9 carbons) and decanoic acid (10 carbons), both of which appear to
have anti-seizure effects. Decanoic acid directly inhibits excitatory neurotransmission in the brain and may contribute to the
anticonvulsant effect of the ketogenic diet.

Figure 2.194 - Fatty acid models. Carboxyl end labeled in red Wikipedia
Numbering
Figure 2.195 shows two different systems for locating double bonds in a fatty acid. The ω system counts carbons starting with
the methyl end (shown in red) while the Δ system counts from the carboxyl end (shown in blue). For example, an ω-3 (omega 3)
fatty acid would have a double bond at the third carbon from the methyl end. In the Δ system, a fatty acid that has a cis double
bond at carbon 6, counting from the carboxyl end, would be written as cis-Δ6.

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 Figure 2.195 - Δ and ω numbering systems for fatty acids Image by Pehr Jacobson

Fatty acids are described as essential fatty acids if they must be in the diet (can’t be synthesized by the organism) and nonessential
fatty acids if the organism can synthesize them. Humans and other animals lack the desaturase enzymes necessary to make double
bonds at positions greater than Δ-9, so fatty acids with double bonds beyond this position must be obtained in the diet. Linoleic
acid and linolenic acid, both fall in this category. Related unsaturated fatty acids can be made from these fatty acids, so the
presence of linoleic and linolenic acids in the diet eliminates the need to have all unsaturated fatty acids in the diet. Both linoleic
and linolenic acid contain 18 carbons, but linoleic acid is an ω-6 fatty acid, whereas linolenic acid is an ω-3 fatty acid. Notably, ω-6
fatty acids tend to be proinflammatory, whereas ω-3 fatty acids are lesser so.

Figure 2.196 - Structure of a fat/oil

Fats/oils
Fats and oils are the primary energy storage forms of animals and are also known as triacylglycerols and triglycerides, since they
consist of a glycerol molecule linked via ester bonds to three fatty acids (Figure 2.196). Fats and oils have the same basic structure.
We give the name fat to those compounds that are solid at room temperature and the name oil to those that are liquid at room
temperature. Note that biological oils are not the same as petroleum oils.
Increasing the number of unsaturated fatty acids (and the amount of unsaturation in a given fatty acid) in a fat decreases the melting
temperature of it. Organisms like fish, which live in cool environments, have fats with more unsaturation and this is why fish oil
contains polyunsaturated fatty acids.
Adipocytes

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 Figure 2.197 - Lipase action on a fat Image by Aleia Kim
Fats are stored in the body in specialized cells known as adipocytes. Enzymes known as lipases release fatty acids from fats by
hydrolysis reactions (Figure 2.197). Triacylglycercol lipase (pancreatic - Figure 2.198) is able to cleave the first two fatty acids
from the fat. A second enzyme, monoacylglycerol lipase, cleaves the last fatty acid. Fats can be synthesized by replacing the
phosphate on phosphatidic acid with a fatty acid.

Figure 2.198 - Pancreatic lipase

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Glycerophospholipids
Glycerophospholipids (phosphoglycerides) are important components of the lipid bilayer of cellular membranes.
Phosphoglycerides are structurally related to fats, as both are derived from phosphatidic acid (Figure 2.199). Phosphatidic acid is a
simple glycerophospholipid that is usually converted into phosphatidyl compounds. These are made by esterifying various groups,
such as ethanolamine, serine, choline, inositol, and others (Figure 2.200) to the phosphate of phosphatidic acid. All of these
compounds form lipid bilayers in aqueous solution , due to their amphiphilic nature.

Figure 2.199 - Structure of phosphatidic acid. R1 and R2 are alkyl groups of fatty acids.
Phosphatidylethanolamines
Since all glycerolipids can have a variety of fatty acids at positions 1 and 2 on the glycerol, they all are families of compounds. The
phosphatidylethanolamines are found in all living cells and are one of the most common phosphatides, making up about 25% of
them. They are common constituents of brain tissue and in the spinal cord, making up as much as 45% of the total phospholipids.
Phosphatidylethanolamines are asymmetrically distributed across membranes, being preferentially located on the inner leaflet
(closest to the cytoplasm) of the plasma membrane. Metabolically, phosphatidylethanloamines are precursors of
phosphatidylcholines. Phosphatidylserines Phosphatidylserines are another group of phosphatidyl compounds that are preferentially
distributed across the lipid bilayer of the plasma membrane. Like the phosphatidylethanolamines, phosphatidylserines are
preferentially located on the inner leaflet of the plasma membrane. When apoptosis (cell suicide) occurs, the preferential
distribution is lost and the phosphatidylserines appear on the outer leaflet where they serve as a signal to macrophages to bind and
destroy the cell.

Figure 2.200 - Four common components of phosphatides Wikipedia

Phosphatidylcholines
Phosphatidylcholines (Figure 2.201) are another group of important membrane components. They tend to be found more
commonly on the outer leaflet of the plasma membrane. Nutritionally, the compounds are readily obtained from eggs and soybeans.
Phosphatidylcholines are moved across membranes by Phosphatidylcholine transfer protein (PCTP). This protein, which is
sensitive to the levels of phosphatidylcholines, acts to stimulate the activity of a thioesterase (breaks thioester bonds, such as acyl-
CoAs) and activates PAX3 transcription factors.

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 Figure 2.201 - Phosphatidylcholine

Cardiolipins
Cardiolipins are an unusual set of glycerophospholipids in containing two diacylglycerol backbones joined in the middle by a
diphosphoglycerol (Figure 2.202). It is an important membrane lipid, constituting about 20% of the inner mitochondrial membrane
and is found in organisms from bacteria to humans. In both plants and animals, it is found almost totally in the inner mitochondrial
membrane.

Figure 2.202 - Cardiolipin

The molecules appear to be required for both Complex IV and Complex III of the electron transport chain to maintain its structure.
The ATP synthase enzyme (Complex V) of the oxidative phosphorylation system also binds four molecules of cardiolipin. It has
been proposed that cardiolipin functions as a proton trap in the process of proton pumping by Complex IV.

Figure 2.203 - Cardiolipin oxidation and the release of cytochrome C in apoptosis

Cardiolipin also plays a role in apoptosis. As shown in Figure 2.203, oxidation of cardiolipin by a cardiolipin-specific oxygenase
causes cardiolipin to move from the inner mitochondrial membrane to the outer one, helping to form a permeable pore and
facilitating the transport of cytochrome c out of the intermembrane space and into the cytoplasm - a step in the process of
apoptosis.

Figure 2.204 - Diacylglycerol

Diacylglycerol

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Diacylglycerol (also called diglyceride and DAG - Figure 2.204) is an important intermediate in metabolic pathways. It is
produced, for example, in the first step of the hydrolysis of fat and is also produced when membrane lipids, such as PIP2
(phosphatidylinositol-4,5-bisphosphate) are hydrolyzed by phospholipase C in a signaling cascade.
DAG is itself a signaling compound, binding to protein kinase C to activate it to phosphorylate substrates. Synthesis of DAG
begins with glycerol-3-phosphate, which gains two fatty acids from two acyl-CoAs to form phosphatidic acid. Dephosphorylation
of phosphatidic acid produces DAG. DAG can also be rephosphorylated by DAG kinase to re-make phosphatidic acid or another
fatty acid can be added to make fat.
Inositol

Figure 2.205 - Inositol

Though technically not a lipid itself, inositol is found in many lipids. Inositol is a derivative of cyclohexane containing six
hydroxyl groups - one on each carbon (Figure 2.205. It has nine different stereoisomers of which one, cis-1,2,3,5-trans-4,6-
cyclohexanehexol (called myo-inositol) is the most common. It has a sweet taste (half that of sucrose).

Figure 2.206 - Phytic acid

Numerous phosphorylated forms of the compound exist, from a single phosphate to six (one on each carbon). Phytic acid, for
example, in plants, has six phosphates (Figure 2.206) that it uses to store phosphate. Inositol is produced from glucose and was
once considered vitamin B8, but is made by the body in adequate amounts, so it is not now considered a vitamin. Phosphorylated
forms of inositol are found in phosphoinositides, such as PIP2 and PIP3, both of which are important in signaling processes. Some
of these include insulin signaling, fat catabolism, calcium regulation, and assembly of the cytoskeleton.
Phosphoinositides

Figure 2.207 - Structure of PIP2

Compounds based on phosphatidylinositol (PI) are often called phosphoinositides. These compounds have important roles in
signaling and membrane trafficking. Hydroxyls on carbons 3,4, and 5 of the inositol ring are targets for phosphorylation by a
variety of kinases. Seven different combinations are used. Steric hindrance inhibits phosphorylation of carbons 2 or 6. Naming of
these phosphorylated compounds follows generally as PI(#P)P, PI(#P, #P)P, or PI(#P, #P, #P)P where #P refers to the number of the

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carbon where a phosphate is located. For example, PI(3)P refers to a phosphatidyl compound with a phosphate added to carbons 3
of the inositol ring, whereas PI(3,4,5)P is a phosphatidyl compound with a phosphate added to carbons 3,4,and 5.
Phosphatidylinositol-4,5- bisphosphate

Figure 2.208 - Phosphatidylinositol-4- phosphate

Phosphatidylinositol-4,5-bisphosphate (PIP2 - Figure 2.207) is a phospholipid of plasma membranes that functions in the
phospholipase C signaling cascade. In this signaling pathway, hydrolysis catalyzed by phospholipase C releases inositol-1,4,5-
trisphosphate (IP3) and diacylglycerol. Synthesis of PIP2 begins with phosphatidylinositol, which is phosphorylated at position 4
followed by phosphorylation at position 5 by specific kinases.
PIP2 can be phosphorylated to form the signaling molecule known as phosphatidylinositol (3,4,5)-trisphosphate (PIP3). Along with
PIP3, PIP2 serves as a docking phospholipid for the recruitment of proteins that play roles in signaling cascades. Binding of PIP2 is
also required by inwardly directed potassium channels.
Phosphatidylinositol (3,4,5)- trisphosphate
Phosphatidylinositol (3,4,5)-trisphosphate (PIP3) is an important molecule for the activation of signaling proteins, such as AKT,
which activates anabolic signaling pathways related to growth and survival. PIP3 can be dephosphorylated by phosphatase PTEN
to yield PIP2 and can be synthesized from PIP2 by kinase action of Class I PI 3- kinases. Kinase activity to synthesize PIP3 results
in movement of PIP3-binding proteins to the plasma membrane. They include Akt/ PKB, PDK1, Btk1, and ARNO and each is
activated by binding to PIP3.
Plasmalogens

Figure 2.209 - Plasmalogen - A vinyl ether lipid Wikipedia

A special class of the glycerophospholipids are the plasmalogens (Figure 2.209). They differ in containing a vinyl ether linkage at
position 1 of glycerol, in contrast to other glycerophopsholipids, which have an ester linkage at this position. Position 2 of each is
an ester. The precursor for the ether linkage is typically a 16 or 18 carbon saturated alcohol or an 18 carbon unsaturated alcohol.
At the phosphate tail, the most commonly attached groups are ethanolamine or choline. Plasmalogens are found abundantly in
humans in heart (30-40% of choline phospholipids). 30% of the glycerophospholipids in brain are plasmalogens and 70% of the
ethanolamine lipids of the myelin sheath of nerve cells are plasmalogens.
Though their function is not understood, it is believed that plasmalogens may provide some protection against reactive oxygen
species and have roles in signaling.
Lecithin
Lecithin is a generic term for a combination of lipid substances that includes phosphoric acid, glycerol, glycolipids, triglycerides,
and phospholipids. Lecithin is a wetting agent helpful with emulsification and encapsulation and is even used as an anti-sludge
additive in motor lubricants. Lecithin is used in candy bars to keep cocoa and cocoa butter from separating. Though considered safe

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as a food ingredient, lecithin can be converted by gut bacteria to trimethylamine-N-oxide which may contribute to cholesterol
deposition and atherosclerosis.
Sphingolipids

Figure 2.210 - Sphingosine and a ceramide made from it Wikipedia

Fatty acids are also components of a broad class of molecules called sphingolipids. Sphingolipids are structurally similar to
glycerophospholipids, though they are synthesized completely independently of them starting with palmitic acid and the amino acid
serine. Sphingolipids are named for the amino alcohol known as sphingosine (Figure 2.210), though they are not directly
synthesized from it. Figure 2.211 shows the generalized structure of sphingolipids.

Figure 2.211 Schematic structure of a sphingolipid

If the R-group is a hydrogen, the molecule is called a ceramide. When the R-group is phosphoethanolamine the resulting molecule
is sphingomyelin, an important component of the myelin sheath and lipid membranes. If a single, simple sugar is instead added, a
cerebroside is created (Figure 2.212). Addition of a complex oligosaccharide creates a ganglioside.
Complex sphingolipids may play roles in cellular recognition and signaling. Sphingolipids are found most abundantly in plasma
membrane and are almost completely absent from mitochondrial and endoplasmic reticulum membranes. In animals, dietary
sphingolipids have been linked to reduced colon cancer, reductions in LDLs, and increases in HDLs. Like the
glycerophospholipids, sphingolipids are amphiphilic. Most sphingolipids except sphingomyelin do not contain phosphate.

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 Figure 2.212 - Categories of Sphingolipid Wikipedia
Eicosanoids

Figure 2.213 - Arachidonic acid drawn as straight (top) and bent (bottom)
Fatty acids made from omega-6 and omega-3 fatty acids include three important fatty acids containing 20 carbons. They include
arachidonic acid (an ω-6 fatty acid with four double bonds (Δ-5,8,11,14) - Figure 2.213), eicosapentaenoic acid (an ω-3 fatty acid
with five double bonds, and dihomo-γ-linolenic acid (an ω-6 fatty acid with three double bonds). The class of compounds known as
eicosanoids is made by oxidation of these compounds. Subclasses include include prostaglandins, prostacyclins, thromboxanes,
lipoxins, leukotrienes, and endocannabinoids (Figures 2.214-2.219). Eicosanoids play important roles affecting inflammation,
immunity, mood, and behavior.
Prostaglandins

Figure 2.214 - Prostaglandin PGH2

A collection of molecules acting like hormones, prostaglandins are derived from arachidonic acid and have many differing (even
conflicting) physiological effects. These include constriction or dilation of vascular smooth muscle cells, induction of labor,
regulation of inflammation, and action on the thermoregulatory center of the hypothalamus to induce fever, among others.
Prostaglandins are grouped with the thromboxanes (below) and prostacyclins (below), as prostanoids. The prostanoids, which all
contain 20 carbons are a subclass of the eicosanoids. Prostaglandins are found in most tissues of higher organisms. They are
autocrine or paracrine compounds produced from essential fatty acids. The primary precursor of the prostaglandins is the fatty acid
known as arachidonic acid and the prostaglandin made from it is known as PGH2 (Figure 2.214), which, in turn is a precursor of
other prostaglandins, as well as the prostacyclins and thromboxanes.
Interesting prostaglandins

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PGD2 - inhibits hair follicle growth, vasodilator, causes bronchial constriction, higher in lungs of asthmatics than others.

Figure 2.215 Prostaglandin E

PGE2 (Figure 2.215) - exerts effects in labor (soften cervix, uterine contraction), stimulates bone resorption by osteoclasts, induces
fever, suppresses T-cell receptor signaling, vasodilator, inhibits release of noradrenalin from sympathetic nerve terminals. It is a
potent activator of the Wnt signaling pathway.
A prostaglandin can have opposite effects, depending on which receptor it binds to. Binding of PGE2 to the EP1 receptor causes
bronchoconstriction and smooth muscle contraction, whereas binding of the same molecule to the EP2 receptor causes
bronchodilation and smooth muscle relaxation.

Figure 2.216 - Prostaglandin F2α

PGF2α (Figure 2.216)- uterine contractions, induces labor, bronchoconstriction.

PGI2 - vasodilation, bronchodilation, inhibition of platelet aggregation.
Thromboxanes

Figure 2.217 Thromboxane A2 2

Thromboxanes play roles in clot formation and named for their role in thrombosis. They are potent vasoconstrictors and facilitate
platelet aggregation. They are synthesized in platelets, as well. The anti-clotting effects of aspirin have their roots in the inhibition
of synthesis of PGH2, which is the precursor of the thromboxanes. The most common thromboxanes are A2 (Figure 2.217) and B2.
Prostacyclin

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 Figure 2.218 - Prostacyclin
Prostacyclin (also known as prostaglandin I2 or PGI2 - Figure 2.218) counters the effects of thromboxanes, inhibiting platelet
activation and acting as vasodilators. It is produced from PGH2 by action of the enzyme prostacyclin synthase.
Leukotrienes

Figure 2.219 - Leukotriene A4 (LTA4)

Another group of eicosanoid compounds are the leukotrienes (Figure 2.219). Like prostaglandins, leukotrienes are made from
arachidonic acid. The enzyme catalyzing their formation is a dioxygenase known as arachidonate 5-lipoxygenase. Leukotrienes are
involved in regulating immune responses. They are found in leukocytes and other immunocompetent cells, such as neutrophils,
monocytes, mast cells, eosinophils, and basophils. Leukotrienes are associated with production of histamines and prostaglandins,
which act as mediators of inflammation. Leukotrienes also trigger contractions in the smooth muscles of the bronchioles. When
overproduced, they may pay a role in asthma and allergic reactions. Some treatments for asthma aim at inhibiting production or
action of leukotrienes.
Cholesterol

Figure 2.220 - Structure of cholesterol

Arguably, no single biomolecule has generated as much discussion and interest as has cholesterol (Figure 2.220). Certainly, from
the perspective of the Nobel Prize committee, no small molecule even comes close, with 13 people having been awarded prizes for
work on it. Evidence for cholesterol’s importance comes from the study of brain tissue where it comprises 10-15% of the dry mass.
Membrane flexibility

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 Figure 2.221 - Sitosterol - A phytosterol
In animal cells, cholesterol provides for membrane flexibility that allows for cellular movement that is in contrast to plant and
bacterial cells with fixed structures. Cholesterol is made in many cells of the body, with the liver making the greatest amount. The
anabolic pathway leading to synthesis of cholesterol is known as the isoprenoid pathway and branches of it lead to other molecules
including other fat-soluble vitamins.

Figure 2.222 - Margarine - A common source of trans fat Wikipedia

Cholesterol is only rarely found in prokaryotes (Mycoplasma, which requires it for growth, is an exception) and is found in only
trace amounts in plants. Instead, plants synthesize similar compounds called phytosterols (Figure 2.221). On average, the body of a
150 pound adult male makes about 1 gram of cholesterol per day, with a total content of about 35 grams.
Packaging

Figure 2.223 - Cholesterol - Ball and stick model

Cholesterol’s (and other lipids’) hydrophobicity requires special packaging into lipoprotein complexes (called chylomicrons,
VLDLs, IDLs, LDLs, and HDLs) for movement in the lymph system and bloodstream. Though cholesterol can be made by cells,
they also take it up from the blood supply by absorbing cholesterol-containing LDLs directly in a process called receptor-mediated
endocytosis.
Oxidative damage to LDLs can lead to formation of atherosclerotic plaques and this is why cholesterol has gotten such a negative
image in the public eye. The liver excretes cholesterol through the bile for elimination into the digestive system, but the compound
is recycled there.
Reducing cholesterol levels

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 Figure 2.224 - Ezetimibe - An inhibitor of cholesterol absorption
Strategies for reducing cholesterol in the body focus primarily on three areas - reducing consumption, reducing endogenous
synthesis, and reducing the recycling. Dietary considerations, such as saturated fat versus unsaturated fat consumption are currently
debated. Dietary trans fats, though, correlate with incidence of coronary heart disease. Consumption of vegetables may provide
some assistance with reducing levels of cholesterol recycled in the digestive system, because plant phytosterols compete with
cholesterol for reabsorption and when this happens, a greater percentage of cholesterol exits the body in the feces. Drugs related to
penicillin are also used to inhibit cholesterol recycling. One of these is ezetimibe, shown in Figure 2.224.

Figure 2.225 - All-trans retinol

Genetic defects in the cholesterol movement system are a cause of the rare disease known as familial hypercholesterolemia in
which the blood of afflicted individuals contains dangerously high levels of LDLs. Left untreated, the disease is often fatal in the
first 10-20 years of life. While LDLs have received (and deserve) a bad rap, another group of lipoprotein complexes known as the
HDLs (high density lipoprotein complexes) are known as “good cholesterol” because their levels correlate with removal of debris
(including cholesterol) from arteries and reduce inflammation.
Membrane function
In membranes, cholesterol is important as an insulator for the transmission of signals in nerve tissue and it helps to manage fluidity
of membranes over a wide range of temperatures. Stacked in the lipid bilayer, cholesterol decreases a membrane’s fluidity and its
permeability to neutral compounds, as well as protons and sodium ions. Cholesterol may play a role in signaling by helping with
construction of lipid rafts within the cell membrane.
Vitamin A

Figure 2.226 - 11-cis retinal

Vitamin A comes in three primary chemical forms, retinol (storage in liver - Figure 2.225), retinal (role in vision - Figure 2.226),
and retinoic acid (roles in growth and development). All vitamin A forms are diterpenoids and differ only in the chemical form of
the terminal group. Retinol is mostly used as the storage form of the vitamin.
Retinol is commonly esterified to a fatty acid and kept in the liver. In high levels, the compound is toxic. Retinol is obtained in the
body by hydrolysis of the ester or by reduction of retinal. Importantly, neither retinal nor retinol can be made from retinoic acid.
Retinoic acid is important for healthy skin and teeth, as well as bone growth. It acts in differentiation of stem cells through a
specific cellular retinoic acid receptor.
Sources

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 Figure 2.227 - β-Carotene
Good sources of vitamin A are liver and eggs, as well as many plants, including carrots, which have a precursor, β-carotene (Figure
2.227) from which retinol may be made by action of a dioxygenase.
Light sensitivity The conjugated double bond system in the side chain of vitamin A is sensitive to light and can flip between cis and
trans forms on exposure to it. It is this response to light that makes it possible for retinal to have a role in vision in the rods and
cones of the eyes. Here, the aldehyde form (retinal) is bound to the protein rhodopsin in the membranes of rod and cone cells.

Figure 2.228 - Color sensitivity for cones and rods Image by Aleia Kim
When exposed to light of a particular wavelength, the “tail” of the retinal molecule will flip back and forth from cis to trans at the
double bond at position 11 of the molecule. When this happens, a nerve signal is generated that signals the brain of exposure to
light. Slightly different forms of rhodopsin have different maximum absorption maxima allowing the brain to perceive red, green
and blue specifically and to assemble those into the images we see (Figure 2.228). Cones are the cells responsible for color vision,
whereas rods are mostly involved in light detection in low light circumstances.
Deficiency and surplus
Deficiency of vitamin A is common in developing countries and was inspiration for the design and synthesis of the
geneticallymodified golden rice, which is used as a source of vitamin A to help prevent blindness in children. Overdose of vitamin
A, called hypervitaminosis A is dangerous and can be fatal. Excess vitamin A is also suspected to be linked to osteoporosis. In
smokers, excess vitamin A is linked to an increased rate of lung cancer, but non-smokers have a reduced rate.
Vitamin D

Figure 2.229 - Cholecalciferol - Vitamin D3

The active form of vitamin D plays important roles in the intestinal absorption of calcium and phosphate and thus in healthy bones.
Technically, vitamin D isn’t even a vitamin, as it is a compound made by the body. Rather, it behaves more like a hormone.

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Derived from ultimately from cholesterol, vitamin D can be made in a reaction catalyzed by ultraviolet light. In the reaction, the
intermediate 7-dehydrocholesterol is converted to cholecalciferol (vitamin D3) by the uv light (Figure 2.229). The reaction occurs
most readily in the bottom two layers of the skin shown in Figure 2.230.

Figure 2.230 - Layers of the skin. Outside is at top.

Forms of vitamin D
Five different compounds are referred to as vitamin D. They are
Vitamin D1 - A mixture of ergocalciferol and lumisterol
Vitamin D2 - Ergocalciferol
Vitamin D3 - Cholecalciferol Vitamin
D4 - 22-Dihydroergocalciferol Vitamin
D5 - Sitocalciferol
Vitamin D3 is the most common form used in vitamin supplements and it and vitamin D2 are commonly obtained in the diet, as
well. The active form of vitamin D, calcitriol (Figure 2.231), is made in the body in controlled amounts. This proceeds through two
steps from cholecalciferol. First, a hydroxylation in the liver produces calcidiol and a second hydroxylation in the kidney produces
calcitriol. Monocyte macrophages can also synthesize vitamin D and they use is as a cytokine to stimulate the innate immune
system.
Mechanism of action
Calcitriol moves in the body bound to a vitamin D binding protein, which delivers it to target organs. Calcitriol inside of cells acts
by binding a vitamin D receptor (VDR), which results in most of the vitamin’s physiological effects. After binding calcitriol, the
VDR migrates to the nucleus where it acts as a transcription factor to control levels of expression of calcium transport proteins (for
example) in the intestine. Most tissues respond to VDR bound to calcitriol and the result is moderation of calcium and phosphate
levels in cells.
Deficiency/excess

Figure 2.231 - Calcitriol - Active form of vitamin D

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Deficiency of vitamin D is a cause of the disease known as rickets, which is characterized by soft, weak bones and most commonly
is found in children. It is not common in the developed world, but elsewhere is of increasing concern.
Excess of vitamin D is rare, but has toxic effects, including hypercalcemia, which results in painful calcium deposits in major
organs. Indications of vitamin D toxicity are increased urination and thirst. Vitamin D toxicity can lead to mental retardation and
many other serious health problems.
Vitamin E

Figure 2.232 α-tocopherol - The most biologically active form of vitamin E Figure
Vitamin E comprises a group of two compounds (tocopherols and tocotrienols - Figure 2.232) and stereoisomers of each. It is
commonly found in plant oils. The compounds act in cells as fat-soluble antioxidants. α-tocopherol (Figure 2.233), the most active
form of the vitamin, works 1) through the glutathione peroxidase protective system and 2) in membranes to interrupt lipid
peroxidation chain reactions. In both actions, vitamin E reduces levels of reactive oxygen species in cells.

Figure 2.233 - Structure of tocotrienols

Action

Figure 2.234 - Lipid peroxidation reactions

Vitamin E scavenges oxygen radicals (possessing unpaired electrons) by reacting with them to produce a tocopheryl radical. This
vitamin E radical can be converted back to its original form by a hydrogen donor. Vitamin C is one such donor. Acting in this way,
Vitamin E helps reduce oxidation of easily oxidized compounds in the lipid peroxidation reactions (Figure 2.234).
Vitamin E also can affect enzyme activity. The compound can inhibit action of protein kinase C in smooth muscle and
simultaneously activate catalysis of protein phosphatase 2A to remove phosphates, stopping smooth muscle growth.
Deficiency/excess
Deficiency of vitamin E can lead to poor conduction of nerve signals and other issues arising from nerve problems. Low levels of
the vitamin may be a factor in low birth weights and premature deliveries. Deficiency, however, is rare, and not usually associated
with diet.
Excess Vitamin E reduces vitamin K levels, thus reducing the ability to clot blood. Hypervitaminosis of vitamin E in conjunction
with aspirin can be life threatening. At lower levels, vitamin E may serve a preventative role with respect to atherosclerosis by
reducing oxidation of LDLs, a step in plaque formation.

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Vitamin K

Figure 2.235 - Forms of vitamin K Image by Pehr Jacobson

Like the other fat-soluble vitamins, Vitamin K comes in multiple forms (Figure 2.235) and is stored in fat tissue in the body. There
are two primary forms of the vitamin - K1 and K2 and the latter has multiple sub-forms . Vitamins K3, K4, and K5 are made
synthetically, not biologically.

Figure 2.236 - Recycling of vitamin K Wikipedia

Action
Vitamin K is used as a co-factor for enzymes that add carboxyl groups to glutamate side chains of proteins to increase their affinity
for calcium. Sixteen such proteins are known in humans. They include proteins involved in blood clotting (prothrombin (called
Factor II), Factors VII, IX, and X), bone metabolism (osteocalcin, also called bone Gla protein (BGP), matrix Gla protein (MGP),
and periostin) and others.
Modification of prothrombin is an important step in the process of blood clotting (see HERE). Reduced levels of vitamin K result
in less blood clotting, a phenomenon sometimes referred to as blood thinning. Drugs that block recycling of vitamin K (Figure
2.236) by inhibiting the vitamin K epoxide reductase, produce lower levels of the vitamin and are employed in treatments for
people prone to excessive clotting. Warfarin (coumadin) is one such compound that acts in this way and is used therapeutically.
Individuals respond to the drug differentially, requiring them to periodically be tested for levels of clotting they possess, lest too
much or too little occur.
Sources

Figure 2.237 - Steroid numbering scheme Image by Pehr Jacobson

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Vitamin K1 is a stereoisomer of the plant photosystem I electron receptor known as phylloquinone and is found abundantly in
green, leafy vegetables. Phylloquinone is one source of vitamin K, but the compound binds tightly to thylakoid membranes and
tends to have low bioavailability. Vitamin K2 is produced by microbes in the gut and is a primary source of the vitamin. Infants in
the first few days before they establish their gut flora and people taking broad spectrum antibiotics may have reduced levels, as a
result. Dietary deficiency is rare in the absence of damage to the small bowel. Others at risk of deficiency include people with
chronic kidney disease and anyone suffering from a vitamin D deficiency. Deficiencies produce symptoms of easy bruising, heavy
menstrual bleeding, anemia, and nosebleeds.
Steroids
Steroids, such as cholesterol are found in membranes and act as signaling hormones in traveling through the body.
Steroid hormones are all made from cholesterol and are grouped into five categories - mineralocorticoids (21 carbons),
glucocorticoids (21 carbons), progestagens (21 carbons), androgens (19 carbons), and estrogens (18 carbons).
Mineralocorticoids
Mineralocorticoids are steroid hormones that influence water and electrolyte balances. Aldosterone (Figure 2.238) is the primary
mineralocorticoid hormone, though other steroid hormones (including progesterone) have some functions like it. Aldosterone
stimulates kidneys to reabsorb sodium, secrete potassium, and passively reabsorb water. These actions have the effect of increasing
blood pressure and blood volume. Mineralocorticoids are produced by the zona glomerulosa of the cortex of the adrenal gland.
Glucocorticoids

Figure 2.238 - Aldosterone - A mineralocorticoid

Glucocorticoids (GCs) bind to glucocorticoid receptors found in almost every vertebrate animal cell and act in a feedback
mechanism in the immune system to reduce its activity. GCs are used to treat diseases associated with overactive immune systems.
These include allergies, asthma, and autoimmune dis- Figure 2.237 - Steroid numbering scheme Image by Pehr Jacobson eases.
Cortisol (Figure 2.239) is an important glucocorticoid with cardiovascular, metabolic, and immunologic functions. The synthetic
glucocorticoid known as dexamethasone has medical applications for treating rheumatoid arthritis, bronchospasms (in asthma), and
inflammation due to its increased potency (25-fold) compared to cortisol. Glucocorticoids are produced primarily in the zona
fasciculata of the adrenal cortex.
Progestagens

Figure 2.239 - Cortisol - A glucocorticoid

Progestagens (also called gestagens) are steroid hormones that work to activate the progesterone receptor upon binding to it.
Synthetic progestagens are referred to as progestins. The most common progestagen is progesterone (also called P4 - Figure 2.240)
and it has functions in maintaining pregnancy. Progesterone is produced primarily in the diestrus phase of the estrous cycle by the
corpus luteum of mammalian ovaries. In pregnancy, the placenta takes over most progesterone production.

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Androgens

Figure 2.240 Progesterone - A progestagen

Androgens are steroid hormones that act by binding androgen receptors to stimulate development and maintenance of male
characteristics in vertebrates. Androgens are precursors of estrogens (see below). The primary androgen is testosterone (Figure
2.241). Other important androgens include dihydrotestosterone (stimulates differentiation of penis, scrotum, and prostate in
embryo) and androstenedione (common precursor of male and female hormones).
Estrogens

Figure 2.241 - Testosterone - An androgen

The estrogen steroid hormones are a class of compounds with important roles in menstrual and estrous cycles. They are the most
important female sex hormones. Estrogens act by activating estrogen receptors inside of cells. These receptors, in turn, affect
expression of many genes. The major estrogens in women include estrone (E1), estradiol (E2 - Figure 2.242), and estriol (E3). In
the reproductive years, estradiol predominates. During pregnancy, estriol predominates and during menopause, estrone is the major
estrogen.

Figure 2.242 - Estradiol - An estrogen

Estrogens are made from the androgen hormones testosterone and androstenedione in a reaction catalyzed by the enzyme known as
aromatase. Inhibition of this enzyme with aromatase inhibitors, such as exemestane, is a strategy for stopping estrogen production.
This may be part of a chemotherapeutic treatment when estrogenresponsive tumors are present.
Cannabinoids
Cannabinoids are a group of chemicals that bind to and have effects on brain receptors (cannabinoid receptors), repressing
neurotransmitter release. Classes of these compounds include endocannabinoids (made in the body), phytocannabinoids (made in
plants, such as marijuana), and synthetic cannabinoids (man-made).
Endocannabinoids are natural molecules derived from arachidonic acid. Cannabinoid receptors are very abundant, comprising the
largest number of G-protein- 247 Figure 2.243 - Tetrahydrocannabinol - Active ingredient in marijuana coupled receptors found in
brain. The best known phytocannabinoid is Δ-9- tetrahydrocannabinol (THC), the primary psychoactive ingredient (of the 85
cannabinoids) of marijuana (Figure 2.243).

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 Figure 2.243 - Tetrahydrocannabinol - Active ingredient in marijuana
Anandamide

Figure 2.244 - Anandamide - An endocannabinoid

Anandamide (N-arachidonoylethanolamine - Figure 2.244) is an endocannabinoid neurotransmitter derived from arachidonic acid.
It exerts its actions primarily through the CB1 and CB2 cannabinoid receptors, the same ones bound by the active ingredient in
marijuana, Δ9-tetrahydrocannabinol. Anandamide has roles in stimulating eating/appetite and affecting motivation and pleasure. It
has been proposed to play a role in “runners high,” an analgesic effect experienced from exertion, especially among runners.
Anandamide appears to impair memory function in rats.
Anandamide has been found in chocolate and two compounds that mimic its effects (N-oleoylethanolamine and
Nlinoleoylethanolamine) are present as well. The enzyme fatty acid amide hydrolase (FAAH) breaks down anandamide into free
arachidonic acid and ethanolamine.
Lipoxins
Lipoxins (Figure 2.245) are eicosanoid compounds involved in modulating immune responses and they have anti-inflammatory
effects. When lipoxins appear in inflammation it begins the end of the process. Lipoxins act to attract macrophages to apoptotic
cells at the site of inflammation and they are engulfed. Lipoxins further act to start the resolution phase of the inflammation
process.

Figure 2.245 - Lipoxin A4

At least one lipoxin (aspirin-triggered LX4) has its synthesis stimulated by aspirin. This occurs as a byproduct of aspirin’s
acetylation of COX-2. When this occurs, the enzyme’s catalytic activity is re-directed to synthesis of 15R-hydroxyeicosatetraenoic
acid (HETE) instead of prostaglandins. 15R-HETE is a procursor of 15-epimer lipoxins, including aspirin-triggered LX4.

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 Figure 2.246 - Structure of heme B
Heme
Heme groups are a collection of protein/ enzyme cofactors containing a large heterocyclic aromatic ring known as a porphyrin ring
with a ferrous (Fe++) ion in the middle. An example porphyrin ring with an iron (found in Heme B of hemoglobin), is shown in
Figure 2.246. When contained in a protein, these are known collectively as hemoproteins (Figure 2.247).
Heme, of course, is a primary component of hemoglobin, but it is also found in other proteins, such as myoglobin, cytochromes,
and the enzymes catalase and succinate dehydrogenase. Hemoproteins function in oxygen transport, catalysis, and electron
transport. Heme is synthesized in the liver and bone marrow in a pathway that is conserved across a wide range of biology.

Figure 2.247 - Heme embedded in the succinate dehydrogenase hemoprotein Wikipedia

Porphobilinogen
Porphobilinogen (Figure 2.248) is a pyrrole molecule involved in porphyrin metabolism. It is produced from aminolevulinate by
action of the enzyme known as ALA dehydratase. Porphobilinogen is acted upon by the enzyme porphobilinogen deaminase.
Deficiency of the latter enzyme (and others in porphyrin metabolism) can result in a condition known as porphyria, which results in
accumulation of porphobilinogen in the cytoplasm of cells.

Figure 2.248 - Porphobilinogen

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The disease can manifest itself with acute abdominal pain and numerous psychiatric issues. Both Vincent van Gogh and King `
George III are suspected to have suffered from porphyria, perhaps causing the “madness of King George III.” Porphyria is also
considered by some to be the impetus for the legend of vampires seeking blood from victims, since the color of the skin in non-
acute forms of the disease can be miscolored, leading some to perceive that as a deficiency of hemoglobin and hence the “thirst”
for blood imagined for vampires.
Dolichols

Figure 2.249 - Structure of dolichol pyrophosphate

Dolichol is a name for a group of non-polar molecules made by combining isoprene units together. Phosphorylated forms of
dolichols play central roles in the N-glycosylation of proteins. This process, which occurs in the endoplasmic reticulum of
eukaryotic cells, begins with a membrane-embedded dolichol pyrophosphate (Figure 2.249) to which an oligosaccharide is attached
(also see HERE). This oligosaccharide contains three molecules of glucose, nine molecules of mannose and two molecules of N-
acetylglucosamine.
Interestingly, the sugars are attached to the dolichol pyrophosphate with the pyrophosphate pointing outwards (away from) the
endoplasmic reticulum, but after attachment, the dolichol complex flips so that the sugar portion is situated on the inside of the
endoplasmic reticulum. There, the entire sugar complex is transferred to the amide of an asparagine side chain of a target protein.
The only asparagine side chains to which the attachment can be made are in proteins where the sequences Asn-X-Ser or Asn-X-Thr
occur. Sugars can be removed/added after the transfer to the protein. Levels of dolichol in the human brain increase with age, but in
neurodegenerative diseases, they decrease.
Terpenes

Figure 2.250 - Pine tree resin - A source of terpenes Wikipedia

Terpenes are members of a class of nonpolar molecules made from isoprene units. Terpenes are produced primarily by plants and
by some insects. Terpenoids are a related group of molecules that contain functional groups lacking in terpenes.
Terpenes have a variety of functions. In plants, they often play a defensive role protecting from insects. The name of terpene comes
from turpentine, which has an odor like some of the terpenes. Terpenes are common components of plant resins (think pine) and
they are widely used in medicines and as fragrances. Hops, for example, gain some of their distinctive aroma and flavor from
terpenes. Not all terpenes, however have significant odor.
Synthesis

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 Figure 2.251 - Three monoterpenes

Terpenes, like steroids, are synthesized starting with simple building blocks known as isoprenes. There are two of them -
dimethylallyl pyrophosphate and the related isopentenyl pyrophosphate and (Figures 2.252 and 2.253) which combine 1-2 units at a
time to make higher order structures. Terpene synthesis overlaps and includes steroid synthesis.

Figure 2.252 - Dimethylallyl pyrophosphate

Terpenes and terpenoids are classified according to how many isoprene units they contain. They include hemiterpenes (one unit),
monoterpenes (two units), sesquiterpenes (three units), diterpenes (four units), sesterterpenes (five units), triterpenes (six units),
sesquarterpenes (seven units), tetraterpenes (eight units), polyterpenes (many units). Another class of terpene-containing molecules,
the norisoterpenoids arise from peroxidase-catalyzed reactions on terpene molecules.

Figure 2.253 - Isopentenyl pyrophosphate

Examples
Common terpenes include monoterpenes of terpineol (lilacs), limonene (citrus), myrcene (hops), linalool (lavender), and pinene
(pine). Higher order terpenes include taxadiene (diterpene precursor of taxol), lycopene (tetraterpenes), carotenes (tetraterpenes),
and natural rubber (polyterpenes).
Steroid precursors geranyl pyrophosphate (monoterpene derivative), farnesyl pyrophosphate (sesquiterpene derivative), and
squalene (triterpene) are all terpenes or derivatives of them. Vitamin A and phytol are derived from diterpenes.
Caffeine

Figure 2.254 - Lycopene - A tetraterpene

Caffeine is the world’s most actively consumed psychoactive drug (Figure 2.255). A methylxanthine alkaloid, caffeine is closely
related to adenine and guanine and this is responsible for many effects on the body. Caffeine blocks the binding of adenosine on its
receptor and consequently prevents the onset of drowsiness induced by adenosine. Caffeine readily crosses the blood-brain barrier
and stimulates release of neurotransmitters. Caffeine stimulates portions of the autonomic nervous system and inhibits the activity
of phosphodiesterase. The latter has the result of raising cAMP levels in cells, which activates protein kinase A and activates
glycogen breakdown, inhibits TNF-α and leukotriene synthesis, which results in reduction of inflammation and innate immunity.

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 Figure 2.255 - Caffeine
Caffeine also has effects on the cholinergic system (acetylcholinesterase inhibitor), is an inositol triphosphate receptor 1 antagonist,
and is a voltage independent activator of ryanodin receptors (a group of calcium channels found in skeletal muscle, smooth muscle,
and heart muscle cells).
The half-life of caffeine in the body varies considerably. In healthy adults, it has a half-life of about 3-7 hours. Nicotine decreases
the half-life and contraceptives and pregnancy can double it. The liver metabolizes caffeine, so the health of the liver is a factor in
the halflife. CYP1A2 of the cytochrome P450 oxidase enzyme is primarily responsible. Caffeine is a natural pesticide in plants,
paralyzing predator bugs.
Lipoprotein complexes and lipid movement in the body
Lipoprotein complexes are combinations of apolipoproteins and lipids bound to them that solubilize fats and other non-polar
molecules, such as cholesterol, so they can travel in the bloodstream between various tissues of the body. The apolipoproteins
provide the emulsification necessary for this. Lipoprotein complexes are formed in tiny “balls” with the water soluble
apolipoproteins on the outside and non-polar lipids, such as fats, cholesteryl esters, and fat soluble vitamins on the inside.
They are categorized by their densities. These include (from highest density to the lowest) high density lipoproteins (HDLs), Low
Density Lipoproteins (LDLs), Intermediate Density Lipoproteins (IDLs), Very Low Density Lipoproteins (VLDLs) and the
chylomicrons. These particles are synthesized in the liver and small intestines.
Apolipoproteins

Figure 2.256 - Apolipoproteins

Each lipoprotein complex contain a characteristic set of apolipoproteins, as shown in Figure 2.256. ApoC-II and ApoC-III are
notable for their presence in all the lipoprotein complexes and the roles they play in activating (ApoC-II) or inactivating (ApoC-III)
lipoprotein lipase. Lipoprotein lipase is a cellular enzyme that catalyzes the breakdown of fat from the complexes. ApoE (see
below) is useful for helping the predict the likelihood of the occurrence of Alzheimer's disease in a patient.

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Gene editing

Figure 2.257 - ApoA-I

ApoB-48 and ApoB-100 are interesting in being coded by the same gene, but a unique mRNA sequence editing event occurs that
converts one into the other. ApoB-100 is made in the liver, but ApoB-48 is made in the small intestine. The small intestine contains
an enzyme that deaminates the cytidine at nucleotide #2153 of the common mRNA. This changes it to a uridine and changes the
codon it is in from CAA (codes for glutamine) to UAA (stop codon). The liver does not contain this enzyme and does not make the
change in the mRNA. Consequently, a shorter protein is synthesized in the intestine (ApoB-48) than the one that is made in the
liver (ApoB-100) using the same gene sequence in the DNA.
Movement
The movement of fats in the body is important because they are not stored in all cells. Only specialized cells called adipocytes store
fat. There are three relevant pathways in the body for moving lipids. As described below, they are 1) the exogenous pathway; 2) the
endogenous pathway, and 3) the reverse transport pathway.
Exogenous pathway

Figure 2.258 - Schematic diagram of a chylomicron Image by Aleia Kim

Dietary fat entering the body from the intestinal system must be transported, as appropriate, to places needing it or storing it. This
is the function of the exogenous pathway of lipid movement in the body. All dietary lipids (fats, cholesterol, fat soluble vitamins,
and other lipids) are moved by it. In the case of dietary fat, it begins its journey after ingestion first by being solubilized by bile
acids in the intestinal tract. After passing through the stomach, pancreatic lipases clip two fatty acids from the fat, leaving a
monoacyl glycerol. The fatty acids and monoacyl glycerol are absorbed by intestinal cells (enterocytes) and reassembled back into
a fat, and then this is mixed with phospholipids, cholesterol esters, and apolipoprotein B-48 and processed to form chylomicrons
(Figures 2.258 & 2.259) in the Golgi apparatus and smooth endoplasmic reticulum.
Exocytosis

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 Figure 2.259 - Another perspective of a chylomicron WIkipedia
These are exocytosed from the cell into lymph capillaries called lacteals. The chylomicrons pass through the lacteals and enter the
bloodstream via the left subclavian vein. Within the bloodstream, lipoprotein lipase breaks down the fats causing the chylomicron
to shrink and become what is known as a chylomicron remnant. It retains its cholesterol and other lipid molecules.
The chylomicron remnants travel to the liver where they are absorbed (Figure 2.260). This is accomplished by receptors in the liver
that recognize and bind to the ApoE of the chylomicrons. The bound complexes are then internalized by endocytosis, degraded in
the lysosomes, and the cholesterol is disbursed in liver cells.
Endogenous pathway
The liver plays a central role in managing the body’s needs for lipids. When lipids are needed by the body or when the capacity of
the liver to contain more lipids than is supplied by the diet, the liver packages up fats and cholesteryl esters into Very Low Density
Lipoprotein (VLDL) complexes and exports them via the endogenous pathway. VLDL complexes contain ApoB-100, ApoC-I,
ApoC-II, ApoC-III, and ApoE apolipoproteins. VLDLs enter the blood and travel to muscles and adipose tissue where lipoprotein
lipase is activated by ApoC-II. In the muscle cells, the released fatty acids are taken up and oxidized. By contrast, in the adipoctyes,
the fatty acids are taken up and reassembled back into triacylglycerides (fats) and stored in fat droplets. Removal of fat from the
VLDLs causes them to shrink, first to Intermediate Density Lipoprotein (IDL) complexes (also called VLDL remnants) and then to
Low Density Lipoprotein (LDL) complexes.

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 Figure 2.260 - Movement of lipids in the body - Green = exogenous pathway; Blue = endogenous pathway; Purple = reverse
transport pathway Image by Aleia Kim
Shrinking of VLDLs is accompanied by loss of apolipoproteins so that LDLs are comprised primarily of ApoB-100. This
lipoprotein complex is important because cells have receptors for it to bind and internalize it by receptor-mediated endocytosis
(Figure 2.261). Up until this point, cholesterol and cholesteryl esters have traveled in chylomicrons, VLDLs, and IDLs as fat has
been stripped stripped away. For cholesterol compounds to get into the cell from the lipoprotein complexes, they must be
internalized by cells and that is the job of receptormediated endocytosis.
Reverse transport pathway
Another important consideration of the movement of lipids in the body is the reverse transport pathway (Figure 2.260). It is also
called the reverse cholesterol transport pathway, since cholesterol is the primary molecule involved. This pathway involves the last
class of lipoprotein complexes known as the High Density Lipoproteins (HDLs). In contrast to the LDLs, which are commonly
referred to as “bad cholesterol” (see below also), the HDLs are known as “good cholesterol.”

Figure 2.261 - The process of receptor-mediated endocytosis Image by Aleia Kim

HDLs are synthesized in the liver and small intestine. They contain little or no lipid when made (called depleted HDLs), but serve
the role of “scavenger” for cholesterol in the blood and from remnants of other (damaged) lipoprotein complexes in the blood. To
perform its task, HDLs carry the enzyme known as lecithincholesterol acyl transferase (LCAT), which they use to form cholesteryl
esters using fatty acids from lecithin (phosphatidylcholine) and then they internalize them.

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The cholesterol used for this purpose comes from the bloodstream, from macrophages, and from foam cells (macrophage-LDL
complexes - Figure 2.262). Addition of cholesteryl esters causes the HDL to swell and Figure 2.261 - The process of receptor-
mediated endocytosis Image by Aleia Kim when it is mature, it returns its load of cholesterol back to the liver or, alternatively, to
LDL molecules for endocytosis. HDLs have the effect of lowering levels of cholesterol and it is for that reason they are described
as “good cholesterol.”
Regulation of lipid transfer

Figure 2.262 - Foam cell aggregate Wikipedia

It is important that cells get food when they need it so some control of the movement of nutrients is critical. The liver, which plays
the central role in modulating blood glucose levels, is also important for performing the same role for lipids. It accomplishes this
task the use of specialized LDL receptors on its surface. Liver LDL receptors bind LDLs that were not taken up by other cells in
their path through the bloodstream. High levels of LDLs are a signal to the liver to reduce the creation of VLDLs for release.
People with the genetic disease known as familial hypercholesterolemia, which manifests with dangerously high levels of LDLs,
lack properly functioning LDL receptors on their liver cells.Figure 2.263 - Progression of atherosclerosis Wikipedia Figure 2.263 -
Progression of atherosclerosis Wikipedia Figure 2.263 - Progression of atherosclerosis Wikipedia

Figure 2.263 - Progression of atherosclerosis Wikipedia

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In sufferers of this disease, the liver never gets the signal that the LDL levels are high. In fact, to the liver, it appears that all VLDLs
and LDLs are being taken up by peripheral tissues, so it creates more VLDLs to attempt to boost levels. Untreated, the disease used
to be fatal early, but newer drugs like the statins have significantly increased life spans of patients. Cellular needs for the contents
of LDLs are directly linked to the levels of synthesis of LDL receptors on their membranes. As cells are needing more cholesterol,
their synthesis of components for receptors goes up and it decreases as need diminishes.
Good cholesterol / bad cholesterol
It is commonly accepted that “high cholesterol” levels are not healthy. This is due, at least indirectly, to the primary carriers of
cholesterol, the LDLs. A primary function of the LDLs is to deliver cholesterol and other lipids directly into cells by receptor
mediated endocytosis (Figure 2.237). High levels of LDLs, though, are correlated with formation of atherosclerotic plaques (Figure
2.263 & 2.264) and incidence of atherosclerosis, leading to the description of them as “bad cholesterol.” This is because when LDL
levels are very high, plaque formation begins. It is thought that reactive oxygen species (higher in the blood of smokers) causes
partial oxidation of fatty acid groups in the LDLs. When levels are high, they tend to accumulate in the extracellular matrix of the
epithelial cells on the inside of the arteries. Macrophages of the immune system take up the damaged LDLs (including the
cholesterol).
Since macrophages can’t control the amount of cholesterol they take up, cholesterol begins to accumulate in them and they take on
appearance that leads to their being described as “foam cells.” With too much cholesterol, the foam cells, however, are doomed to
die by the process of programmed cell death (apoptosis). Accumulation of these, along with scar tissue from inflammation result in
formation of a plaque. Plaques can grow and block the flow of blood or pieces of them can break loose and plug smaller openings
in the blood supply, ultimately leading to heart attack or stroke.
Good cholesterol
On the other hand, high levels of HDL are inversely correlated with atherosclerosis and arterial disease. Depleted HDLs are able to
remove cholesterol from foam cells. This occurs as a result of contact between the ApoA-I protein of the HDL and a transport
protein on the foam cell (ABC-G1). Another transport protein in the foam cell, ABCA-1 transports extra cholesterol from inside the
cell to the plasma membrane where it is taken up into the HDL and returned to the liver or to LDLs by the reverse transport
cholesterol pathway.

Figure 2.264 - Actual carotid artery plaque Wikipedia

Deficiency of the ABCA-1 gene leads to Tangier disease. In this condition, HDLs are almost totally absent because they remain
empty as a result of not being able to take up cholesterol from foam cells, so they are destroyed by the body.
ApoE and Alzheimer’s disease
ApoE is a component of the chylomicrons and is also found in brain, macrophages, kidneys, and the spleen. In humans, it is found
in three different alleles, E2, E3, and E4. The E4 allele (present at about 14% of the population) is associated with increased
likelihood of contracting Alzheimer's disease. People heterozygous for the allele are 3 times as likely to contract the disease and

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those homozygous for it are 15 times as likely to do so. It is not known why this gene or allele is linked to the disease. The three
alleles differ only slightly in amino acid sequence, but the changes do cause notable structural differences. The E4 allele is
associated with increased calcium ion levels and apoptosis after injury. Alzheimer’s disease is associated with accumulation of
aggregates of the β- amyloid peptide. ApoE does enhance the proteolytic breakdown of it and the E4 isoform is not as efficient in
these reactions as the other isoforms.

This page titled 2.8: Structure and Function - Lipids and Membranes is shared under a CC BY-NC-SA license and was authored, remixed, and/or
curated by Kevin Ahern, Indira Rajagopal, & Taralyn Tan.

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2.2: Structure & Function - Amino Acids
Source: BiochemFFA_2_1.pdf. The entire textbook is available for free from the authors at
http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy
All of the proteins on the face of the earth are made up of the same 20 amino acids. Linked together in long chains called
polypeptides, amino acids are the building blocks for the vast assortment of proteins found in all living cells.

"It is one of the more striking generalizations of biochemistry ...that the twenty amino acids and the four bases, are, with minor
reservations, the same throughout Nature." - Francis Crick
All amino acids have the same basic structure, which is shown in Figure 2.1. At the “center” of each amino acid is a carbon called
the α carbon and attached to it are four groups - a hydrogen, an α- carboxyl group, an α-amine group, and an R-group, sometimes
referred to as a side chain. The α carbon, carboxyl, and amino groups are common to all amino acids, so the R-group is the only
unique feature in each amino acid. (A minor exception to this structure is that of proline, in which the end of the R-group is
attached to the α-amine.) With the exception of glycine, which has an R-group consisting of a hydrogen atom, all of the amino
acids in proteins have four different groups attached to them and consequently can exist in two mirror image forms, L and D. With
only very minor exceptions, every amino acid found in cells and in proteins is in the L configuration.

Figure 2.1 - General amino acid structure

There are 22 amino acids that are found in proteins and of these, only 20 are specified by the universal genetic code. The others,
selenocysteine and pyrrolysine use tRNAs that are able to base pair with stop codons in the mRNA during translation. When this
happens, these unusual amino acids can be incorporated into proteins. Enzymes containing selenocysteine, for example, include
glutathione peroxidases, tetraiodothyronine 5' deiodinases, thioredoxin reductases, formate dehydrogenases, glycine reductases,
and selenophosphate synthetase. Pyrrolysine-containing proteins are much rarer and are mostly confined to archaea.

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Essential and non-essential
Nutritionists divide amino acids into two groups - essential amino acids (must be in the diet because cells can’t synthesize them)
and non-essential amino acids (can be made by cells). This classification of amino acids has little to do with the structure of amino
acids. Essential amino acids vary considerable from one organism to another and even differ in humans, depending on whether they
are adults or children. Table 2.1 shows essential and non-essential amino acids in humans.
Some amino acids that are normally nonessential, may need to be obtained from the diet in certain cases. Individuals who do not
synthesize sufficient amounts of arginine, cysteine, glutamine, proline, selenocysteine, serine, and tyrosine, due to illness, for
example, may need dietary supplements containing these amino acids.

Table 2.1 - Essential and non-essential amino acids

Non-protein amino acids
There are also α-amino acids found in cells that are not incorporated into proteins. Common ones include ornithine and citrulline.
Both of these compounds are intermediates in the urea cycle. Ornithine is a metabolic precursor of arginine and citrulline can be
produced by the breakdown of arginine. The latter reaction produces nitric oxide, an important signaling molecule. Citrulline is the
metabolic byproduct. It is sometimes used as a dietary supplement to reduce muscle fatigue.
R-group chemistry

Table 2.2 - Amino acid categories (based on R-group properties)

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We separate the amino acids into categories based on the chemistry of their R-groups. If you compare groupings of amino acids in
different textbooks, you will see different names for the categories and (sometimes) the same amino acid being categorized
differently by different authors. Indeed, we categorize tyrosine both as an aromatic amino acid and as a hydroxyl amino acid. It is
useful to classify amino acids based on their R-groups, because it is these side chains that give each amino acid its characteristic
properties. Thus, amino acids with (chemically) similar side groups can be expected to function in similar ways, for example,
during protein folding.

Non-polar amino acids

Alanine (Ala/A) is one of the most abundant amino acids found in proteins, ranking second only to leucine in occurrence. A D-
form of the amino acid is also found in bacterial cell walls. Alanine is non-essential, being readily synthesized from pyruvate. It
is coded for by GCU, GCC, GCA, and GCG.
Glycine (Gly/G) is the amino acid with the shortest side chain, having an R-group consistent only of a single hydrogen. As a
result, glycine is the only amino acid that is not chiral. Its small side chain allows it to readily fit into both hydrophobic and
hydrophilic environments.

Figure 2.2 - Amino acid side chain properties Wikipedia

Glycine is specified in the genetic code by GGU, GGC, GGA, and GGG. It is nonessential to humans.
Isoleucine (Ile/I) is an essential amino acid encoded by AUU, AUC, and AUA. It has a hydrophobic side chain and is also chiral
in its side chain.
Leucine (Leu/L) is a branched-chain amino acid that is hydrophobic and essential. Leucine is the only dietary amino acid
reported to directly stimulate protein synthesis in muscle, but caution is in order, as 1) there are conflicting studies and 2)
leucine toxicity is dangerous, resulting in "the four D's": diarrhea, dermatitis, dementia and death . Leucine is encoded by six
codons: UUA,UUG, CUU, CUC, CUA, CUG.
Methionine (Met/M) is an essential amino acid that is one of two sulfurcontaining amino acids - cysteine is the other.
Methionine is non-polar and encoded solely by the AUG codon. It is the “initiator” amino acid in protein synthesis, being the
first one incorporated into protein chains. In prokaryotic cells, the first methionine in a protein is formylated.

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 Figure 2.3 - Non-polar amino acids
Proline (Pro/P) is the only amino acid found in proteins with an R-group that joins with its own α-amino group, making a
secondary amine and a ring. Proline is a non-essential amino acid and is coded by CCU, CCC, CCA, and CCG. It is the least
flexible of the protein amino acids and thus gives conformational rigidity when present in a protein. Proline’s presence in a
protein affects its secondary structure. It is a disrupter of α-helices and β-strands. Proline is often hydroxylated in collagen (the
reaction requires Vitamin C - ascorbate) and this has the effect of increasing the protein’s conformational stability. Proline
hydroxylation of hypoxia-inducible factor (HIF) serves as a sensor of oxygen levels and targets HIF for destruction when
oxygen is plentiful.
Valine (Val/V) is an essential, non-polar amino acid synthesized in plants. It is noteworthy in hemoglobin, for when it replaces
glutamic acid at position number six, it causes hemoglobin to aggregate abnormally under low oxygen conditions, resulting in
sickle cell disease. Valine is coded in the genetic code by GUU, GUC, GUA, and GUG.

Carboxyl Amino Acids

Aspartic acid (Asp/D) is a non-essential amino acid with a carboxyl group in its Rgroup. It is readily produced by
transamination of oxaloacetate. With a pKa of 3.9, aspartic acid’s side chain is negatively charged at physiological pH. Aspartic
acid is specified in the genetic code by the codons GAU and GAC.
Glutamic acid (Glu/E), which is coded by GAA and GAG, is a non-essential amino acid readily made by transamination of α-
ketoglutarate. It is a neurotransmitter and has an R-group with a carboxyl group that readily ionizes (pKa = 4.1) at physiological
pH.

Figure 2.4 - Carboxyl amino acids

Amine amino acids

Arginine (Arg/R) is an amino acid that is, in some cases, essential, but non-essential in others. Premature infants cannot
synthesize arginine. In addition, surgical trauma, sepsis, and burns increase demand for arginine. Most people, however, do not
need arginine supplements. Arginine’s side chain contains a complex guanidinium group with a pKa of over 12, making it
positively charged at cellular pH. It is coded for by six codons - CGU, CGC, CGA, CGG, AGA, and AGG.

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 Histidine (His/H) is the only one of the proteinaceous amino acids to contain an imidazole functional group. It is an essential
amino acid in humans and other mammals. With a side chain pKa of 6, it can easily have its charge changed by a slight change
in pH. Protonation of the ring results in two NH structures which can be drawn as two equally important resonant structures.

Figure 2.5 - Amine amino acids

Lysine (Lys/K) is an essential amino acid encoded by AAA and AAG. It has an Rgroup that can readily ionize with a charge of
+1 at physiological pH and can be posttranslationally modified to form acetyllysine, hydroxylysine, and methyllysine. It can
also be ubiquitinated, sumoylated, neddylated, biotinylated, carboxylated, and pupylated, and. O-Glycosylation of
hydroxylysine is used to flag proteins for export from the cell. Lysine is often added to animal feed because it is a limiting
amino acid and is necessary for optimizing growth of pigs and chickens.

Aromatic amino acids

Figure 2.6 - Aromatic amino acids

Phenylalanine (Phe/ F) is a non-polar, essential amino acid coded by UUU and UUC. It is a metabolic precursor of tyrosine.
Inability to metabolize phenylalanine arises from the genetic disorder known as phenylketonuria. Phenylalanine is a component
of the aspartame artificial sweetener.
Tryptophan (Trp/W) is an essential amino acid containing an indole functional group. It is a metabolic precursor of serotonin,
niacin, and (in plants) the auxin phytohormone. Though reputed to serve as a sleep aid, there are no clear research results
indicating this.
Tyrosine (Tyr/Y) is a non-essential amino acid coded by UAC and UAU. It is a target for phosphorylation in proteins by
tyrosine protein kinases and plays a role in signaling processes. In dopaminergic cells of the brain, tyrosine hydroxylase
converts tyrosine to l-dopa, an immediate precursor of dopamine. Dopamine, in turn, is a precursor of norepinephrine and
epinephrine. Tyrosine is also a precursor of thyroid hormones and melanin.

Hydroxyl amino acids

Serine (Ser/S) is one of three amino acids having an R-group with a hydroxyl in it (threonine and tyrosine are the others). It is
coded by UCU, UCC, UCA, UGC, AGU, and AGC. Being able to hydrogen bond with water, it is classified as a polar amino

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 acid. It is not essential for humans. Serine is precursor of many important cellular compounds, including purines, pyrimidines,
sphingolipids, folate, and of the amino acids glycine, cysteine, and tryptophan. The hydroxyl group of serine in proteins is a
target for phosphorylation by certain protein kinases. Serine is also a part of the catalytic triad of serine proteases.

Figure 2.7 - Hydroxyl amino acids

Threonine (Thr/T) is a polar amino acid that is essential. It is one of three amino acids bearing a hydroxyl group (serine and
tyrosine are the others) and, as such, is a target for phosphorylation in proteins. It is also a target for Oglycosylation of proteins.
Threonine proteases use the hydroxyl group of the amino acid in their catalysis and it is a precursor in one biosynthetic pathway
for making glycine. In some applications, it is used as a pro-drug to increase brain glycine levels. Threonine is encoded in the
genetic code by ACU, ACC, ACA, and ACG.
Tyrosine - see HERE.

Figure 2.8 - Amino acid properties Wikipedia

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Other amino acids
Asparagine (Asn/N) is a non-essential amino acid coded by AAU and AAC. Its carboxyamide in the R-group gives it polarity.
Asparagine is implicated in formation of acrylamide in foods cooked at high temperatures (deep frying) when it reacts with
carbonyl groups. Asparagine can be made in the body from aspartate by an amidation reaction with an amine from glutamine.
Breakdown of asparagine produces malate, which can be oxidized in the citric acid cycle.
Cysteine (Cys/C) is the only amino acid with a sulfhydryl group in its side chain. It is nonessential for most humans, but may be
essential in infants, the elderly and individuals who suffer from certain metabolic diseases. Cysteine’s sulfhydryl group is
readily oxidized to a disulfide when reacted with another one. In addition to being found in proteins, cysteine is also a
component of the tripeptide, glutathione. Cysteine is specified by the codons UGU and UGC.

Figure 2.9 - Other amino acids

Glutamine (Gln/Q) is an amino acid that is not normally essential in humans, but may be in individuals undergoing intensive
athletic training or with gastrointestinal disorders. It has a carboxyamide side chain which does not normally ionize under
physiological pHs, but which gives polarity to the side chain. Glutamine is coded for by CAA and CAG and is readily made by
amidation of glutamate. Glutamine is the most abundant amino acid in circulating blood and is one of only a few amino acids
that can cross the blood-brain barrier.
Selenocysteine (Sec/U) is a component of selenoproteins found in all kingdoms of life. It is a component in several enzymes,
including glutathione peroxidases and thioredoxin reductases. Selenocysteine is incorporated into proteins in an unusual scheme
involving the stop codon UGA. Cells grown in the absence of selenium terminate protein synthesis at UGAs. However, when
selenium is present, certain mRNAs which contain a selenocysteine insertion sequence (SECIS), insert selenocysteine when
UGA is encountered. The SECIS element has characteristic nucleotide sequences and secondary structure base-pairing patterns.
Twenty five human proteins contain selenocysteine.
Pyrrolysine (Pyl/O) is a twenty second amino acid, but is rarely found in proteins. Like selenocysteine, it is not coded for in the
genetic code and must be incorporated by unusual means. This occurs at UAG stop codons. Pyrrolysine is found in
methanogenic archaean organisms and at least one methane-producing bacterium. Pyrrolysine is a component of methane-
producing enzymes.

Ionizing groups
pKa values for amino acid side chains are very dependent upon the chemical environment in which they are present. For example,
the R-group carboxyl found in aspartic acid has a pKa value of 3.9 when free in solution, but can be as high as 14 when in certain
environments inside of proteins, though that is unusual and extreme. Each amino acid has at least one ionizable amine group (α-
amine) and one ionizable carboxyl group (α- carboxyl). When these are bound in a peptide bond, they no longer ionize. Some, but
not all amino acids have R-groups that can ionize. The charge of a protein then arises from the charges of the α-amine group, the α-
carboxyl group. and the sum of the charges of the ionized R-groups. Titration/ionization of aspartic acid is depicted in Figure 2.10.
Ionization (or deionization) within a protein’s structure can have significant effect on the overall conformation of the protein and,
since structure is related to function, a major impact on the activity of a protein.

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 Figure 2.10 - Titration curve for aspartic acid Image by Penelope Irving
Most proteins have relatively narrow ranges of optimal activity that typically correspond to the environments in which they are
found (Figure 2.11). It is worth noting that formation of peptide bonds between amino acids removes ionizable hydrogens from
both the α- amine and α- carboxyl groups of amino acids. Thus, ionization/ deionization in a protein arises only from 1) the amino
terminus; 2) carboxyl terminus; 3) R-groups; or 4) other functional groups (such as sulfates or phosphates) added to amino acids
post-translationally - see below.

Carnitine
Not all amino acids in a cell are found in proteins. The most common examples include ornithine (arginine metabolism), citrulline
(urea cycle), and carnitine (Figure 2.12). When fatty acids destined for oxidation are moved into the mitochondrion for that
purpose, they travel across the inner membrane attached to carnitine. Of the two stereoisomeric forms, the L form is the active one.
The molecule is synthesized in the liver from lysine and methionine.

Figure 2.12 - L-Carnitine

From exogenous sources, fatty acids must be activated upon entry into the cytoplasm by being joined to coenzyme A. The CoA
portion of the molecule is replaced by carnitine in the intermembrane space of the mitochondrion in a reaction catalyzed by
carnitine acyltransferase I. The resulting acylcarnitine molecule is transferred across the inner mitochondrial membrane by the
carnitineacylcarnitine translocase and then in the matrix of the mitochondrion, carnitine acyltransferase II replaces the carnitine
with coenzyme A (Figure 6.88).

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 Figure 2.11 - Enzyme activity changes as pH changes Image by Aleia Kim

Catabolism of amino acids

We categorize amino acids as essential or non-essential based on whether or not an organism can synthesize them. All of the amino
acids, however, can be broken down by all organisms. They are, in fact, a source of energy for cells, particularly during times of
starvation or for people on diets containing very low amounts of carbohydrate. From a perspective of breakdown (catabolism),
amino acids are categorized as glucogenic if they produce intermediates that can be made into glucose or ketogenic if their
intermediates are made into acetyl-CoA. Figure 2.13 shows the metabolic fates of catabolism of each of the amino acids. Note that
some amino acids are both glucogenic and ketogenic.

Figure 2.13 - Catabolism of amino acids. Some have more than one path. Image by Pehr Jacobson
Post-translational modifications
After a protein is synthesized, amino acid side chains within it can be chemically modified, giving rise to more diversity of
structure and function (Figure 2.14). Common alterations include phosphorylation of hydroxyl groups of serine, threonine, or
tyrosine. Lysine, proline, and histidine can have hydroxyls added to amines in their R-groups. Other modifications to amino acids
in proteins include addition of fatty acids (myristic acid or palmitic acid), isoprenoid groups, acetyl groups, methyl groups, iodine,
carboxyl groups, or sulfates. These can have the effects of ionization (addition of phosphates/sulfates), deionization (addition of
acetyl group to the R-group amine of lysine), or have no effect on charge at all. In addition, N-linked- and O-linkedglycoproteins
have carbohydrates covalently attached to side chains of asparagine and threonine or serine, respectively.
Some amino acids are precursors of important compounds in the body. Examples include epinephrine, thyroid hormones, Ldopa,
and dopamine (all from tyrosine), serotonin (from tryptophan), and histamine (from histidine).

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 Figure 2.14 - Post-translationally modified amino acids. Modifications shown in green. Image by Penelope Irving

Figure 2.15 - Phosphorylated amino acids

Building Polypeptides
Although amino acids serve other functions in cells, their most important role is as constituents of proteins. Proteins, as we noted
earlier, are polymers of amino acids.
Amino acids are linked to each other by peptide bonds, in which the carboxyl group of one amino acid is joined to the amino group
of the next, with the loss of a molecule of water. Additional amino acids are added in the same way, by formation of peptide bonds
between the free carboxyl on the end of the growing chain and the amino group of the next amino acid in the sequence. A chain
made up of just a few amino acids linked together is called an oligopeptide (oligo=few) while a typical protein, which is made up
of many amino acids is called a polypeptide (poly=many). The end of the peptide that has a free amino group is called the N-
terminus (for NH2), while the end with the free carboxyl is termed the C-terminus (for carboxyl).

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 Figure 2.16 Formation of a peptide bond
As we’ve noted before, function is dependent on structure, and the string of amino acids must fold into a specific 3-D shape, or
conformation, in order to make a functional protein. The folding of polypeptides into their functional forms is the topic of the next
section.

This page titled 2.2: Structure & Function - Amino Acids is shared under a CC BY-NC-SA license and was authored, remixed, and/or curated by
Kevin Ahern, Indira Rajagopal, & Taralyn Tan.

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2.3: Structure & Function- Proteins I
Source: BiochemFFA_2_2.pdf. The entire textbook is available for free from the authors at
http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy
Proteins are the workhorses of the cell. Virtually everything that goes on inside of cells happens as a result of the actions of
proteins. Among other things, protein enzymes catalyze the vast majority of cellular reactions, mediate signaling, give structure
both to cells and to multicellular organisms, and exert control over the expression of genes. Life, as we know it, would not exist if
there were no proteins. The versatility of proteins arises because of their varied structures.
Proteins are made by linking together amino acids, with each protein having a characteristic and unique amino acid sequence. To
get a sense for the diversity of proteins that can be made using 20 different amino acids, consider that the number of different
combinations possible with 20 amino acids is 20n, where n=the number of amino acids in the chain. It becomes apparent that even a
dipeptide made of just two amino acids joined together gives us 202 = 400 different combinations. If we do the calculation for a
short peptide of 10 amino acids, we arrive at an enormous 10,240,000,000,000 combinations. Most proteins are much larger than
this, making the possible number of proteins with unique amino acid sequences unimaginably huge.

Levels of Structure
The significance of the unique sequence, or order, of amino acids, known as the protein’s primary structure, is that it dictates the 3-
D conformation the folded protein will have. This conformation, in turn, will determine the function of the protein. We shall
examine protein structure at four distinct levels (Figure 2.17) - 1) how sequence of the amino acids in a protein (primary structure)
gives identity and characteristics to a protein (Figure 2.18); 2) how local interactions between one part of the polypeptide backbone
and another affect protein shape (secondary structure); 3) how the polypeptide chain of a protein can fold to allow amino acids to
interact with each other that are not close in primary structure (tertiary structure); and 4) how different polypeptide chains interact
with each other within a multi-subunit protein (quaternary structure).

Figure 2.17 - Four Levels of Protein Structure

At this point, we should provide a couple of definitions. We use the term polypeptide to refer to a single polymer of amino acids. It
may or may not have folded into its final, functional form. The term protein is sometimes used interchangeably with polypeptide,
as in “protein synthesis”. It is generally used, however, to refer to a folded, functional molecule that may have one or more subunits
(made up of individual polypeptides). Thus, when we use the term protein, we are usually referring to a functional, folded
polypeptide or peptides. Structure is essential for function. If you alter the structure, you alter the function - usually, but not always,
this means you lose all function. For many proteins, it is not difficult to alter the structure.

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Proteins are flexible, not rigidly fixed in structure. As we shall see, it is the flexibility of proteins that allows them to be amazing
catalysts and allows them to adapt to, respond to, and pass on signals upon binding of other molecules or proteins. However,
proteins are not infinitely flexible. There are constraints on the conformations that proteins can adopt and these constraints govern
the conformations that proteins display.
Subtle changes

Figure 2.18 - Sequence of a simple polypeptide Wikipedia

Even very tiny, subtle changes in protein structure can give rise to big changes in the behavior of proteins. Hemoglobin, for
example, undergoes an incredibly small structural change upon binding of one oxygen molecule, and that simple change causes the
remainder of the protein to gain a considerably greater affinity for oxygen that the protein didn’t have before the structural change.
Sequence, structure and function

Figure 2.19 Linking of amino acids through peptide bond formation

As discussed earlier, the number of different amino acid sequences possible, even for short peptides, is very large. No two proteins
with different amino acid sequences (primary structure) have identical overall structure. The unique amino acid sequence of a
protein is reflected in its unique folded structure. This structure, in turn, determines the protein’s function. This is why mutations
that alter amino acid sequence can affect the function of a protein.

Protein Synthesis
Synthesis of proteins occurs in the ribosomes and proceeds by joining the carboxyl terminus of the first amino acid to the amino
terminus of the next one (Figure 2.19). The end of the protein that has the free α-amino group is referred to as the amino terminus
or N-terminus. The other end is called the carboxyl terminus or C-terminus , since it contains the only free α-carboxyl group. All of
the other α-amino groups and α-carboxyl groups are tied up in forming peptide Figure 2.19 Linking of amino acids through peptide
bond formation bonds that join adjacent amino acids together. Proteins are synthesized starting with the amino terminus and ending
at the carboxyl terminus.

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 Figure 2.20 - Cis vs trans orientation of R-groups around peptide bond Image by Aleia Kim
Schematically, in Figure 2.18, we can see how sequential R-groups of a protein are arranged in an alternating orientation on either
side of the polypeptide chain. Organization of R-groups in this fashion is not random. Steric hindrance can occur when consecutive
R-groups are oriented on the same side of a peptide backbone (Figure 2.20)

Primary Structure
Primary structure is the ultimate determinant of the overall conformation of a protein. The primary structure of any protein arrived
at its current state as a result of mutation and selection over evolutionary time. Primary structure of proteins is mandated by the
sequence of DNA coding for it in the genome. Regions of DNA specifying proteins are known as coding regions (or genes).
The base sequences of these regions directly specify the sequence of amino acids in proteins, with a one-to-one correspondence
between the codons (groups of three consecutive bases) in the DNA and the amino acids in the encoded protein. The sequence of
codons in DNA, copied into messenger RNA, specifies a sequence of amino acids in a protein. (Figure 2.21).

Figure 2.21 - From RNA to amino acids - the genetic code Wikipedia
The order in which the amino acids are joined together in protein synthesis starts defining a set of interactions between amino acids
even as the synthesis is occurring. That is, a polypeptide can fold even as it is being made. The order of the R-group structures and
resulting interactions are very important because early interactions affect later interactions. This is because interactions start
establishing structures - secondary and tertiary. If a helical structure (secondary structure), for example, starts to form, the
possibilities for interaction of a particular amino acid Rgroup may be different than if the helix had not formed (Figure 2.22). R-

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group interactions can also cause bends in a polypeptide sequence (tertiary structure) and these bends can create (in some cases)
opportunities for interactions that wouldn’t have been possible without the bend or prevent (in other cases) similar interaction
possibilities.

Secondary Structure
As protein synthesis progresses, interactions between amino acids close to each other begin to occur, giving rise to local patterns
called secondary structure. These secondary structures include the well known α- helix and β-strands. Both were predicted by Linus
Pauling, Robert Corey, and Herman Branson in 1951. Each structure has unique features.
α-helix

Figure 2.22 - The α-helix. Hydrogen bonds (dotted lines) between the carbonyl oxygen and the amine hydrogen stabilize the
structure. Image by Aleia Kim
The α-helix has a coiled structure, with 3.6 amino acids per turn of the helix (5 helical turns = 18 amino acids). Helices are
predominantly right handed - only in rare cases, such as in sequences with many glycines can left handed α- helices form. In the α-
helix, hydrogen bonds form between C=O groups and N-H groups in the polypeptide backbone that are four amino acids distant.
These hydrogen bonds are the primary forces stabilizing the α-helix.

Figure 2.23 - α-helices in a protein with a leucine zipper structural domain. The α-helices are shown in blue and green and are
bound to a DNA double helix in brown.

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We use the terms rise, repeat, and pitch to describe the parameters of any helix. The repeat is the number of residues in a helix
before it begins to repeat itself. For an α-helix, the repeat is 3.6 amino acids per turn of the helix. The rise is the distance the helix
elevates with addition of each residue. For an α-helix, this is 0.15 nm per amino acid. The pitch is the distance between complete
turns of the helix. For an α-helix, this is 0.54 nm. The stability of an α-helix is enhanced by the presence of the amino acid
aspartate.

Figure 2.24 - α-helix sculpture outside Linus Pauling’s boyhood home Wikipedia

Figure 2.25 - Helical Wheel Representation of an α-Helix. The one letter genetic code is used. The helix starts at Serine #77 at the
right and ends at lysine #92 in the lower right. Hydrophobic amino acids are shown in yellow and ionizing amino acids are shown
in blue. Hydrophobic amino acids tend to interact with each other and not with ionizing amino acids. Wikipedia
β strand/sheet

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 Figure 2.26 - β strand
A helix is, of course, a three-dimensional object. A flattened form of helix in two dimensions is a common description for a β-
strand. Rather than coils, β-strands have bends and these are sometimes referred to as pleats, like the pleats in a curtain. β-strands
can be organized to form elaborately organized structures, such as sheets, barrels, and other arrangements.
Higher order β-strand structures are sometimes called supersecondary structures), since they involve interactions between amino
acids not close in primary sequence. These structures, too, are stabilized by hydrogen bonds between carbonyl oxygen atoms and
hydrogens of amine groups in the polypeptide backbone (Figure 2.28). In a higher order structure, strands can be arranged parallel
(amino to carboxyl orientations the same) or anti-parallel (amino to carboxyl orientations opposite of each other (in Figure 2.27, the
direction of the strand is shown by the arrowhead in the ribbon diagrams).
Turns

Figure 2.27 - Ribbon depictions of supersecondary β-sheets (A-D) and α-helix arrangements (E-F) Image by Aleia Kim
Turns (sometimes called reverse turns) are a type of secondary structure that, as the name suggests, causes a turn in the structure of
a polypeptide chain. Turns give rise to tertiary structure ultimately, causing interruptions in the secondary structures (α- helices and
β-strands) and often serve as connecting regions between two regions of secondary structure in a protein. Proline and glycine play
common roles in turns, providing less flexibility (starting the turn) and greater flexibility (facilitating the turn), respectively.

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 Figure 2.28 - Components of a β-sheet in a parallel arrangement. H-bonds in yellow. Image by Aleia Kim
There are at least five types of turns, with numerous variations of each giving rise to many different turns. The five types of turns
are
• δ-turns - end amino acids are separated by one peptide bond
• γ-turns - separation by two peptide bonds
•β-turns - separation by three peptide bonds
•α-turns - separation by four peptide bonds
•π-turns - separation by five bonds
Of these, the β-turns are the most common form and the δ-turns are theoretical, but unlikely, due to steric limitations. Figure 2.29
depicts a β- turn.
310 helices

Figure 2.29 - β-turn. R-groups are shown in orange, hydrogens in yellow, carbons in charcoal, nitrogens in purple, and oxygens in
green. A stabilizing hydrogen bond is indicated with the dotted line. Image by Aleia Kim
In addition to the α-helix, β-strands, and various turns, other regular, repeating structures are seen in proteins, but occur much less
commonly. The 310 helix is the fourth most abundant secondary structure in proteins, constituting about 10-15% of all helices. The
helix derives its name from the fact that it contains 10 amino acids in 3 turns. It is right-handed. Hydrogen bonds form between

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amino acids that are three residues apart. Most commonly, the 310 helix appears at the amine or carboxyl end of an α-helix. Like the
α-helix, the 310 helix is stabilized by the presence of aspartate in its sequence.

Figure 2.30 - Top view of a 310 Helix. Carbonyl groups are in red and pointed upwards. Note the almost perfect 3-fold symmetry
Wikipedia

Figure 2.31 - Resonance of the peptide bond Wikipedia

Figure 2.32 - π helix Wikipedia

π-helices

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 Figure 2.33 - Planes (light blue) defined by the double-bonded character of the peptide bond Image by Aleia Kim
A π-helix may be thought of as a special type of α- helix. Some sources describe it as an α-helix with an extra amino acid stuck in
the middle of it (Figure 2.32). π-helices are not exactly rare, occurring at least once in as many as 15% of all proteins. Like the α-
helix, the π-helix is right-handed, but where the α-helix has 18 amino acids in 5 turns, the π-helix has 22 amino acids in 5 turns. π-
helices typically do not stretch for very long distances. Most are only about 7 amino acids long and the sequence almost always
occurs in the middle of an α-helical region.
Ramachandran plots

Figure 2.34 - ω, ψ, and φ rotational angles in a peptide Image by Aleia Kim

In 1963, G.N. Ramachandran, C. Ramakrishnan, and V. Sasisekharan described a novel way to describe protein structure. If one
considers the backbone of a polypeptide chain, it consists of a repeating set of three bonds. Sequentially (in the amino to carboxyl
direction) they are 1) a rotatable bond (ψ) between α-carbon and α-carboxyl preceding the peptide bond (see HERE), 2) a non-
rotatable peptide bond (ω) between the α-carboxyl and α-amine groups), and 3) a rotatable bond (φ) between the α-amine and α-
carbon following the peptide bond (see HERE). Note in Figures 2.33 and 2.34 that the amino to carboxyl direction is right to left.
The presence of the carbonyl oxygen on the α-carboxyl group allows the peptide bond to exist as a resonant structure, meaning that
it behaves some of the time as a double bond. Double bonds cannot, of course, rotate, but the bonds on either side of it have some
freedom of rotation. The φ and ψ angles are restricted to certain values, because some angles will result in steric hindrance. In
addition, each type of secondary structure has a characteristic range of values for φ and ψ.

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 Figure 2.35 - Theoretical Ramachandran plot Image by Penelope Irving
Ramachandran and colleagues made theoretical calculations of the energetic stability of all possible angles from 0° to 360° for each
of the φ and ψ angles and plotted the results on a Ramachandran Plot (also called a φ-ψ plot), delineating regions of angles that
were theoretically the most stable (Figure 2.35).
Three primary regions of stability were identified, corresponding to φ-ψ angles of β-strands (top left), right handed α- helices
(bottom left), and lefthanded α-helices (upper right). The plots of predicted stability are remarkably accurate when compared to φ-ψ
angles of actual proteins.
Secondary structure prediction

Table 2.3 - Relative tendencies of each amino acid to be in a secondary structure. Higher values indicate greater tendency Image by
Penelope Irving
By comparing primary structure (amino acid sequences) to known 3D protein structures, one can tally each time an amino acid is
found in an α-helix, β-strand/sheet, or a turn. Computer analysis of thousands of these sequences allows one to assign a likelihood
of any given amino acid appearing in each of these structures. Using these tendencies, one can, with up to 80% accuracy, predict
regions of secondary structure in a protein based solely on amino acid sequence.
This is seen in Table 2.3. Occurrence in primary sequence of three consecutive amino acids with relative tendencies higher than one
is an indicator that that region of the polypeptide is in the corresponding secondary structure. An online resource for predicting

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secondary structures called PSIPRED is available HERE.
Hydrophobicity

Table 2.4 - Hydropathy Scores

The chemistry of amino acid Rgroups affects the structures they are most commonly found in. Subsets of their chemical properties
can give clues to structure and, sometimes, cellular location. A prime example is the hydrophobicity (wateravoiding tendencies) of
some Rgroups. Given the aqueous environment of the cell, such R-groups are not likely to be on the outside surface of a folded
protein.
However, this rule does not hold for regions of protein that may be embedded within the lipid bilayers of cellular/ organelle
membranes. This is because the region of such proteins that form the transmembrane domains are are buried in the hydrophobic
environment in the middle of the lipid bilayer.
Not surprisingly, scanning primary sequences for specifically sized/spaced stretches of hydrophobic amino acids can help to
identify proteins found in membranes. Table 2.4 shows hydrophobicity values for R-groups of the amino acids. In this set, the scale
runs from positive values (hydrophobic) to negative values (hydrophilic). A KyteDoolittle Hydropathy plot for the RET
protooncogene membrane protein is shown in Figure 2.36. Two regions of the protein are very hydrophobic as can be seen from the
peaks near amino acids 5-10 and 630-640. Such regions might be reasonably expected to be situated either within the interior of the
folded protein or to be part of transmembrane domains.
Random coils

Figure 2.36 Kyte-Doolittle hydropathy plot for the RET protooncogene Wikipedia

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Some sections of a protein assume no regular, discernible structure and are sometimes said to lack secondary structure, though they
may have hydrogen bonds. Such segments are described as being in random coils and may have fluidity to their structure that
results in them having multiple stable forms. Random coils are identifiable with spectroscopic methods, such as circular dichroism
Wikipedia and nuclear magnetic resonance (NMR) in which distinctive signals are observed. See also metamorphic proteins
(HERE) and intrinsically disordered proteins (HERE).

Figure 2.37 - Ribbon depiction of a β-hairpin. Shown are two β strands in turquoise interacting with each other.
Supersecondary structure
Another element of protein structure is harder to categorize because it incorporates elements of secondary and tertiary structure.
Dubbed supersecondary structure (or structural motifs), these structures contain multiple nearby secondary structure components
arranged in a specific way and that appear in multiple proteins. Since there are many ways of making secondary structures from
different primary structures, so too can similar motifs arise from different primary sequences. An example of a structural motif is
shown in Figure 2.37.
Tertiary structure

Figure 2.38 - Folding of a polypeptide chain

Proteins are distinguished from each other by the sequence of amino acids comprising them. The sequence of amino acids of a
protein determines protein shape, since the chemical properties of each amino acid are forces that give rise to intermolecular
interactions to begin to create secondary structures, such as α-helices and β-strands. The sequence also defines turns and random
coils that play important roles in the process of protein folding.
Since shape is essential for protein function, the sequence of amino acids gives rise to all of the properties a protein has. As protein
synthesis proceeds, individual components of secondary structure start to interact with each other, giving rise to folds that bring
amino acids close together that are not near each other in primary structure (Figure 2.38). At the tertiary level of structure,
interactions among the R-groups of the amino acids in the protein, as well as between the polypeptide backbone and amino acid
side groups play a role in folding.
Globular proteins

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 Figure 2.39 - Unfolding (denaturation) of a protein Wikipedia
Folding gives rise to distinct 3-D shapes in proteins that are non-fibrous. These proteins are called globular. A globular protein is
stabilized by the same forces that drive its formation. These include ionic interactions, hydrogen bonding, hydrophobic forces,
ionic bonds, disulfide bonds and metallic bonds. Treatments such as heat, pH changes, detergents, urea and mercaptoethanol
overpower the stabilizing forces and cause a protein to unfold, losing its structure and (usually) its function (Figure 2.39). The
ability of heat and detergents to denature proteins is why we cook our food and wash our hands before eating - such treatments
denature the proteins in the microorganisms on our hands. Organisms that live in environments of high temperature (over 50°C)
have proteins with changes in stabilizing forces - additional hydrogen bonds, additional salt bridges (ionic interactions), and
compactness may all play roles in keeping these proteins from unfolding.
Protein stabilizing forces
Before considering the folding process, let us consider some of the forces that help to stabilize proteins.
Hydrogen bonds

Figure 2.40 - Hydrogen bonds (dotted lines) between two molecules of acetic acid
Hydrogen bonds arise as a result of partially charged hydrogens found in covalent bonds. This occurs when the atom the hydrogen
is bonded to has a greater electronegativity than hydrogen itself does, resulting in hydrogen having a partial positive charge because
it is not able to hold electrons close to itself (Figure 2.40).
Hydrogen partially charged in this way is attracted to atoms, such as oxygen and nitrogen that have partial negative charges, due to
having greater electronegativities and thus holding electrons closer to themselves. The partially positively charged hydrogens are
called donors, whereas the partially negative atoms they are attracted to are called acceptors. (See Figure 1.30).

Figure 2.41 - Hydrogen bonding in liquid water Wikipedia

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Individual hydrogen bonds are much weaker than a covalent bond, but collectively, they can exert strong forces. Consider liquid
water, which contains enormous numbers of hydrogen bonds (Figure 2.41). These forces help water to remain liquid at room
temperature. Other molecules lacking hydrogen bonds of equal or greater molecular weight than water, such as methane or carbon
dioxide, are gases at the same temperature. Thus, the intermolecular interactions between water molecules help to “hold” water
together and remain a liquid. Notably, only by raising the temperature of water to boiling are the forces of hydrogen bonding
overcome, allowing water to become fully gaseous.
Hydrogen bonds are important forces in biopolymers that include DNA, proteins, and cellulose. All of these polymers lose their
native structures upon boiling. Hydrogen bonds between amino acids that are close to each other in primary structure can give rise
to regular repeating structures, such as helices or pleats, in proteins (secondary structure).
Ionic interactions
Ionic interactions are important forces stabilizing protein structure that arise from ionization of R-groups in the amino acids
comprising a protein. These include the carboxyl amino acids (HERE), the amine amino acids as well as the sulfhydryl of cysteine
and sometimes the hydroxyl of tyrosine.
Hydrophobic forces
Hydrophobic forces stabilize protein structure as a result of interactions that favor the exclusion of water. Non-polar amino acids
(commonly found in the interior of proteins) favor associating with each other and this has the effect of excluding water. The
excluded water has a higher entropy than water interacting with the hydrophobic side chains. This is because water aligns itself
very regularly and in a distinct pattern when interacting with hydrophobic molecules.
When water is prevented from having these kinds of interactions, it is much more disordered that it would be if it could associate
with the hydrophobic regions. It is partly for this reason that hydrophobic amino acids are found in protein interiors - so they can
exclude water and increase entropy.
Disulfide bonds

Figure 2.42 - Formation of a disulfide bond

Disulfide bonds, which are made when two sulfhydryl side-chains of cysteine are brought into close proximity, covalently join
together different protein regions and can give great strength to the overall structure (Figures 2.42 & 2.43). An Ode to Protein
Structure by Kevin Ahern The twenty wee amino A's Define a protein many ways Their order in a peptide chain Determines forms
that proteins gain And when they coil, it leaves me merry Cuz that makes structures secondary It's tertiary, I am told That happens
when a protein folds But folded chains are downright scary When put together quaternary They're nature's wonders, that's for sure
Creating problems, making cures A fool can fashion peptide poems But proteins come from ribosoems These joined residues of
cysteine are sometimes referred to as cystine. Disulfide bonds are the strongest of the forces stabilizing protein structure.

Figure 2.43 - Cystine - Two cysteines joined by a disulfide bond

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van der Waals forces
van der Waals forces is a term used to describe various weak interactions, including those caused by attraction between a polar
molecule and a transient dipole, or between two temporary dipoles. van der Waals forces are dynamic because of the fluctuating
nature of the attraction, and are generally weak in comparison to covalent bonds, but can, over very short distances, be significant.
Post-translational modifications
Post-translational modifications can result in formation of covalent bonds stabilizing proteins as well. Hydroxylation of lysine and
proline in strands of collagen can result in cross-linking of these groups and the resulting covalent bonds help to strengthen and
stabilize the collagen.
Folding models
Two popular models of protein folding are currently under investigation. In the first (diffusion collision model), a nucleation event
begins the process, followed by secondary structure formation. Collisions between the secondary structures (as in the β-hairpin in
Figure 2.37) allow for folding to begin. By contrast, in the nucleation-condensation model, the secondary and tertiary structures
form together.
Folding in proteins occurs fairly rapidly (0.1 to 1000 seconds) and can occur during synthesis - the amino terminus of a protein can
start to fold before the carboxyl terminus is even made, though that is not always the case.
Folding process

Figure 2.44 Folding funnel energy model of folding Wikipedia

Protein folding is hypothesized to occur in a “folding funnel” energy landscape in which a folded protein’s native state corresponds
to the minimal free energy possible in conditions of the medium (usually aqueous solvent) in which the protein is dissolved. As
seen in the diagram (Figure 2.44), the energy funnel has numerous local minima (dips) in which a folding protein can become
trapped as it moves down the energy plot. Other factors, such as temperature, electric/magnetic fields, and spacial considerations
likely play roles.
If external forces affect local energy minima during folding, the process and end-product can be influenced. As the speed of a car
going down a road will affect the safety of the journey, so too do energy considerations influence and guide the folding process,
resulting in fully functional, properly folded proteins in some cases and misfolded “mistakes” in others.
Getting stuck
As the folding process proceeds towards an energy minimum (bottom of the funnel in Figure 2.44), a protein can get “stuck” in any
of the local minima and not reach the final folded state. Though the folded state is, in general, more organized and therefore has
reduced entropy than the unfolded state, there are two forces that overcome the entropy decrease and drive the process forward.

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The first is the magnitude of the decrease in energy as shown in the graph. Since ΔG = ΔH -TΔS, a decrease in ΔH can overcome a
negative ΔS to make ΔG negative and push the folding process forward. Favorable (decreased) energy conditions arise with
formation of ionic bonds, hydrogen bonds, disulfide bonds, and metallic bonds during the folding process. In addition, the
hydrophobic effect increases entropy by allowing hydrophobic amino acids in the interior of a folded protein to exclude water, thus
countering the impact of the ordering of the protein structure by making the ΔS less negative.
Structure prediction
Computer programs are very good at predicting secondary structure solely based on amino acid sequence, but struggle with
determining tertiary structure using the same information. This is partly due to the fact that secondary structures have repeating
points of stabilization based on geometry and any regular secondary structure (e.g., α-helix) varies very little from one to another.
Folded structures, though, have an enormous number of possible structures as shown by Levinthal’s Paradox.
Spectroscopy
Because of our inability to accurately predict tertiary structure based on amino acid sequence, proteins structures are actually
determined using techniques of spectroscopy. In these approaches, proteins are subjected to varied forms of electromagnetic
radiation and the ways they interact with the radiation allows researchers to determine atomic coordinates at Angstrom resolution
from electron densities (see X-ray crystallography) and how nuclei spins interact (see NMR).
Levinthal’s paradox
In the late 1960s, Cyrus Levinthal outlined the magnitude of the complexity of the protein folding problem. He pointed out that for
a protein with 100 amino acids, it would have 99 peptide bonds and 198 considerations for φ and ψ angles. If each of these had
only three conformations, that would result in 3198 different possible foldings or 2.95x1094.
Even allowing a reasonable amount of time (one nanosecond) for each possible fold to occur, it would take longer than the age of
the universe to sample all of them, meaning clearly that the process of folding is not occurring by a sequential random sampling
and that attempts to determine protein structure by random sampling were doomed to fail. Levinthal, therefore, proposed that
folding occurs by a sequential process that begins with a nucleation event that guides the process rapidly and is not unlike the
funnel process depicted in Figure 2.44.
Diseases of protein misfolding

Figure 2.45 - Misfolding of the normal PRPc protein induced by PRPsc Image by Penelope Irving
The proper folding of proteins is essential to their function. It follows then that misfolding of proteins (also called proteopathy)
might have consequences. In some cases, this might simply result in an inactive protein. Protein misfolding also plays a role in
numerous diseases, such as Mad Cow Disease, Alzheimers, Parkinson’s Disease, and CreutzfeldJakob disease. Many, but not all,
misfolding diseases affect brain tissue.

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 Figure 2.46 - Cows with Mad Cow Disease lose their ability to stand
Insoluble deposits
Misfolded proteins will commonly form aggregates called amyloids that are harmful to tissues containing them because they
change from being soluble to insoluble in water and form deposits. The process by which misfolding (Figure 2.45) occurs is not
completely clear, but in many cases, it has been demonstrated that a “seed” protein which is misfolded can induce the same
misfolding in other copies of the same protein. These seed proteins are known as prions and they act as infectious agents, resulting
in the spread of disease. The list of human diseases linked to protein misfolding is long and continues to grow. A Wikipedia link is
HERE.
Prions

Figure 2.47 - Diffuse amyloidosis in a blood vessel (red dots) Wikipedia

Prions are infectious protein particles that cause transmissible spongiform encephalopathies (TSEs), the best known of which is
Mad Cow disease. Other manifestations include the disease, scrapie, in sheep, and human diseases, such as CreutzfeldtJakob
disease (CJD), Fatal Familial Insomnia, and kuru. The protein involved in these diseases is a membrane protein called PrP. PrP is
encoded in the genome of many organisms and is found in most cells of the body. PrPc is the name given to the structure of PrP
that is normal and not associated with disease. PrPSc is the name given to a misfolded form of the same protein, that is associated
with the development of disease symptoms (Figure 2.45).
Misfolded
The misfolded PrPSc is associated with the TSE diseases and acts as an infectious particle. A third form of PrP, called PrPres can
be found in TSEs, but is not infectious. The ‘res’ of PrPres indicates it is protease resistant. It is worth noting that all three forms of
PrP have the same amino acid sequence and differ from each other only in the ways in which the polypeptide chains are folded.
The most dangerously misfolded form of PrP is PrPSc, because of its ability to act like an infectious agent - a seed protein that can
induce misfolding of PrPc , thus converting it into PrPSc.
Function

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 Figure 2.48 - One model of prion propagation Wikipedia
The function of PrPc is unknown. Mice lacking the PrP gene do not have major abnormalities. They do appear to exhibit problems
with long term memory, suggesting a function for PrPc . Stanley Prusiner, who discovered prions and coined the term, received the
Nobel Prize in Medicine in 1997 for his work. I think that if I chanced to be on A protein making up a prion I’d twist it and for
goodness sakes Stop it from making fold mistakes
Amyloids
Amyloids are a collection of improperly folded protein aggregates that are found in the human body. As a consequence of their
misfolding, they are insoluble and contribute to some twenty human diseases including important neurological ones involving
prions. Diseases include (affected protein in parentheses) - Alzheimer’s disease (Amyloid β), Parkinson’s disease (α-synuclein),
Huntington’s disease (huntingtin), rheumatoid arthritis (serum amyloid A), fatal familial insomnia (PrPSc), and others.
Amino acid sequence plays a role in amyloidogenesis. Glutamine-rich polypeptides are common in yeast and human prions.
Trinucleotide repeats are important in Huntington’s disease. Where sequence is not a factor, hydrophobic association between β-
sheets can play a role.
Amyloid β

Figure 2.49 - Huntingtin

Amyloid β refers to collections of small proteins (36-43 amino acids) that appear to play a role in Alzheimer’s disease. (Tau protein
is the other factor.) They are, in fact, the main components of amyloid plaques found in the brains of patients suffering from the
disease and arise from proteolytic cleavage of a larger amyloid precursor glycoprotein called Amyloid Precursor Protein, an
integral membrane protein of nerve cells whose function is not known. Two proteases, β-secretase and γ- secretase perform this
function. Amyloid β proteins are improperly folded and appear to induce other proteins to misfold and thus precipitate and form the
amyloid characteristic of the disease. The plaques are toxic to nerve cells and give rise to the dementia characteristic of the disease.
It is thought that aggregation of amyloid β proteins during misfolding leads to generation of reactive oxygen species and that this is
the means by which neurons are damaged. It is not known what the actual function of amyloid β is. Autosomal dominant mutations

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in the protein lead to early onset of the disease, but this occurs in no more than 10% of the cases. Strategies for treating the disease
include inhibition of the secretases that generate the peptide fragments from the amyloid precursor protein.
Huntingtin
Huntingtin is the central gene in Huntington’s disease. The protein made from it is glutamine rich, with 6-35 such residues in its
wild-type form. In Huntington’s disease, this gene is mutated, increasing the number of glutamines in the mutant protein to between
36 and 250. The size of the protein varies with the number of glutamines in the mutant protein, but the wild-type protein has over
3100 amino acids and a molecular weight of about 350,000 Da. Its precise function is not known, but huntingtin is found in nerve
cells, with the highest level in the brain. It is thought to possibly play roles in transport, signaling, and protection against apoptosis.
Huntingtin is also required for early embryonic development. Within the cell, huntingtin is found localized primarily with
microtubules and vesicles.
Trinucleotide repeat
The huntingtin gene contains many copies of the sequence CAG (called trinucleotide repeats), which code for the many glutamines
in the protein. Huntington’s disease arises when extra copies of the CAG sequence are generated when the DNA of the gene is
being copied. Expansion of repeated sequences can occur due to slipping of the polymerase relative to the DNA template during
replication. As a result, multiple additional copies of the trinucleotide repeat may be made, resulting in proteins with variable
numbers of glutamine residues. Up to 35 repeats can be tolerated without problem. The number of repeats can expand over the
course of a person’s lifetime, however, by the same mechanism. Individuals with 36-40 repeats begin to show signs of the disease
and if there are over 40, the disease will be present.
Molecular chaperones
The importance of the proper folding of proteins is highlighted by the diseases associated with misfolded proteins, so it is no
surprise, then, that cells expend energy to facilitate the proper folding of proteins. Cells use two classes of proteins known as
molecular chaperones, to facilitate such folding in cells. Molecular chaperones are of two kinds, the chaperones, and the
chaperonins. An example of the first category is the Hsp70 class of proteins. Hsp stands for “heat shock protein”, based on the fact
that these proteins were first observed in large amounts in cells that had been briefly subjected to high temperatures. Hsps function
to assist cells in stresses arising from heat shock and exposure to oxidizing conditions or toxic heavy metals, such as cadmium and
mercury. However, they also play an important role in normal conditions, where they assist in the proper folding of polypeptides by
preventing aberrant interactions that could lead to misfolding or aggregation. The Hsp70 proteins are found in almost all cells and
use ATP hydrolysis to stimulate structural changes in the shape of the chaperone to accommodate binding of substrate proteins. The
binding domain of Hsp70s contains a β-barrel structure which wraps around the polypeptide chain of the substrate and has affinity
for hydrophobic side chains of amino acids. As shown in Figure 2.50, Hsp70 binds to polypeptides as they emerge from ribosomes
during protein synthesis. Binding of substrate stimulates ATP hydrolysis and this is facilitated by another heat shock protein known
as Hsp40. The hydrolysis of ATP causes the Hsp70 to taken on a closed conformation that helps shield exposed hydrophobic
residues and prevent aggregation or local misfolding.
After protein synthesis is complete, ADP is released and replaced by ATP and this results in release of the substrate protein, which
then allows the full length polypeptide to fold correctly.

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 Figure 2.50 - Action of Hsp70 (blue) to facilitate proper folding of a protein (orange) Image by Aleia Kim
In heat shock
In times of heat shock or oxidative stress, Hsp70 proteins bind to unfolded hydrophobic regions of proteins to similarly prevent
them from aggregating and allowing them to properly refold. When proteins are damaged, Hsp70 recruits enzymes that ubiquitinate
the damaged protein to target them for destruction in proteasomes. Thus, the Hsp70 proteins play an important role in ensuring not
only that proteins are properly folded, but that damaged or nonfunctional proteins are removed by degradation in the proteasome.
Chaperonins
A second class of proteins involved in assisting other proteins to fold properly are known as chaperonins. There are two primary
categories of chaperonins - Class I (found in bacteria, chloroplasts, and mitochondria) and Class II (found in the cytosol of
eukaryotes and archaebacteria). The best studied chaperonins are the GroEL/GroES complex proteins found in bacteria (Figure
2.51).

Figure 2.51 - View from bottom of GroEL (left) and GroEL/ GroES complex (right) Wikipedia
GroEL/GroES may not be able to undo aggregated proteins, but by facilitating proper folding, it provides competition for
misfolding as a process and can reduce or eliminate problems arising from improper folding. GroEL is a double-ring 14mer with a
hydrophobic region that can facilitate folding of substrates 15-60 kDa in size. GroES is a singlering heptamer that binds to GroEL
in the presence of ATP and functions as a cover over GroEL. Hydrolysis of ATP by chaperonins induce large conformational
changes that affect binding of substrate proteins and their folding. It is not known exactly how chaperonins fold proteins. Passive
models postulate the chaperonin complex functioning inertly by preventing unfavorable intermolecular interactions or placing
restrictions on spaces available for folding to occur. Active models propose that structural changes in the chaperonin complex
induce structural changes in the substrate protein.
Protein breakdown

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 Figure 2.52 - 26S proteasome. Active site shown in red Wikipedia
Another protein complex that has an important function in the lifetime dynamics of proteins is the proteasome (Figure 2.52).
Proteasomes, which are found in all eukaryotes and archaeans, as well as some bacteria, function to break down unneeded or
damaged proteins by proteolytic degradation. Proteasomes help to regulate the concentration of some proteins and degrade ones
that are misfolded. The proteasomal degradation pathway plays an important role in cellular processes that include progression
through the cell cycle, modulation of gene expression, and response to oxidative stresses.
Degradation in the proteasome yields short peptides seven to eight amino acids in length. Threonine proteases play important roles.
Breakdown of these peptides yields individual amino acids, thus facilitating their recycling in cells. Proteins are targeted for
degradation in eukaryotic proteasomes by attachment to multiple copies of a small protein called ubiquitin (8.5 kDa - 76 amino
acids). The enzyme catalyzing the reaction is known as ubiquitin ligase. The resulting polyubiquitin chain is bound by the
proteasome and degradation begins. Ubiquitin was named due to it ubiquitously being found in eukaryotic cells.
Ubiquitin

Figure 2.53 - Ubiquitin (lysine side chains shown in yellow) Wikipedia

Ubiquitin (Figure 2.53) is a small (8.5 kDa) multi-functional protein found in eukaryotic cells. It is commonly added to target
proteins by action of ubiquitin ligase enzymes (E3 in Figure 2.54). One (ubiquitination) or many (polyubiquitination) ubiquitin
molecules may be added. Attachment of the ubiquitin is through the side chain of one of seven different lysine residues in
ubiquitin.
The addition of ubiquitin to proteins has many effects, the best known of which is targeting the protein for degradation in the
proteasome. Proteasomal targeting is seen when polyubiquitination occurs at lysines #29 and 48. Polyubiquitination or
monoubiquitination at other lysines can result in altered cellular location and changed protein-protein interactions. The latter may
alter affect inflammation, endocytic trafficking, translation and DNA repair.

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 Figure 2.54 - Pathway for ubiquitination of a target substrate protein Image by Pehr Jacobson
Ubiquitin ligase malfunction
Parkin is a Parkinson’s disease-related protein that, when mutated, is linked to an inherited form of the disease called autosomal
recessive juvenile Parkinson’s disease. The function of the protein is not known, but it is a component of the E3 ubiquitin ligase
system responsible for transferring ubiquitin from the E2 protein to a lysine side chain on the target protein. It is thought that
mutations in parkin lead to proteasomal dysfunction and a consequent inability to break down proteins harmful to dopaminergic
neurons. This results in the death or malfunction of these neurons, resulting in Parkinson’s disease.
Intrinsically disordered proteins

Movie 2.1 - Dynamic movement of cytochrome C in solution Wikipedia

As is evident from the many examples described elsewhere in the book, the 3-D structure of proteins is important for their function.
But, increasingly, it is becoming evident that not all proteins fold into a stable structure. Studies on the so-called intrinsically
disordered proteins (IDPs) in the past cou- ple of decades has shown that many proteins are biologically active, even thought they
fail to fold into stable structures. Yet other proteins exhibit regions that remain unfolded (IDP regions) even as the rest of the
polypeptide folds into a structured form.
Intrinsically disordered proteins and disordered regions within proteins have, in fact, been known for many years, but were
regarded as an anomaly. It is only recently, with the realization that IDPs and IDP regions are widespread among eukaryotic
proteins, that it has been recognized that the observed disorder is a "feature, not a bug".

Movie 2.2 SUMO-1, a protein with intrinsically disordered sections Wikipedia

Comparison of IDPs shows that they share sequence characteristics that appear to favor their disordered state. That is, just as some
amino acid sequences may favor the folding of a polypeptide into a particular structure, the amino acid sequences of IDPs favor
their remaining unfolded. IDP regions are seen to be low in hydrophobic residues and unusually rich in polar residues and proline.
The presence of a large number of charged amino acids in the IDPs can inhibit folding through charge repulsion, while the lack of

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hydrophobic residues makes it difficult to form a stable hydrophobic core, and proline discourages the formation of helical
structures. The observed differences between amino acid sequences in IDPs and structured proteins have been used to design
algorithms to predict whether a given amino acid sequence will be disordered.
What is the significance of intrinsically disordered proteins or regions? The fact that this property is encoded in their amino acid
sequences suggests that their disorder may be linked to their function. The flexible, mobile nature of some IDP regions may play a
crucial role in their function, permitting a transition to a folded structure upon binding a protein partner or undergoing post-
translational modification. Studies on several wellknown proteins with IDP regions suggest some answers. IDP regions may
enhance the ability of proteins like the lac repressor to translocate along the DNA to search for specific binding sites. The
flexibility of IDPs can also be an asset in protein-protein interactions, especially for proteins that are known to interact with many
different protein partners.

Figure 2.55 - Denaturation and renaturation of ribonuclease Wikipedia

For example, p53 has IDP regions that may allow the protein to interact with a variety of functional partners. Comparison of the
known functions of proteins with predictions of disorder in these proteins suggests that IDPs and IDP regions may
disproportionately function in signaling and regulation, while more structured proteins skew towards roles in catalysis and
transport. Interestingly, many of the proteins found in both ribosomes and spliceosomes are predicted to have IDP regions that may
play a part in correct assembly of these complexes. Even though IDPs have not been studied intensively for very long, what little is
known of them suggests that they play an important and underestimated role in cells.
Metamorphic proteins
Another group of proteins that have recently changed our thinking about protein structure and function are the so-called
metamorphic proteins. These proteins are capable of forming more than one stable, folded state starting with a single amino acid
sequence. Although it is true that multiple folded conformations are not ruled out by the laws of physics and chemistry,
metamorphic proteins are a relatively new discovery. It was known, of course, that prion proteins were capable of folding into
alternative structures, but metamorphic proteins appear to be able to toggle back and forth between two stable structures. While in
some cases, the metamorphic protein undergoes this switch in response to binding another molecule, some proteins that can
accomplish this transition on their own. An interesting example is the signaling molecule, lymphotactin. Lymphotactin has two
biological functions that are carried out by its two conformers- a monomeric form that binds the lymphotactin receptor and a
dimeric form that binds heparin. It is possible that this sort of switching is more widespread than has been thought.
Refolding denatured proteins
All information for protein folding is contained in the amino acid sequence of the protein. It may seem curious then that most
proteins do not fold into their proper, fully active form after they have been+++ denatured and the denaturant is removed. A few
do, in fact. One good example is bovine ribonuclease (Figure 2.55). Its catalytic activity is very resistant to heat and urea and
attempts to denature it don’t work very well. However, if one treats the enzyme with β-mercaptoethanol (which breaks disulfide

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bonds) prior to urea treatment and/or heating, activity is lost, indicating that the covalent disulfide bonds help stabilize the overall
enzyme structure and when they are broken, denaturation can readily occur. When the mixture cools back down to room
temperature, over time some enzyme activity reappears, indicating that ribonuclease re-folded under the new conditions.
Interestingly, renaturation will occur maximally if a tiny amount of β-mercaptoethanol is left in the solution during the process. The
reason for this is because β- mercaptoethanol permits reduction (and breaking) of accidental, incorrect disulfide bonds during the
folding process. Without it, these disulfide bonds will prevent proper folds from forming.
Irreversible denaturation
Most enzymes, however, do not behave like bovine ribonuclease. Once denatured, their activity cannot be recovered to any
significant There are not very many ways Inactivating RNase It’s stable when it’s hot or cold Because disulfides tightly hold If you
desire to make it stall Use hot mercaptoethanol extent. This may seem to contradict the idea of folding information being inherent
to the sequence of amino acids in the protein. It does not.
Most enzymes don’t refold properly after denaturation for two reasons. First, normal folding may occur as proteins are being made.
Interactions among amino acids early in the synthesis are not “confused” by interactions with amino acids later in the synthesis
because those amino acids aren’t present as the process starts.
Chaperonins’ role
In other cases, the folding process of some proteins in the cell relied upon action of chaperonin proteins (see HERE). In the absence
of chaperonins, interactions that might result in misfolding occur, thus preventing proper folding. Thus, early folding and the
assistance of chaperonins eliminate some potential “wrong-folding” interactions that can occur if the entire sequence was present
when folding started.
Quaternary structure
A fourth level of protein structure is that of quaternary structure. It refers to structures that arise as a result of interactions between
multiple polypeptides. The units can be identical multiple copies or can be different polypeptide chains. Adult hemoglobin is a
good example of a protein with quaternary structure, being composed of two identical chains called α and two identical chains
called β.
Though the α-chains are very similar to the β- chains, they are not identical. Both of the α- and the β-chains are also related to the
single polypeptide chain in the related protein called myoglobin. Both myoglobin and hemoglobin have similarity in binding
oxygen, but their behavior towards the molecule differ significantly. Notably, hemoglobin’s multiple subunits (with quaternary
structure) compared to myoglobin’s single subunit (with no quaternary structure) give rise to these differences.
References
1. https://en.wikipedia.org/wiki/Van_der_W aals_force 105

This page titled 2.3: Structure & Function- Proteins I is shared under a CC BY-NC-SA license and was authored, remixed, and/or curated by
Kevin Ahern, Indira Rajagopal, & Taralyn Tan.

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 CHAPTER OVERVIEW

3: Membranes
Topic hierarchy
3.1: Basic Concepts in Membranes
3.2: Transport in Membranes
3.3: Other Considerations in Membranes

Thumbnail: The cell membrane, also called the plasma membrane or plasmalemma, is a semipermeable lipid bilayer common to
all living cells. It contains a variety of biological molecules, primarily proteins and lipids, which are involved in a vast array of
cellular processes. It also serves as the attachment point for both the intracellular cytoskeleton and, if present, the cell wall. Image
used with permission (CC BU-SA 3.0; Dhatfield and LadyofHats).

This page titled 3: Membranes is shared under a CC BY-NC-SA license and was authored, remixed, and/or curated by Kevin Ahern, Indira
Rajagopal, & Taralyn Tan.

1
3.1: Basic Concepts in Membranes
Source: BiochemFFA_3_1.pdf. The entire textbook is available for free from the authors at
http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy

Lipid bilayers
The protective membrane around cells contains many components, including cholesterol, proteins, glycolipids,
glycerophospholipids, and sphingolipids. The last two of these will, when mixed vigorously with water, spontaneously form what is
called a lipid bilayer (Figure 3.1), which serves as a protective boundary for the cell that is largely impermeable to the movement of
most materials across it. With the notable exceptions of water, carbon dioxide, carbon monoxide, and oxygen, most polar/ionic
require transport proteins to help them to efficiently navigate across the bilayer. The orderly movement of these compounds is
critical for the cell to be able to 1) get food for energy; 2) export materials; 3) maintain osmotic balance; 4) create gradients for
secondary transport; 5) provide electromotive force for nerve signaling; and 6) store energy in electrochemical gradients for ATP
production (oxidative phosphorylation or photosynthesis). In some cases, energy is required to move the substances (active
transport).

Facilitated Diffusion
In other cases, no external energy is required and they move by diffusion through specific cellular channels. This is referred to as
facilitated diffusion. Before we discuss movement of materials across membranes, it is appropriate we discuss the composition of
cellular membranes. Plasma membranes differ from cell walls both in the materials comprising them and in their flexibility. Cell
walls will be covered near the end of this chapter.

Figure 3.1: Lipid bilayer closeup Image by Aleia Kim

Though some cells do not have cell walls (animal cells) and others do (bacteria, fungi, and plants), there is commonality among
cells in that they all possess plasma membranes. There is also commonality in the components of the membranes, though the
relative amount of constituents varies. Figures 3.1 and 3.2 illustrate the structure and environments of plasma membranes. All
plasma membranes contain a significant amount of amphiphilic substances linked to fatty acids. These include the
glycerophospholipids and the sphingolipids. The fatty acid(s) are labeled as hydrophobic tails in the figures.

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 Figure 3.2: Organization of the lipid bilayer Image by Aleia Kim

Hydrophilic heads
The composition of the hydrophilic heads varies considerably. In glycerophospholipids, a phosphate is always present, of course,
and it is often esterified to another substance to make a phosphatide (Figure 3.3). Common compounds linked to the phosphate (at
the X position) include serine, ethanolamine, and choline. These vary in the their charges so in this way, the charge on the external
or internal surface can be controlled. Cells tend to have more negative charges on the exterior half of the lipid bilayer (called the
outer leaflet) and more positive charges on the interior half (inner leaflet).

Figure 3.3 - Schematic diagram of a phosphatide

Figure 3.4: A sphingolipid. Polar head in black. Nonpolar tail in red Image by Aleia Kim

Sphingolipids
In sphingolipids (Figure 3.4), the hydrophilic head can contain a phosphate linked to ethanolamine or choline and this describes the
structure of sphingomyelin, an important component of neural membranes. Most sphingolipids lack the phosphate and have instead
a hydrophilic head of a single sugar (cerebrosides) or a complex oligosaccharide (gangliosides).
Water exclusion

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In each case, the glycerophospholipid or sphingolipid has one end that is polar and one end that is non-polar. As we saw in the
organization of amino acids with hydrophobic side chains occurring preferentially on the inside of a folded protein to exclude
water, so too do the non-polar portions of these amphiphilic molecules arrange themselves so as to exclude water. Remember that
the cytoplasm of a cell is mostly water and the exterior of the cell is usually bathed in an aqueous layer. It therefore makes perfect
sense that the polar portions of the membrane molecules arrange themselves as they do - polar parts outside interacting with water
and non-polar parts in the middle of the bilayer avoiding/excluding water.

Figure 3.6 - Different lipid bilayer structures Image by Aleia Kim

Figure 3.5 - Similarity of form between a phosphoglyceride and sphingomyelin Image by Aleia Kim

Composition Bias
The plasma membrane has distinct biases of composition relative to its inside and the outside (Figure 3.7). First, glycosylation (of
lipids and proteins) has the sugar groups located almost exclusively on the outside of the cell, away from the cytoplasm (Figure
3.8). Among the membrane lipids, sphingolipids are much more commonly glycosylated than glycerophospholipids. In addition,
some of the glyerophospholipids are found preferentially on one side or the other (Figure 3.7). Phosphatidylserine and
phosphatidylethanolamine are found preferentially within the inner leaflet of the plasma membrane, whereas phosphatidylcholine
tends to be located on the outer leaflet. In the process of apoptosis, the phosphatidylserines appear on the outer leaflet where they
serve as a signal to macrophages to bind and destroy the cell. Sphingolipids are found preferentially in the plasma membrane and
are almost completely absent from mitochondrial and endoplasmic reticulum membranes (Figure 3.9).

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 Figure 3.7: Differential distribution of membrane lipids by inner and outer leaflet Image by Pehr Jacobson

Figure 3.8 - Molecular organization of the lipid bilayer Image by Aleia Kim

Figure 3.9 - Distribution of lipids in organelle membranes Image by Pehr Jacobson

Organelle membranes
Bias of lipid composition also exists with respect to organelle membranes. The unusual diphosphodiglycerolipid known as
cardiolipin, for example, is almost only found in mitochondrial membranes (see HERE) and like phosphatidylserine, its movement
is an important step in apoptosis. In signaling, phosphatidylinositols play important roles providing second messengers upon being
cleaved (see HERE).

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Lateral Diffusion
Movement of lipids within each leaflet of the lipid bilayer occurs readily and rapidly due to membrane fluidity. This type of
movement is called lateral diffusion and can be measured by the technique called FRAP (Figure 3.10, see HERE also). In this
method, a laser strikes and stains a section of the lipid bilayer of a cell, leaving a spot as shown in B. Over time, the stain diffuses
out ultimately across the entire lipid bilayer, much like a drop of ink will diffuse throughout when added to a glass of water. A
measurement of the rate of diffusion gives an indication of the fluidity of a membrane.

Figure 3.10 - Fluorescence recovery after photobleaching (FRAP)

Transverse Diffusion
While the movement in lateral diffusion occurs rapidly, movement of molecules from one leaflet over to the other leaflet occurs
much more slowly. This type of molecular movement is called transverse diffusion and is almost nonexistent in the absence of
enzyme action. Remember that there is a bias of distribution of molecules between leaflets of the membrane, which means that
something must be moving them.
There are three enzymes that catalyze movement of compounds in transverse diffusion. Flippases move membrane
glycerophospholipids/ sphingolipids from outer leaflet to inner leaflet (cytoplasmic side) of cell. Floppases move membrane lipids
in the opposite direction. Scramblases move in either direction.

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 Figure 3.11 - Catalytic action of a flippase, a floppase, and a scramblase

Figure 3.12 - Structure of a flippase Wikipedia

Other components of lipid bilayer

Besides glycerophospholipids and sphingolipids, there are other materials commonly found in lipid bilayers of cellular membranes.
Two important prominent ones are cholesterol (Figure 3.13) and proteins. Besides serving as a metabolic precursor of steroid
hormones and the bile acids, cholesterol’s main role in cells is in the membranes. The flatness and hydrophobicity of the sterol
rings allow cholesterol to interact with the nonpolar portions of the lipid bilayer while the hydroxyl group on the end can interact
with the hydrophilic part.

Membrane fluidity
Cholesterol’s function in the lipid bilayer is complex (Figure 3.13). It influences membrane fluidity. Figure 3.14 shows the phase
transition for a membrane as it is heated, moving from a more “frozen” character to that of a more “fluid” one as the temperature
rises. The mid-point of this transition, referred to as the Tm, is influenced by the fatty acid composition of the lipid bilayer
compounds. Longer and more saturated fatty acids will favor higher Tm values, whereas unsaturation and short fatty acids will
favor lower Tm values. It is for this reason that fish, which live in cool environments, have a higher level of unsaturated fatty acids
in them - to use to make membrane lipids that will remain fluid at ocean temperatures. Interestingly, cholesterol does not change
the Tm value, but instead widens the transition range between frozen and fluid forms of the membrane, allowing it to have a wider
range of fluidity.

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 Figure 3.14 - Membrane transition temperature Image by Aleia Kim

Figure 3.13 - Cholesterol in the lipid bilayer Image by Aleia Kim

Lipid Rafts
Cholesterol is also abundantly found in membrane structures called lipid rafts. Depicted in Figure 3.15, lipid rafts are organized
structures within the membrane typically containing signaling molecules and other integral membrane proteins. Lipid rafts affect
membrane fluidity, neurotransmission, and trafficking of receptors and membrane proteins.

Figure 3.15 - A lipid raft - 1 = Non-raft membrane / 2 = Lipid raft / 3 = Lipid raft associated transmembrane protein / 4 = Non-raft
membrane protein / 5 = Glycosylation modifications (on glycoproteins and glycolipids) / 6 = GPIanchored protein / 7 = Cholesterol
/ 8 = Glycolipid
Features
Distinguishing features of the rafts is that they are more ordered than the bilayers surrounding them, containing more saturated fatty
acids (tighter packing and less disorganization, as a result) and up to 5 times as much cholesterol. They also are relatively rich in
sphingolipids, with as much as 50% greater quantities of sphingomyelin than surrounding areas of the bilayer. The higher
concentration of cholesterol in the rafts may be due to its greater ability to associate with sphingolipids (Figure 3.16). Some groups,
such as prenylated proteins, like RAS, may be excluded from lipid rafts.

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 Figure 3.16 - A sphingolipid (left) associating with cholesterol (right)
Lipid rafts may provide concentrating platforms after individual protein receptors bind to ligands in signaling. After receptor
activation takes place at a lipid raft, the signaling complex would provide protection from nonraft enzymes that could inactivate the
signal. For example, a common feature of signaling systems is phosphorylation, so lipid rafts might provide protection against
dephosphorylation by enzymes called phosphatases. Lipid rafts appear to be involved in many signal transduction processes, such
as T cell antigen receptor signaling, B cell antigen receptor signaling, EGF receptor signaling, immunoglobulin E signaling, insulin
receptor signaling and others. For more on signaling, see HERE.

Barrier
Transport of materials across membranes is essential for a cell to exist. The lipid bilayer is an effective barrier to the entry of most
molecules and without a means of allowing food molecules to enter a cell, it would die. The primary molecules that move freely
across the lipid bilayer are small, uncharged ones, such as H2O, CO2, CO, and O2, so larger molecules, like glucose, that the cell
needs for energy, would be effectively excluded if there were not proteins to facilitate its movement across the membrane.

Figure 3.17 - The lipid bilayer as a barrier Image by Pehr Jacobson

Figure 3.17 depicts the barrier that the lipid bilayer provides to movement across it and the pressures (ionic attraction, in this case)
that can affect movement. Potential energy from charge and concentration differences are harvested by cells for purposes that
include synthesis of ATP, and moving materials against a concentration gradient in a process called active transport.

Membrane proteins
Proteins in a lipid bilayer can vary in quantity enormously, depending on the membrane. Protein content by weight of various
membranes typically ranges between 30 and 75% by weight. Some mitochondrial membranes can have up to 90% protein. Proteins
linked to and associated with membranes come in several types.
Transmembrane proteins

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Transmembrane proteins are integral membrane proteins that completely span from one side of a biological membrane to the other
and are firmly embedded in the membrane (Figure 3.18). Transmembrane proteins can function as docking sites for attachment (to
the extracellular matrix, for example), as receptors in the cellular signaling system, or facilitate the specific transport of molecules
into or out of the cell.

Figure 3.18 - Membrane protein types Image by Aleia Kim

Example of integrated/ transmembrane proteins include those involved in transport (e.g., Na+/K+ ATPase), ion channels (e.g.,
potassium channel of nerve cells) and signal transduction across the lipid bilayer (e.g., GProtein Coupled Receptors).
Peripheral membrane proteins interact with part of the bilayer (usually does not involve hydrophobic interactions), but do not
project through it. A good example is phospholipase A2, which cleaves fatty acids from glycerophospholipids in membranes.
Associated membrane proteins typically do not have external hydrophobic regions, so they cannot embed in a portion of the lipid
bilayer, but are found near them. Such association may arise as a result of interaction with other proteins or molecules in the lipid
bilayer. A good example is ribonuclease.
Anchored membrane proteins
Anchored membrane proteins are not themselves embedded in the lipid bilayer, but instead are attached to a molecule (typically a
fatty acid) that is embedded in the membrane (Figure 3.19). The oncogene family of proteins known as ras are good examples.
These proteins are anchored to the lipid bilayer by attachment to non-polar farnesyl groups catalyzed by the enzyme
farnesyltransferase.

Figure 3.19 - Integral and anchored proteins Image by Aleia Kim

Finer classification
A more detailed classification scheme further categorizes the integral and anchored proteins into six different types (Figure 3.20).
Type I and Type II have only one portion of the protein pass through the membrane. They differ in the orientation of the amine and
carboxyl end with respect to inside/outside. Type I transmembrane proteins have the amino terminus on the outside and carboxy
terminus on the inside, whereas Type II proteins have this reversed. Type III proteins are a single polypeptide chain that has

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multiple regions of it cross back and forth across the membrane, often to form a channel. Type IV is a multi-polypeptide protein
which has multiple crossings of the membrane. Type V transmembrane proteins do not have a part of them that crosses the
membrane, but they are anchored to the membrane by a lipid (such as a fatty acid) embedded in the lipid bilayer. Type VI
transmembrane proteins both have a portion of them that crosses the membrane and they are attached to a lipid embedded in the
lipid bilayer.

Figure 3.20 Types of integral and anchored membrane proteins Image by Aleia Kim

Blood Types
Cells have hundreds-thousands of membrane proteins and the protein composition of a membrane varies with its function and
location. Glycoproteins embedded in membranes play important roles in cellular identification. Blood types, for example, differ
from each other in the structure of the carbohydrate chains projecting out from the surface of the glycoprotein in their membranes
(Figure 3.21).

Figure 3.21

Osmotic Pressure
Membranes provide barriers/boundaries for most molecules, but the permeability of water across a lipid bilayer creates a variable
that must be considered. The variable here is osmotic pressure. Osmotic pressure (loosely) refers to the tendency of a solution to
take in water by the process of osmosis. In Figure 3.22, one can see a visual representation of the concept of the pressure.

Figure 3.22

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A U-shaped tube has at its bottom a semipermeable membrane. Water can pass through the membrane, but sugar molecules
(C6H12O6) cannot. On the left side, sugar is added creating a concentration difference between the right and left chambers. Water
diffuses across the membrane from right to left in an attempt to equalize the concentrations, causing the level of the right side to
decrease and the left side to increase. The pressure resulting from the differences in height is felt at the membrane.
Equalizing concentrations
The liquid on the right does not completely move to the left, though, as might be expected if the only force involved is equalizing
the concentration of sugar across the membrane (no sugar on right = no water). Instead, an equilibrium of sorts of water levels is
reached even though the concentrations don’t equal out. The pressure existing at the membrane then from the differences in level
corresponds to the osmotic pressure of the mixture. The osmotic pressure of a solution is the pressure difference needed to halt the
flow of solvent across a semipermeable membrane. Osmotic pressure can also be thought of as the pressure required to counter
osmosis. The osmotic pres- Figure 3.21 - Blood types arise from cell surface glycoproteins Figure 3.22 - Osmotic pressure. Water
diffuses leftwards to try to equalize the solute concentration. The pressure realized at the membrane in the right figure is the
osmotic pressure sure of a dilute solution mathematically behaves like the ideal gas law
nRT
Posmotic = (3.1.1)
V

where n is the number of moles, R is the gas constant, T is the temperature in Kelvin, and V is the volume.
It is more convenient in solutions to work with molarity, so
∗
Posmotic = M R T (3.1.2)

where M is the molarity of the dissolved molecules, R* is the gas constant expressed in (L atm)/(K mol), and T is the temperature.
The Greek letter Π is used to refer to the Posmoticterm, so
∗
Π = MR T (3.1.3)

Remember when calculating the molarity to include the molarity of each particle. For example, when one dissolves sucrose in
solution, it does not split into smaller particles, so
M olarityP articles = M olaritySucrose (3.1.4)

However, for salts, like KOH, which forms two ions in solution (K+ and OH- ),
MolarityParticles= 2* MolarityKOH.
Significant consideration
Osmotic pressure is a significant consideration for cells. Consider the fact that water can move freely across cellular membranes,
but most of the contents of the cell, such as proteins, DNA, ions, sugars, etc., cannot. Second, the concentration of these
compounds inside the cell is different than the concentration of them outside of the cell. Third, since water can move through the
lipid bilayer, it will tend to move in the direction that will tend to equalize solute.
Hypotonic, hypertonic, isotonic
We consider three situations (Figure 3.23). First, if the concentration of solutes is greater inside the cell than outside, water will
tend to move into the cell, causing the cell to swell. This circumstance is called hypotonic. Conversely, if the solute concentration is
greater outside the cell than inside of it, water will exit the cell and the cell with shrink. This is a hypertonic situation. Last, if the
concentrations of solutes into and outside of the cell is equal, this is called an isotonic solution. Here, no movement of water occurs
across the cell membrane and the cell retains its size.

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 Figure 3.23 - The effect of three different osmotic conditions on red blood cells concentrations on either side of the membrane
If the osmotic pressure is greater than the forces holding together a cellular membrane, the cell will rupture. Because of this, some
cells have built in defenses to prevent problems. Plant cells, for example have a fairly rigid cell wall that resists expansion in
hypotonic solutions (Figure 3.24). Bacteria also have a cell wall that provides protection.

This page titled 3.1: Basic Concepts in Membranes is shared under a CC BY-NC-SA license and was authored, remixed, and/or curated by Kevin
Ahern, Indira Rajagopal, & Taralyn Tan.

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3.2: Transport in Membranes
Source: BiochemFFA_3_2.pdf. The entire textbook is available for free from the authors at
http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy

Movement of materials across membranes

As noted earlier, it is essential for cells to be able to uptake nutrients. This function along with movement of ions and other
substances is provided by proteins/protein complexes that are highly specific for the compounds they move.
Selective movement of ions by membrane proteins and the ions’ extremely low permeability across the lipid bilayer are important
for helping to maintain the osmotic balance of the cell and also for providing for the most important mechanism for it to make ATP
- the process of oxidative phosphorylation.
Terminology
A protein involved in moving only one molecule across a membrane is called a uniport (Figure 3.25). Proteins that move two
molecules in the same direction across the membrane are called symports (also called synporters, synports, or symporters). If two
molecules are moved in opposite directions across the bilayer, the protein is called an antiport. Proteins involved in moving ions are
called ionophores.
If the action of a protein in moving ions across a membrane results in a net change in charge, the protein is described as
electrogenic and if there is no change in charge the protein is described as electroneutral (Figure 3.26). When the driving force for
movement through the membrane protein is simply diffusion, the process is called facilitated diffusion or passive transport and
when the process requires other energy input, the process is called active transport.

Figure 3.25 - A uniport, a symport, and an antiport

Figure 3.26 - Electroneutral and electrogenic transporters - Image by Aleia Kim

Channels and transporters

With respect to movement of materials through membrane proteins, there is a difference between channels (sometimes called
pores) and transporters. Channels largely provide openings with some specificity and molecules pass through them at close to the
rate of diffusion. They usually involve movement of water or ions. Examples would be the sodium or potassium channels of nerve
cells. Transporters have high specificity and transfer rates that are orders of magnitude slower. Transport proteins include the
sodium-potassium pump, the sodium-calcium exchanger, and lactose permease, amongst many others).
Facilitated diffusion
As noted, the driving force for facilitated diffusion is concentration, meaning that in facilitated diffusion, materials will only move
from a higher concentration to a lower concentration and that at the end of the process, the concentration of materials on each side
of a bilayer will be equal (Figure 3.28). This may work well in many cases.
For example, the blood concentration of glucose is sufficiently high that red blood cells can use facilitated diffusion as a means of
acquiring glucose. Other cells, further removed from the blood supply where the glucose concentration is lower, must use active
transport mechanisms because there is not a sufficient concentration of glucose to provide cells with the glucose they need.

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 Figure 3.27 - Ion channel and transporter proteins

Figure 3.28 - In diffusion, solutes move from high concentration to lower concentration and eventually equalize

Ion channels
Ion channels are pore-forming membrane proteins in the membranes of all cells that regulate movement of selected ions across a
membrane (Figures 3.29 & 3.30). They help to establish the resting membrane potential and to affect action potentials and other
electrical signals. They are very important in the process of nerve transmission. Ion channels control the flow of ions across
secretory and epithelial cells, and consequently help to regulate cell volume by affecting osmotic pressure.
Ion channels are essential features of almost all cells, functioning as selective “tunnels” that restrict movement through them to ions
with specific characteristics (typically size). The size of the opening is very narrow (usually one or two atoms wide) and is able to
select even against ions that are too small.

Figure 3.29 - Schematic structure of an ion channel protein - 1 = General structure / 2 = Entrance / 3 = Selectivity filter / 4 =
Effective diameter / 5 = Controllable opening

Figure 3.30 - Two types of ion channel - single and double gates - Image by Pehr Jacobson
Control mechanisms
Ion channels are controlled by mechanisms that include voltage, ligands, light, temperature, and mechanical deformation (stretch
activated). Ligand-gated ion channels (LGICs) are transmembrane proteins which open to selectively allow ions such as Na+, K+,

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Ca++, or Cl− to pass through the membrane in response to the binding of a ligand messenger.
Sound waves cause mechanical deformation of hair cells in the ear. This results in the opening of ion channels and initiation of a
nerve signal to the brain.
Sodium ion channels in the tongue for sugar receptors open in response to binding of sucrose, allowing sodium concentration in the
nerve cell to increase and initiate a nerve signal to the brain. In this case, the default for the gate is to be closed and it opens in
response to binding of a ligand (sucrose).
In light sensing cells of the eye, calcium gates are open by default, but stimulation by light causes them to close, triggering a series
of events that result in a signal being sent the brain about the perception of light. Thus, in this case, the stimulus (light) causes an
open channel to close.
Moving the other direction, nerve signals originating in the brain travel to muscle tissue and through a complicated set of
exchanges, result in the opening of calcium gates of muscle cells, increasing the concentration of calcium and stimulating muscular
contraction (see HERE).
Voltage gated channels are essential for transmission of nerve signals, a process discussed in more depth HERE.
Ion movement through channels
The ability of ion channels to select against ions too large is intuitive - the size of the opening in the ion channel simply isn’t big
enough for a larger ion to fit through the opening. Potassium, for example, passes through sodium channels rarely because the
opening is too small.
Potassium channels that are selective for potassium ions must be big enough to allow potassium to enter, but if size were the only
selection means, then sodium ions would also readily pass through potassium channels, since sodium ions (0.95 Å) are smaller than
potassium ions (1.33 Å). In order for potassium channels to select against sodium ions and favor potassium ions, other
considerations come into play.

Figure 3.31 - A potassium channel closed (left) and open (right) - David Goodsell - "Molecule of the Month: Potassium Channels".
(from Wikipedia)
Hydration shell
To understand this unique selectivity, it is important to understand how ions move through channels. Before an ion can pass
through a channel, it must first be dissociated from (stripped of) the water molecules in its hydration shell - water molecules
surrounding ions in aqueous solutions (Figure 3.32). This process requires an input of energy. The initial energy required to strip
the water molecules from the hydration shell has been compared to the activation energy of an enzymatic reaction.

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 Figure 3.32 - Sodium surrounded by water molecules in a hydration shell
Comparable to enzymes
Just as enzymes lower the activation energy of enzymatic reactions and thus allow them to more readily occur, so too do channel
proteins lower the energy requirements for a molecule to traverse a lipid bilayer. In the absence of the channel protein, the
dehydration energy is mostly prohibitive for most polar molecules to occur, so very few make it across the lipid bilayer without the
channel protein. This is why ion channel/transport proteins are so important to the cell.
After the water has been stripped, the ion can pass through the channel and when it arrives at the other side of the channel, the
diffusing ion becomes rehydrated, thus regaining the energy that was required initially to strip away the water molecules from the
ion.
Selectivity of the potassium channel
The potassium channel (Figure 3.33) uses the dimensions of the potassium ion precisely to shepherd it through the channel. The
sodium ion, which has different dimensions has a more difficult time making it through the channel despite its smaller size. The
reason this is rooted in the energy required for dehydration.
For potassium ions, after the water has been stripped off, precisely positioned carbonyl groups along the channel help to stabilize
the ion as it moves. The sodium ion, on the other hand is too small and does not make efficient connections with carbonyl groups
and thus has a more difficult path. Because of this, the energy difference between dehydration and rehydration of a sodium ion in a
potassium channel is energetically unfavorable (requires net input of energy) but the same process for a potassium ion is
energetically favorable (results in a net gain of energy).

Figure 3.33 - A potassium ion (center) transiting the potassium channel - David Goodsell

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 Movie 3.1 - Gramicidin A Wikipedia (animated gif, download to view)
Energy factor
Thus the selection in favor of potassium and against sodium ions in a potassium channel is based on energy, not physical size,
whereas in the selection of sodium ions over potassium ions in a sodium channel, size is the primary consideration.
Ion balance
The movement of ions across a lipid bilayer is tightly regulated, and with good reason. Maintaining a proper balance of ions inside
and outside of cells is important for maintaining osmotic balance. It is also important inside and outside of organelles like the
mitochondria and chloroplasts for energy generation. If the ionic balance of a cell is sufficiently disturbed by an uncontrolled
ionophore, a cell may die.
Gramicidin
Gramicidins (Movie 3.1) are antibiotic polypeptides synthesized by the soil bacterium known as Bacillus brevis. These small
pentadecapeptides (15 amino acids) are synthesized by the bacterium to kill other bacteria.
When released by the Bacillus brevis, the gramicidins insert themselves in the membranes of Gram positive bacteria and allow the
movement of sodium ions into the target cells, ultimately killing them. Gramicidins can also cause hemolysis in humans so they
cannot be used internally, but instead are used topically.
Aquaporins
Aquaporins are pore-containing integral membrane proteins that selectively permit passage of water molecules in and out of the
cell, while preventing ions and other solutes from moving (Figures 3.34 & 3.35). Some aquaporins called aquaglyceroporins, also
transport other small uncharged entities, such as glycerol, ammonia, urea, and CO2, across the membrane,. The water pores are
completely impermeable to charged molecules, such as protons, which is important for the preserving the membrane's
electrochemical potential difference.

Figure 3.34 - View of aquaporin from the side - Wikipedia

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 Figure 3.35 - View of aquaporin from the top - David Goodsell
Porins
Porins are proteins containing a β-barrel structure that crosses the cell membrane/wall and acts as a pore/channel through which
specific molecules diffuse. Porins are found in the outer membrane of Gram-negative bacteria and some Gram-positive bacteria,
mitochondria, and chloroplasts.
Porins typically transport only one group of molecules or, in some cases, one specific molecule. Antibiotics, such as β-lactam and
fluoroquinolone pass through porins to reach the cytosol of Gram negative bacteria. Bacteria may develop resistance to these
antibiotics when a mutation occurs to the porin involved that results in exclusion of the antibiotics that would otherwise pass
through.

Transporter proteins
Not all facilitated transport occurs through ion channel proteins. Transporter proteins, as noted earlier (HERE and Figure 3.27)
facilitate movement of materials across a lipid bilayer, but are slower than ion channels. Figure 3.36 illustrates a transporter protein
in action. As can be seen, transporter proteins rely on a specific receptor site for proper recognition of the molecule to be moved.
Binding of the proper molecule causes a conformational change in the shape of the protein (an eversion) which results in a flipping
of the open side of the protein to the other side of the lipid bilayer. In this way, the molecule is moved. Like ion channels,
transporter proteins facilitate movement of materials in either direction, driven only by the concentration difference between one
side and the other.

Figure 3.36 - Facilitated diffusion with a transporter protein - Wikipedia

Active transport
All of the transport mechanisms described so far are driven solely by a concentration gradient - moving from higher concentrations
in the direction of lower concentrations. These movements can occur in either direction and, as noted, result in equal concentrations
on either side of the bilayer, if allowed to go to completion. Many times, however, cells must move materials against a
concentration gradient and when this occurs, another source of energy is required. This process is known as active transport.
A good definition of active transport is that in active transport, at least one molecule is being moved against a concentration
gradient. A common, but not exclusive, energy source is ATP (see Na+/K+ ATPase), but other energy sources are also employed.
For example, the sodium-glucose transporter uses a sodium gradient as a force for actively transporting glucose into a cell. Thus, it
is important to know that not all active transport uses ATP energy.

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 Figure 3.37 - An overview of active transport by the Na+K+ ATPase

Figure 3.38 - Sequential steps in the active transport of ions by theNa+K+ ATPase - Wikipedia

Na+/K+ ATPase
An important integral membrane transport protein is the Na+/K+ ATPase antiport (Figures 3.37 and 3.38), which moves three
sodium ions out of the cell and two potassium ions into the cell with each cycle of action. In each case, the movement of ions is
against the concentration gradient. Since three positive charges are moved out for each two positive charges moved in, the system
is electrogenic.
The protein uses the energy of ATP to create ion gradients that are important both in maintaining cellular osmotic pressure and (in
nerve cells) for creating the sodium and potassium gradients necessary for signal transmission. Failure of the system to function
results in swelling of the cell due to movement of water into the cell through osmotic pressure. The transporter expends about one
fifth of the ATP energy of animal cells. The cycle of action occurs as follows:
1. Pump binds ATP followed by binding of 3 Na+ ions from cytoplasm of cell
2. ATP hydrolysis results in phosphorylation of aspartate residue of pump. ADP is released
3. Phosphorylated pump undergoes conformational change to expose Na+ ions to exterior of cell. Na+ ions are released.
4. Pump binds 2 extracellular K+ ions.
5. Pump dephosphorylates causing it to expose K+ ions to cytoplasm as pump returns to original shape.
6. Pump binds 3 Na+ ions, binds ATP and releases 2 K+ ions to restart process
The Na+/K+ ATPase is classified as a P-type ATPase. This category of pump is notable for having a phosphorylated aspartate
intermediate and is present across the biological kingdoms - bacteria, archaeans, and eukaryotes.
ATPase types
ATPases have roles in either the synthesis or hydrolysis of ATP and come in several different forms.
F-ATPases (F1FO-ATPases) are present in mitochondria, chloroplasts and bacterial plasma membranes and are the prime ATP
synthesizers for these systems. Each uses a proton gradient as its energy source for ATP production. Complex V of the
mitochondrion is an F-type ATPase.
V-ATPases (V1VO-ATPases) are mostly found in vacuoles of eukaryotes . They utilize energy from ATP hydrolysis to transport
solutes and protons into vacuoles and lysosomes, thus lowering their pH values.

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The V-type and F-type ATPases are very similar in structure. The V-type (Figure 3.39) uses ATP to pump protons into vacuoles and
lysosomes, whereas F-types use proton gradients of the mitochondria and chloroplasts to make ATP.
A-ATPases (A1AO-ATPases) are found in archaeans and are similar to F-ATPases in function.
P-ATPases (E1E2-ATPases) are in bacteria, fungi and in eukaryotic plasma membranes and organelles. They transport a
diversity of ions across membranes. Each has a common mechanism of action which include autophosphorylation of a
conserved aspartic acid side chain within it. Examples of P-type ATPases include the Na+/K+ ATPase and the calcium pump.
E-ATPases are enzymes found on the cell surface. They hydrolyze a range of extracellular nucleoside triphosphates, including
ATP.

Figure 3.39 - Schematic structure of V-ATPases- Wikipedia

Nerve transmission
Now that you have seen how the Na+/K+ ATPase functions, it is appropriate to discuss how nerve cells use ion gradients created
with it to generate and transmit nerve signals. Neurons are cells of the nervous system that use chemical and electrical signals to
rapidly transmit information across the body (Figure 3.40). The sensory nerve system links receptors for vision, hearing, touch,
taste, and smell to the brain for perception. Motor neurons run from the spinal cord to muscle cells. These neurons have a cell body
and a very long, thin extension called an axon, that stretches from the cell body in the spinal cord all the way to the muscles they
control. Nerve impulses travel down the axon to stimulate muscle contraction.
Signals travel through neurons, ultimately arriving at junctions with other nerve cells or target cells such as muscle cells. Note that
neurons do not make physical contact with each other or with muscle cells. The tiny space between two neurons or between a
neuron and a muscle cell is called the synaptic cleft. At the synaptic cleft, the neuron releases neurotransmitters that exit the nerve
cell and travel across the junction to a recipient cell where a response is generated. That response may be creating another nerve
signal, if the adjacent cell is a nerve cell or it may be a muscular contraction if the recipient is a muscle cell (Figure 3.41).
In considering information movement via nerve cells, then, we will discuss two steps - 1) creation and propagation of a signal in a
nerve cell and 2) action of neurotransmitters exiting a nerve cell and transiting a synaptic junction.

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 Figure 3.40 - Anatomy of a nerve cell - Image by Pehr Jacobson
Signal source
Creation of a nerve signal begins with a stimulus to the nerve cell. In the case of muscle contraction, the motor cortex of the brain
sends signals to the appropriate motor neurons, stimulating them to generate a nerve impulse. How is such an impulse generated?
Resting potential
In the unstimulated state, all cells, including nerve cells, have a small voltage difference (called the resting potential) across the
plasma membrane, arising from unequal pumping of ions across the membrane. The Na+/K+ ATPase, for example, pumps sodium
ions out of the cell and potassium ions into cells. Since three sodium ions get pumped out for every two potassium ions pumped in,
a charge and chemical gradient is created. It is the charge gradient that gives rise to the resting potential.
Altering the gradients of ions across membranes provide the driving force for nerve signals. This happens as a result of opening and
closing of gated ion channels. Opening of gates to allow ions to pass through the membrane swiftly changes the ionic balance
across the membrane resulting in a new voltage difference called the action potential. It is the action potential that is the impetus of
nerve transmission.
Initiation of signal
The signal generated by a motor neuron begins with opening of sodium channels in the membrane of the nerve cell body causing a
rapid influx of sodium ions into the nerve cell. This step, called depolarization (Figure 3.42), triggers an electrochemical signal -
the action potential. Remember that the Na+/K+ ATPase has created a large sodium gradient, so sodium ions rush into the cell
when sodium channels open. After the initial depolarization, potassium channel gates, responding to the depolarization, open,
allowing potassium ions to rapidly diffuse out of the cell (remember K+ ions are more abundant inside of the cell). This phase is
called the repolarization phase and during it, the sodium gates close.
The rapid exit of potassium ions causes the voltage difference to “overshoot” the resting potential and potassium gates close. This
followed by the so-called refractory period, when the Na+/K+ ATPase begins its work to re-establish the original conditions by
pumping sodium ions out and potassium ions into the nerve cell. Eventually, the system recovers and the resting potential is re-
established. The initiating end of the nerve cell is then ready for another signal.
Propagation of action potential
What we have described here is only the initiation of the nerve signal in one part of the nerve cell. For the signal to be received, the
action potential must travel the entirety of the length of the nerve cell (the axon) and cause a chemical signal to be released into the
synaptic cleft to get to its target. Propagating the nerve signal (action potential) in the original nerve cell is the function of all of the
rest of the gated ion channels (Figure 3.43) positioned on the sides of the nerve cell. The sodium and potassium gates involved in
propagation of the signal all act in response to voltage changes created by the electrochemical gradient moving down the nerve cell
(Figure 3.44). Remember that opening of the initial gates at initiation of the signal created an influx of sodium ions and an efflux of
potassium ions.

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Figure 3.41 - (1) The action potential reaches the axon terminal; (2) voltage-dependent calcium gates open - calcium enters the
axon terminal; (3) neurotransmitter vesicles fuse with the presynaptic membrane and release acetylcholine (ACh); (4) ACh binds to
postsynaptic receptors on the sarcolemma; (5) ion channels open in response and allow sodium ions to flow into the muscle cell;
(6) The flow of sodium ions into the muscle cell generates an action potential which travels to the myofibril and results in muscle
contraction. - Wikipedia

Figure 3.42 - Depolarization and repolarization of a nerve cell - Wikipedia

Figure 3.43 - Voltage gated ion channels - Wikipedia

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 Figure 3.44 - Action potential moving across a synaptic junction of two nerve cells - Image by Pehr Jacobson
Moving signal
This chemical and electrical change that creates the action potential leaves the end of the nerve cell where it started and travels
down the axon towards the other end of the nerve cell. Along the way, it encounters more sodium and potassium gated channels. In
each case, these respond simply to the voltage change of the action potential and open and close, exactly in the same way the gates
opened to start the signal. Thus, a rapid wave of increasing sodium ions and decreasing potassium ions moves along the nerve cell,
propagated (and amplified) by gates opening and closing as the ions and charges move down the nerve cell. Eventually, the ionic
tidal wave reaches the end of the nerve cell (axon terminal) facing the synaptic cleft.
Crossing the synaptic cleft
For the signal to be received by the intended target (postsynaptic cell) from the originating neuron (presynaptic neuron), it must
cross the synaptic cleft and stimulate the neighboring cell (Figure 3.45). Communicating information across a synaptic cleft is the
job of neurotransmitters. These are small molecules synthesized in nerve cells that are packaged in membrane vesicles called
synaptic vesicles in the nerve cell. Neurotransmitters come in all shapes and chemical forms, from small chemicals like
acetylcholine to peptides like neuropeptide Y. The most abundant neurotransmitter is glutamate, which acts at over 90% of the
synapses in the human brain.

Figure 3.45 - A) Pre-synaptic neuron; B) post-synaptic neuron; 1) Mitochondria; 2) synaptic vesicle with neurotransmitters; 3)
autoreceptor; 4) synapse with neurotransmitter released (serotonin); 5) postsynaptic receptors activated by neurotransmitter; 6)
calcium channel; 7) exocytosis of a vesicle; 8) recaptured neurotransmitter. Wikipedia
Movie 3.2 - Movement of an action potential down a nerve cell - Wikipedia

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Into the cleft
As the action potential in the presynaptic neuron approaches the axon terminus, synaptic vesicles begin to fuse with the membrane
and their neurotransmitter contents spill into the synaptic cleft. Once in the cleft, the neurotransmitters diffuse, some of them
reaching receptors on the postsynaptic cell. Binding of the neurotransmitter to the receptors on the membrane of the postsynaptic
cell stimulates a response.
For motor neurons, the postsynaptic cell will be a muscle cell, and the response will be muscle contraction/relaxation. At this point,
the originating nerve cell has done its job and communicated its information to its immediate target. If the postsynaptic cell is a
nerve cell, the process repeats in that cell until it gets to its destination.

Na+/glucose transporter
Absorbing nutrients from the digestive system is necessary for animal life. The sodium/glucose transport protein is an electrogenic
symporter that moves glucose into intestinal cells. It is found in the intestinal mucosa and the proximal tubule of the nephron of the
kidney. The sodium/glucose transport system functions in the latter to promote reabsorption of glucose.
The pump works in conjunction with the Na+/K+ transport system. The gradient of sodium ions built up by the Na+/K+ pump is
used as an energy source to drive movement of glucose into cells (see Figure 3.38). Use of an ion gradient established by a separate
pump is known as secondary active transport. For intestinal mucosa, the pump transports glucose out of the gut and into gut cells.
Later, the glucose is exported out the other side of the gut cells to the interstitial space for use in the body.

Figure 3.46 - The sodium/glucose pump (right) and the Na+/K+ ATPase (left). The sodium gradient generated by the Na+/K+
ATPase is used by the sodium/glucose pump to import glucose into the cell. Wikipedia

Calcium pumps
Calcium ions are necessary for muscular contraction and play important roles as signaling molecules within cells. In addition, when
calcium concentrations rise too high, DNA in chromosomes can precipitate. Calcium concentration in cells is therefore managed
carefully. It is kept very low in the cytoplasm as a result of action of pumps, both in the plasma membrane, which pump calcium
outwards from the cytoplasm and in organelles, such as the endoplasmic reticulum (sarcoplasmic reticulum of muscle cells), which
pump calcium out of the cytoplasm and into these organelles.
Opening of calcium channels, then, increases calcium concentration quickly in the cytoplasm resulting in a quick response, whether
the intention is signaling or contraction of a muscle. After the response is generated, the calcium is pumped back out of the
cytoplasm by the respective calcium pumps.
Some calcium pumps use ATP as an energy source to move calcium and others use ion gradients, such as sodium for the same
purpose.
Na+/Ca++ transporter
One calcium pump of interest uses the sodium gradient as an energy source. It is the sodium/calcium pump. This electrogenic
antiport system uses sodium’s movement into the cell as a driving force to move calcium out of the cell, although its direction can
reverse in some circumstances. The pump is a high capacity system to move a lot of calcium quickly, moving up to 5000 calcium
ions per second and is found in many tissues with many functions.
Digitalis
One important function of the Na+/Ca++ pump occurs in heart cells. Ca++ is important for contraction of heart muscle. Calcium
efflux from the cells is the normal operation of the pump, however, during the upstroke of the cycle, there is a large movement of
sodium ions into the heart cell. When this occurs, the pump reverses and pumps Na+ out and Ca++ in briefly. Since calcium helps

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stimulate contraction of cardiac muscle, this can help make the heart beat stronger and is the focus of the use of digitalis to treat
congestive heart failure.
Digitalis blocks the sodium-potassium ATPase and interferes with the sodium ion gradient. As noted above, when the Na+ gradient
is oriented in the wrong direction, calcium is pumped in. Digitalis is therefore used to treat congestive heart failure because it
increases the concentration of calcium in the heart cells, favoring more forceful beats.

ABC transporters
ABC transporters are another class of transmembrane proteins that use ATP energy to transport things against concentration
gradients (Figures 3.47 & 3.48). This protein superfamily includes hundreds of proteins (48 in humans alone) and spans all extant
phyla from prokaryotes to humans. These proteins function not only in membrane transport, but also in processes that include DNA
repair and the process of translation.

Figure 3.47 - Mechanism of an ABC importer

Figure 3.48 - Another view of an ABC exporter. NBD = Nucleotide Binding Domain
Transport
Substances that ABC transporters move across membranes include metabolic products, lipids, sterols, and drugs. ABC transporters
function in multi-drug resistance of many cells, and provide resistance to antibiotics in bacteria as well as resistance to
chemotherapy in higher cells by exporting drugs used to treat both of these types of cells.
ABC transporters are divided into three main groups - 1) importers (prokaryotes only); 2) exporters (prokaryotes and eukaryotes),
and 3) non-transporters with roles in DNA repair and translation. All ABC transport proteins have four protein domains - two that
are cytoplasmic and two that are membrane bound. They are alternately open to the cytoplasmic or extracellular (or periplasmic)
regions and this is controlled by hydrolysis of ATP.
Disease
ABC transporters have roles in cystic fibrosis and other inherited human diseases. They are very involved in development of
resistance to multiple drugs by a diverse group of cells. ABC transporters provide multi-drug resistance by expelling drug(s) from
cells. ABCB1 protein, for example, pumps tumor suppression drugs out of the cell. Another ABC transporter known as Pgp
transports organic cationic or neutral compounds.
Cystic fibrosis
Cystic fibrosis (CF) is a an autosomal recessive genetic disorder arising from mutations in both copies of the gene for the cystic
fibrosis transmembrane conductance regulator (CFTR) protein. This ABC transporter system, which moves chloride and
thiocyanate ions across epithelial tissue membranes exerts its effect mostly in the lungs but the pancreas, liver, kidneys, and
intestine are all also affected by it.
Function
CFTR has roles in the production of sweat, mucus, and digestive fluids. Manifestations of the disease include breathing difficulty
and overproduction of mucus in the lungs. When CFTR is functional, these fluids are normally thin, but when the gene is non-
functional, they become much thicker and are points of infection.
CFTR contains two ATP-hydrolyzing domains and two cell membrane-crossing domains with 6 α-helices each. It can be activated
by phosphorylation by a cAMP-dependent protein kinase. The carboxyl end of CFTR is linked to the cytoskeleton by a PDZ
domain.

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Lactose permease
Another integral membrane protein performing active transport is lactose permease. It facilitates the movement of the sugar lactose
across the lipid bilayer of the cell membrane (Figures 3.49- 3.51). The transport mechanism is classified as a secondary active
transport since it exploits the inwardly directed H+ electrochemical gradient as an energy source. When lactose is transported into
cells, it is broken down into its substituent monosaccharide sugars - glucose and galactose - for energy creation.
The enzyme catalyzing this reaction is known as lactase and deficiency of it in humans leads to lactose intolerance (see HERE).

Figure 3.49 - Mechanism of action of lactose permease - Note that the proton acts by altering the charge of a carboxyl group -
Image by Aleia Kim

Figure 3.50 - Structure of lactose permease - Wikipedia

Figure 3.51 - Lactose (galactoside) permease is a secondary transporter, using a proton gradient as an energy source to pump
lactose into the cell - Image by Aleia Kim

GLUTs
GLUTs (GLUcose Transport proteins) are uniport, type III integral membrane proteins that participate in the transport of glucose
across membranes into cells. GLUTs are found in all phyla and are abundant in humans, with 12 GLUT genes. GLUT1, in
erythrocytes is well-studied. Through GLUT 1, glucose enters and passes through it via facilitated diffusion at a rate that is 50,000
higher than in its absence. GLUTs of various types are found in different cells of the body. The one in red blood cells is known as
GLUT 1 and has 12 membrane-spanning hydrophobic helices.
Though the structure of GLUT 1 is not known, it is speculated that the 12 helices form a chamber able to form hydrophilic bonds
with glucose to facilitate its passage.

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GLUT 1 levels in erythrocytes go up as glucose levels decrease and decrease when glucose levels go down. GLUT 1 can also
transport ascorbate (vitamin C) in addition to glucose in mammals (such as humans) that do not produce their own vitamin C.
Glut 4
GLUT 4 is regulated by insulin and is found primarily in adipose and striated muscle tissue. Insulin alters intracellular trafficking
pathways in response to increases in blood sugar to favor movement of various GLUT proteins (including GLUT 4) from
intracellular vesicles to the cell membrane, thus stimulating uptake of the glucose. GLUT 4 is also found in the hippocampus
where, if trafficking is disrupted, the result can be depressive behavior and cognitive dysfunction.
For all of the GLUT proteins, a key to keeping the glucose in the cell is phosphorylation of it by the glycolysis enzyme,
hexokinase, in the cytoplasm. Phosphorylated molecules cannot enter GLUTs and don’t have an easy means of exiting the cell.

Figure 3.52 - GLUTs found in cells - Image by Aleia Kim

Distance Ed
To the tune of “Mister Ed”
Metabolic Melodies Website HERE
A course is a source,
of course, of course
Of all of the knowledge that we endorse
A major force for better/worse is the campus Distance Ed
It’s true to outsource a college course
There are a few standards to be enforced
The long and short’s we reinforce the campus Distance Ed
Bridge
A classroom class meets every week the same time every day
But Distance Ed is most unique - its flexible schedule’s okay
E-course is a source, of course, of course
Of online assistance for lab reports
You’re not enrolled in an online course?
Then sign up for this!
“You’ll love Distance Ed”
Recording by David Simmons
Lyrics by Kevin Ahern
Recording by David Simmons Lyrics by Kevin Ahern
313
It's one o'clock and
Ahern's talkin'

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Henderson and
Hasselbalch and
pKa's and
Buffers I should know
This song's for BB three five oh
I hope that maybe
He'll think the way we
Wrote our answers
Wasn't crazy
I really need the
Partial credit - so
This song's for BB three five oh
It's really groovy
That it improves me
Watching lectures
In Quicktime movies
I really need to
Go and download those
Podcasts for BB three five oh
This Song's For BB 3-5-0
To the tune of "This Land is My Land"
Metabolic Melodies Website HERE
I'm feeling manic
I'm in a panic
I'd better study
My old organic
It has reactions
That I need to know
This song's for BB three five oh
I know he said it
That's why I dread it
'cause I skipped Friday's
Extra credit
'twil pro'bly haunt me
That lowly ze-ro
Grade in BB three five oh
It could be steric
Or esoteric
That carbons get so
Anomeric
I'm too hysteric
Better let it go
This song's for BB three five oh
Recording by Tim Karplus
Lyrics by Kevin Ahern
Recording by Tim Karplus Lyrics by Kevin Ahern

This page titled 3.2: Transport in Membranes is shared under a CC BY-NC-SA license and was authored, remixed, and/or curated by Kevin
Ahern, Indira Rajagopal, & Taralyn Tan.

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3.3: Other Considerations in Membranes
Source: BiochemFFA_3_3.pdf. The entire textbook is available for free from the authors at
http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy
There are many functions and factors relating to cell membranes that don’t fit into broad categories. Those items will be the focus
of this section.

Endocytosis
Besides transporter proteins and ion channels, another common way for materials to get into cells is by the process of endocytosis.
Endocytosis is an alternate form of active transport for getting materials into cells. Some of these processes, such as phagocytosis,
are able to import much larger particles than would be possible via a transporter protein. Like transporter proteins, endocytosis uses
energy for the purpose (though it is not as visible as with protein transporters), but unlike protein transporters, the process is not
nearly as specific for individual molecules.
As a result, the process usually involves the importation of many different molecules each time it occurs. The list of compounds
entering cells in this way includes LDLs and their lipid contents, but it also include things like iron (packaged in transferrin),
vitamins, hormones, proteins, and even some viruses sneak in by this means. There are three types of endocytosis we will consider
(Figure 3.53).

Figure 3.53 - Three types of endocytosis

Receptor mediated endocytosis

The process of receptor mediated endocytosis is a relatively specific means of bringing molecules into cells because it requires the
incoming material to be somehow associated with a specific cell surface receptor. In the example of Figure 3.53, the receptor is the
cellular LDL receptor. Clathrin-coated invaginations, as shown in the figure are known as “coated pits.” The mechanism proceeds
with an inward budding of the plasma membrane receptor (coated vesicles). Binding of the ligand (ApoB-100 of the LDL, for
example, in Figure 3.54) to the LDL receptor leads to formation of a membrane invagination. The absorbed LDL particle fuses to
form an early endosome (Figure 3.55) and contents are subsequently sorted and processed for use by the cell.

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 Figure 3.54 - Overview of clathrin-based receptor mediated endocytosis - Image by Aleia Kim

Figure 3.55 - Clathrin-mediated endocytosis of receptors - Wikipedia

The components from the coated vesicle are recycled to the plasma membrane for additional actions. Receptor mediated
endocytosis can also play a role in internalization of cellular receptors that function in the process of signaling. Here, a receptor
bound to a ligand is brought into the cell and may ultimately generate a response in the nucleus.
While receptor mediated endocytosis of receptors can have the effect of communicating a signal inwards to the cell, it can also
reduce the total amount of signaling occurring, since the number of receptors on the cell surface is decreased by the process.

Non-clathrin endocytosis
There are three types of endocytosis occurring in cells that do not involve clathrin. They are 1) caveolae-based endocytosis, 2)
macropinocytosis, and 3) phagocytosis. Caveolae-based endocytosis is based on a receptor molecule known as caveolin. Caveolins
are a class of integral membrane proteins that compartmentalize and concentrate signaling molecules in the process of endocytosis.
They are named for the cave-like caveolae structures of the plasma membrane where they are found.

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Caveolins
Caveolins have affinity for cholesterol and associate with it in the membrane of cells, causing the formation of membrane
invaginations of about 50 nm. The caveolin proteins can oligomerize and this is important for the coating and formation of the
cave-like structures.
There are three caveolin genes found in vertebrate cells, CAV1, CAV2, and CAV3. Down-regulation of caveolin-1 results in less
efficient cellular migration in vitro. Caveolins are implicated in both formation and suppression of tumors. High expression of them
inhibits cancer-related growth factor signaling pathways, but some caveolin-expressing cancer cells are more aggressive and
metastatic, possible due to an enhanced capacity for anchorage-independent growth.

Macropinocytosis
A phenomenon known as “cell drinking,” macropinocytosis literally involves a cell “taking a gulp” of the extracellular fluid. It
does this, as shown in Figure 3.56, by a simple invagination of ruffled surface features of the plasma membrane. A pocket results,
which pinches off internally to create a vesicle containing extracellular fluid and dissolved molecules. Within the cytosol, this
internalized vesicle will fuse with endosomes and lysosomes. The process is non-specific for materials internalized.

Figure 3.56 - Macropinocytosis

Phagocytosis
Phagocytosis is a process whereby relatively large particles (0.75 µm in diameter) are intenalized. Cells of the immune system,
such as neutrophils, macrophages, and others, use phagocytosis to internalize cell debris, apoptotic cells, and microorganisms.

Figure 3.57 - Generalized scheme for phagocytosis of a bacterium - Wikipedia

The process operates through specific receptors on the surface of the cell and phagocytosing cell engulfs its target by growing
around it. The internalized structure is known as a phagosome, which quickly merges with a lysosome to create a phagolysosome
(Figure 3.58), which subjects the engulfed particle to toxic conditions to kill it, if it is a cell, and/or to digest it into smaller pieces.
In some cases, as shown in the figure, soluble debris may be released by the phagocytosing cell.

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 Figure 3.58 - Phagocytosis by a neutrophil (yellow) of an Anthrax bacillus (orange) - Wikipedia

Endosomes
Internalized material from endocytosis that doesn’t involve phagocytosis passes through an internalized structure called an
endosome. Endosomes are membrane bounded structures inside of eukaryotic cells that play a role in endocytosis (Figure 3.59).
They have a sorting function for material internalized into the cell, providing for retrieval of materials not destined for destruction
in the lysosomes. LDLs, for example, are targeted after endocytosis to the endosomes for processing before part of them is
delivered to the lysosome. The endosomes can also receive molecules from the trans-Golgi network. These can be delivered to the
lysosomes, as well, or redirected back to the Golgi. Endosomes come in three forms - 1) early, 2) late, and 3) recycling.

Figure 3.59 - Internalization of the epidermal growth factor receptor (EGFR) into endosomes. Early (E) and late (M) endosomes
and lysosomes (L) are labeled. - Wikipedia

Exocytosis
The process of exocytosis is used by cells to export molecules out of cells that would not otherwise pass easily through the plasma
membrane. In the process, secretory vesicles fuse with the plasma membrane and release their contents extracellularly. Materials,
such as proteins and lipids embedded in the membranes of the vesicles become a part of the plasma membrane when fusion
between it and the vesicles occurs.

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Membrane fusion
Fusion is a membrane process where two distinct lipid bilayers merge their hydrophobic cores, producing one interconnected
structure. Membrane fusion involving vesicles is the mechanism by which the processes of endocytosis and exocytosis occur.
When the fusion proceeds through both leaflets of both bilayers, an aqueous bridge results and the contents of the two structures
mix. Common processes involving membrane fusion (Figure 3.60) include fertilization of an egg by a sperm, separation of
membranes in cell division, transport of waste products, and neurotransmitter release (Figure 3.61). Artificial membranes such as
liposomes can also fuse with cellular membranes. Fusion is also important for transporting lipids from the point of synthesis inside
the cell to the membrane where they are used. Entry of pathogens can also be governed by fusion, as many bilayer-coated viruses
use fusion proteins in entering host cells.

Figure 3.60 - Cell membrane fusions - Image by Pehr Jacobson

Figure 3.61 - Release of neurotransmitters (small circles) from presynaptic neuron A to postsynaptic neuron B. 1 = Mitochondrion /
2 = Synaptic vesicle with neurotransmitter / 3 = Autoreceptor / 4 = Synaptic cleft / 5 = Neurotransmitter receptor / 6 = Calcium
channel / 7 = Fused vesicle releasing neurotransmitter / 8 = Neurotransmitter re-uptake pump - Wikipedia

SNARE proteins
Mediation of fusion of vesicles in exocytosis is carried out by proteins known as SNAREs (Soluble NSF Attachment Protein
REceptor). This large superfamily of proteins spans a wide biological range, from yeast to mammals. Common vesicle fusions
occur when synaptic vesicles dock with neurons (Figure 3.61) and release neurotransmitters. These are well-studied. The SNAREs
involved in this process can be proteolytically cleaved by bacterial neurotoxins that give rise to the conditions of botulism and
tetanus.
SNAREs are found in two locations. v-SNAREs are found in the membranes of transport vesicles during the budding process,
whereas t-SNARES can be found in the membranes of targeted compartments.
The act of vesicle fusion coincides with increases of intracellular calcium. Fusion of synaptic vesicles in neurotransmission results
in activation of voltage-dependent calcium channels in the targeted cell. Influx of calcium helps to stimulate vesicle fusion. In the

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endocrine system, binding of hormones to G protein coupled receptors activate the IP3/DAG system to increase levels of calcium.
In the process of membrane fusion (Figure 3.62), the v-SNAREs of a secretory vesicle (upper left) interact with the t-SNAREs of a
target membrane (bottom). The v- and t-SNAREs “zipper” themselves together to bring the membrane vesicle and the target
membrane closer together.
Zippering also causes flattening and lateral tension of the curved membrane surfaces, favoring hemifusion of the outer layers of
each membrane. Continued tension results in subsequent fusion of the inner membranes as well, yielding opening of the contents of
the vesicle chamber to its target (usually outside the cell).

Figure 3.62 - Proteins involved in vesicle fusion in neurotransmission. A SNARE complex between α-helices of synaptobrevin,
syntaxin and SNAP-25 intertwine and “zip” membranes together. Synaptotagmin is a calcium sensor regulating the process of
zipping - Wikipedia

Shuttles
Another way to transport items across a membrane for which there is no specific transport system available is the use of shuttles.
Shuttles are important when there is no transport mechanism for moving material across a membrane for which no transport system
exists.
A great example is NADH. NADH is an important electron carrier that is produced in the cytoplasm and mitochondria of the cell.
NADH produced in the mitochondrion goes directly to the electron transport system and delivers electrons to Complex I. NADH
produced in the cytoplasm (such as from glycolysis) does not have this option, since the inner membrane of the mitochondrion is
impermeable to the molecule and no transporter exists to move it across. The important part of the NADH is its electron cargo, so
cells have evolved two ways to move the electrons into the mitochondrial matrix apart from NADH.
Both methods involve shuttles. In each case, an acceptor molecule receives electrons from NADH and the reduced form of the
acceptor molecule is transported. It gets transported into the matrix where it is oxidized (electrons are lost) and then donated to the
electron transport system.
Glycerol phosphate shuttle
The first of these methods is the least efficient, but it is rapid. It found commonly in muscles which have needs for rapid energy and
brain tissue. This shuttle is referred to as the glycerol phosphate shuttle and is shown in Figure 3.63. It operates in the
intermembrane space between the inner and outer mitochondrial membranes. The outer mitochondrial membrane is very porous,
allowing many materials to pass freely through it. In the intermembrane space, the cytoplasmic enzyme, glyceraldehyde-3-
phosphate dehydrogenase (cGPD) catalyzes transfer of electrons from NADH to dihdydroxyacetone phosphate (#2 in the figure),
yielding NAD+ and glyceraldehyde-3-phosphate (#1 in the figure). The glyceraldehyde-3-phosphate then binds to a
glyceraldehyde-3-phosphate dehydrogenase (mGPD) embedded in the outer portion of the inner mitochondrial membrane. mGPD
catalyzes the transfer of electrons from glyceraldehyde-3-phosphate to FAD, producing dihycroxyacetone phosphate and FADH2.
FADH2 then transfers its electrons to the electron transport system through CoQ (Q above), forming CoQH2 (QH2 above). As will
be discussed in the section on electron transport, this is not an efficient shuttle system because it does not result in production of as
much ATP as occurs when electrons are transferred to NAD+ instead of FAD.

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 Figure 3.63 - Glycerol phosphate shuttle system in the intermembrane space of a mitochondrion - Image by Pehr Jacobson
Malate-aspartate shuttle
A more efficient system of transferring electrons is the malate-aspartate shuttle and it is shown in Figure 3.64. As is apparent in the
figure, this shuttle involves more steps than the glycerol phosphate shuttle, but the advantage of the malate-aspartate shuttle is that
it is more efficient. NADH outside of the mitochondrion transfers its electrons to the shuttle and then NADH is re-made on the
inside of the shuttle. No energy is expended in the process.

Figure 3.64 - The malate aspartate shuttle - Image by Aleia Kim

When NADH accumulates in the cytoplasm, it moves to the intermembrane space where the enzyme malate dehydrogenase
catalyzes the transfer of electrons to oxaloacetate to yield NAD+ and malate. A transport system for malate moves malate into the
mitochondrial matrix in exchange for α-ketoglutarate.
Inside the mitochondrion, malate is reoxidized to oxaloacetate and electrons are given to NAD+ to recreate NADH. NADH then
donates electrons to Complex I of the electron transport system. That’s really all there is to the shuttle. The remaining steps are
simply to balance the equation of the process. Oxaloacetate accepts an amine group from glutamic acid to yield aspartic acid and α-
ketoglutarate. Aspartate then moves out of the mitochondrion through an antiport transport protein that swaps it for glutamate. A
series of reactions in the intermembrane space balance the equation.
It is easy to get lost in the mess of balancing equations. The most important thing to understand here is that oxaloacetate accepts
electrons on the outside to become malate which is the carrier of electrons across the membrane. Once inside the matrix, malate is
converted back to oxaloacetate and its electrons are given to NAD+, forming NADH. Everything else is simple equation balancing.
Acetyl-CoA shuttle
Another kind of shuttle also involves the mitochondrion and in this case, the item being moved is a molecule, not a pair of
electrons. The molecule of interest here is acetyl-CoA, for which no transport system operates, but which is needed in the
cytoplasm for fatty acid synthesis when the cell has abundant energy.

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Acetyl-CoA is mostly in the mitochondrion and so long as the citric acid cycle is operating efficiently, its concentration is relatively
stable. However, when the citric acid cycle slows, acetyl-CoA and the citrate made from it in the cycle begin to accumulate.
A membrane transport system for citrate exists, so it gets moved out to the cytoplasm. In the cytoplasm, an enzyme known as
citrate lyase cleaves citrate to acetyl-CoA and oxaloacetate. Oxaloacetate can be reduced to malate and moved back into the
mitochondrion.
As for acetyl-CoA, the more of that cells have in the cytoplasm, the more likely they will begin making fatty acids and fat, since
acetyl-CoA is the starting material for fatty acid synthesis, which occurs in the cytoplasm. When does this process occur? As noted
above, it occurs when the citric acid cycle stops and this occurs when levels of NADH and FADH2 increase. These, of course,
increase when one is not burning off as many calories as one is consuming as a byproduct of respiratory control. Lack of exercise
leads to higher levels of reduced electron carriers.

Cell junctions
Cells in multicellular organisms are in close contact with each other and links between them are called junctions. In vertebrate
organisms, there are three main types of cell junctions and one of them (gap junctions) is important for movement of materials
between cells. The three types are
1. Gap junctions
2. Adherens junctions, (Anchoring Junctions, desmosomes and hemidesmosomes)
3. Tight junctions
Cell junctions in multicellular plants are structured differently from those in vertebrates and are called plasmodesmata. They too
function in exchange of materials between individual cells.
Gap junctions
Gap junctions are specialized structures made up of two sets of structures called connexons (one from each cell - see Figure 3.65)
directly link the cytoplasms of the connected cells. Gap junctions are regulated to control the flow of molecules, ions, and electrical
impulses between cells. In plants, similar structures known as plasmodesmata traverse the cell wall (Figure 3.66) and perform
similar functions.

Figure 3.65 - Structure of connexons joining two cells. Bundles of six copies of connexin proteins in the plasma membrane of each
cell comprise the connexon structures

Figure 3.66 - Two means of intercellular communication in plant cells - apoplastic pathway (through cell wall) and symplastic
pathway (through the plasodesma)

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Adherens junctions
Adherens junctions (Figure 3.67) are protein complexes on the cytoplasmic side of the cell membranes of epithelial and endothelial
tissues that link cells to each other or to the extracellular matrix. They correspond to the fascia adherens found in non-
epithelial/non-endothelial cells.

Figure 3.67 - Adherens junction

Adherens junctions contain the following proteins - 1) α-catenin (binds cadherin through β-catenin); 2) β-catenin (attachment for α-
catenin to cadherin; 3) γ-catenin (binds to cadherin); 4) cadherins (group of transmembrane proteins that dimerize with cadherins
on adjacent cells; 5) p120 (also called Δ-catenin - binds to cadherin); 6) plakoglobin (catenin family protein homologous to and
acting like β-catenin); 7) actin; 8) actinin; and 9) vinculin. Adherens junctions may help to maintain the actin contractile ring which
forms in the process of cytokinesis.
Tight junctions
Tight junctions (Figure 3.68) are a network of protein strands that seal cells together and restrict the flow of ions in the spaces
between them. The effect of their structure is to restrict the movement of materials through tissues by requiring them to pass
through cells instead of around them. Tight junctions join together the cytoskeletons of cells and through their structure maintain
their apical and basolateral polarity.

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 Figure 3.68 - Tight junctions
GPI anchors
Membrane proteins attached to glycosylphosphatidylinositol (also known as a GPI anchor) are referred to as being glypiated. The
proteins, which play important roles in many biochemical processes, are attached to the GPI anchor at their carboxyl terminus.
Phospholipases, such as phospholipase C can cut the bond between the protein and the GPI, freeing the protein from the outer cell
membrane. Proteins destined to be glypiated have two signal sequences. They are 1) An N-terminal signal sequence and 2) A C-
terminal signal sequence that is recognized by a GPI transamidase (GPIT). The N-terminal signal sequences is responsible for
directing co-translational transport into the endoplasmic reticulum. The C-terminal sequence is recognized by GPI transamidase,
which links the carboxy terminus of a protein to the GPI anchor.

Liposomes
The spontaneous ability of phosphoglycerolipid and sphingolipid compounds to form lipid bilayers is exploited in the formation of
artificial membranous structures called liposomes (Figure 3.69). Liposomes are useful for delivering their contents into cells via
membrane fusion. In the figure, items targeted for delivery to cells would be encased in the middle circular region of the liposome
and when the liposome fuses with the cell membrane, it will deliver the contents directly into the cytoplasm.

Figure 3.70 - Formation of liposomes from phospholipids in water - Image by Pehr Jacobson

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 Figure 3.69 - A glypiated protein linked to a membrane-embedded inositol-based molecule. The protein portion is above the red
phosphate ion - Image by Pehr Jacobson

Hydropathy index
The interior portion of the lipid bilayer is very hydrophobic, which makes it very restrictive to movement of ions and polar
substances across it. This property also places limitations on the types of amino acids that will interact with it as well. Because of
this, transmembrane protein domains found in integral membrane proteins are biased in the amino acids that interact with either the
lipid bilayer or the aqueous material on either side of it.

Figure 3.71 - Hydropathy index for amino acids. More positive values indicate higher hydrophobicity. Wikipedia
Hydrophobic amino acids are found within the bilayer, whereas hydrophilic amino acids are found predominantly on the surfaces.
An additional clue to identifying membrane crossing regions of a protein is that tryptophan or tyrosine is commonly positioned at
non-polar/polar interface(s) of the lipid bilayer for integral proteins. Such an organization of amino acids can be recognized by
computer analysis of amino acid sequences using what is called a hydropathy index/score (Figure 3.71). Though the names and the
scorings vary, the idea is to assign a number (usually positive) to amino acid side chains with higher hydrophobicity and negative to
those that are ionic. With these scores, a computer program can easily find the average scores of short amino acid segments (say 3
amino acids long) and then plot those values on a graph of hydrophobicity index versus position in polypeptide chain. Doing that
for a transmembrane protein such as glycophorin results in the plot shown in Figure 3.72. It is apparent in the analysis that this is a
transmembrane protein that has seven domains crossing the lipid bilayer, as labeled.

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 Figure 3.72 - Hydropathy index plot for glycophorin. Each lipid bilayer-crossing domain noted with a number - Image by Pehr
Jacobson

Cell walls
Cells walls are found in many cells, including plants, fungi, and bacteria, but are not found in animal cells. They are designed to
provide strength and integrity and at least some protection against bursting from osmotic pressure (Figures 3.73-3.75).

Figure 3.73 - Plant cell wall. Direction of the cytoplasm is down

Figure 3.74 - Cell walls of diatoms - Wikipedia

Gram positive bacteria (Figure 3.75) have the simplest cell wall design. Moving from outside the cell towards the cytoplasm there
is an outer peptidoglycan layer for the cell wall followed by a periplasmic space, a plasma membrane, and then the cytoplasm.
Gram negative bacteria add a second protective layer external to all of this, so for them, starting at the outside and moving inwards,
one encounters an outer lipopolysaccharide layer, a periplasmic space, the peptidoglycan cell wall, a second periplasmic space, a
plasma membrane and then the cytoplasm.

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 Figure 3.75 - Gram positive versus Gram negative bacteria cell coverings - Wikipedia
Herbaceous plants have a rigid outer cell wall (primarily composed of cellulose, hemicellulose, and pectin) and an inner plasma
membrane. Woody plants add a second level of wall with lignin between the cellulosic wall and the plasma membrane of
herbaceous plants.
BB Wonderland
To the tune of “Winter Wonderland”
Metabolic Melodies Website HERE
Milam Hall - It’s 12:30
And Ahern’s gettin’ wordy
He walks to and fro’
While not talkin’ slow
Givin’ it to B-B-4-5-0
I was happy when the term got started
Lecture notes and videos galore
MP3s got added to my iPod
But recitations sometimes were a bore
And exams bit me roughly
When the curve turned out ugly
I don’t think it’s so
My scores are too low
Slidin’ by in B-B-4-5-0
Final-LY there’s an examination
On December 9th at 6:00 pm
I’ll have my card packed with information
So I don’t have to memorize it then
And I’ll feel like a smarty
With my jam-packed note-cardy
Just one more to go
And then ho-ho-ho
I’ll be done with B-B-4-5-0

3.3.13 https://bio.libretexts.org/@go/page/7817
Recording by David Simmons
Lyrics by Kevin Ahern
Recording by David Simmons Lyrics by Kevin Ahern
334
Thank God There's a Video
To the tune of "Thank God I'm a Country Boy"
Metabolic Melodies Website HERE
There's a bundle of things a student oughta know
And Ahern's talk isn't really very slow
Learnin' ain't easy / the lectures kinda blow
Thank God there's a video
Well we’ve gone through the cycles and their enzymes too
Studying the regulation everything is new
I gotta admit that I haven’t got a clue
What am I gonna do?
So I got me a note card and bought me a Stryer
Got the enzymes down and the names he requires
I hope that I can muster up a little more desire
Thank God there's a video
Just got up to speed about the N-A-D
Protons moving through Complex Vee
Electrons dance in the cytochrome C
Gotta hear the MP3
Fatty acid oxidation makes acetyl-CoA
Inside the inner matrix of the mitochondri-ay
It's very complicated, I guess I gotta say
Thank God there's a video
So I got me a note card and bought me a Stryer
Got the enzymes down and the names he requires
I hope that I can muster up a little more desire
Thank God there's a video
Replication's kind of easy in a simple kind of way
Copyin' the bases in the plasmid DNAs
Gs goes with Cs and Ts go with As
Thanks to polymerase
And the DNA's a template for the RNA
Helices unwinding at T-A-T-A
Termination happens, then the enzyme goes away
Don't forget the poly-A
So I got me a note card and bought me a Stryer
Got the enzymes down and the names he requires
I think that I can muster up a little more desire
Thank God there's a video
Recording by David Simmons

3.3.14 https://bio.libretexts.org/@go/page/7817
Lyrics by Kevin Ahern
Recording by David Simmons Lyrics by Kevin Ahern

This page titled 3.3: Other Considerations in Membranes is shared under a CC BY-NC-SA license and was authored, remixed, and/or curated by
Kevin Ahern, Indira Rajagopal, & Taralyn Tan.

3.3.15 https://bio.libretexts.org/@go/page/7817
 CHAPTER OVERVIEW

4: Catalysis
Topic hierarchy
4.1: Basic Principles of Catalysis
4.2: Control of Enzymatic Activity
4.3: Mechanisms of Catalysis
4.4: Blood Clotting

Thumbnail: Enzyme changes shape by induced fit upon substrate binding to form enzyme-substrate complex. Hexokinase has a
large induced fit motion that closes over the substrates adenosine triphosphate and xylose. Binding sites in blue, substrates in black
and Mg2+ cofactor in yellow. (PDB: 2E2N, 2E2Q). Image used with permission (CC BY 4.0l Thomas Shafee)

This page titled 4: Catalysis is shared under a CC BY-NC-SA license and was authored, remixed, and/or curated by Kevin Ahern, Indira
Rajagopal, & Taralyn Tan.

1
4.1: Basic Principles of Catalysis
A printable version of this section is here: BiochemFFA_4_1.pdf. The entire textbook is available for free from the authors
at http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy
If there is a magical component to life, an argument can surely be made for it being catalysis. Thanks to catalysis, reactions that can
take hundreds of years to complete in the uncatalyzed “real world,” occur in seconds in the presence of a catalyst. Chemical
catalysts, such as platinum, can speed reactions, but enzymes (which are simply super-catalysts with a “twist,” as we shall see) put
chemical catalysts to shame (Figure 4.1). To understand enzymatic catalysis, it is necessary first to understand energy. Chemical
reactions follow the universal trend of moving towards lower energy, but they often have a barrier in place that must be overcome.
The secret to catalytic action is reducing the magnitude of that barrier.

Figure 4.1 - Rate enhancement for several enzymes Image by Aleia Kim
Before discussing enzymes, it is appropriate to pause and discuss an important concept relating to chemical/biochemical reactions.
That concept is equilibrium and it is very often misunderstood. The “equi" part of the word relates to equal, as one might expect,
but it does not relate to absolute concentrations. What happens when a biochemical reaction is at equilibrium is that the
concentrations of reactants and products do not change over time. This does not mean that the reactions have stopped. Remember
that reactions are reversible, so there is a forward reaction and a reverse reaction: if you had 8 molecules of A, and 4 of B at the
beginning, and 2 molecules of A were converted to B, while 2 molecules of B were simultaneously converted back to A, the
number of molecules of A and B remain unchanged, i.e., the reaction is at equilibrium. However, you will notice that this does not
mean that there are equal numbers of A and B molecules.

Concentration Matters
So, contrary to the perceptions of many students, the concentrations of products and reactants are not equal at equilibrium, unless
the ΔG°’ for a reaction is zero, because when this is the case,
[Products]
ΔG = ln( ) (4.1.1)
[Reactants]

since the ΔG°’ is zero. Because ΔG itself is zero at equilibrium, then

[P roducts] = [Reactants]. (4.1.2)

This is the only circumstance where

[P roducts] = [Reactants] (4.1.3)

at equilibrium. Reiterating, at equilibrium, the concentrations of reactant and product do not change over time. That is, for a
reaction A ⇌ B[A] at time zero when equilibrium is reached, [A] , will be the same 5 minutes later (assuming A and B are
T0

chemically stable). Thus,

[A]T = [A]T +5 (4.1.4)
0

Similarly,
[B]T = [B]T +5 (4.1.5)
0

For that matter, at any amount of time X after equilibrium has been reached,
[A]T 0 = [A]T + 5 = [A]T X (4.1.6)

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and
[B]T 0 = [B]T + 5 = [B]T X (4.1.7)

However, unless ΔG°’ = 0, it is wrong to say [A]T0 = [B]T0 As we study biochemical reactions and reaction rates, it is important
to remember that 1) reactions do not generally start at equilibrium; 2) all reactions move in the direction of equilibrium; and 3)
reactions in cells behave just like those in test tubes - they do not begin at equilibrium, but they move towards it. Dynamic
reactions The reactions occurring in cells, though, are very dynamic and complex. In a test tube, they can be studied one at a time.
In cells, the product of one reaction is often the substrate for another one. Reactions in cells are interconnected in this way, giving
rise to what are called metabolic pathways. There are, in fact, thousands of different interconnected reactions going on
continuously in cells.
Attempts to study a single reaction in the chaos of a cell is daunting to say the least. For this reason, biochemists isolate enzymes
from cells and study reactions individually. It is with this in mind that we begin our consideration of the phenomenon of catalysis
by describing, first, the way in which enzymes work.

Activation energy
Figure 4.2 schematically depicts the energy changes that occur during the progression of a simple reaction. In order for the reaction
to proceed, an activation energy must be overcome in order for the reaction to occur.

Figure 4.2 - Energy changes during the course of an uncatalyzed reaction. Image by Aleia Kim
In Figure 4.3, the activation energy for a catalyzed reaction is overlaid. As you can see, the reactants start at the same energy level
for both catalyzed and uncatalyzed reactions and that the products end at the same energy for both as well. The catalyzed reaction,
however, has a lower energy of activation (dotted line) than the uncatalyzed reaction. This is the secret to catalysis - overall ΔG for
a reaction does NOT change with catalysis, but the activation energy is lowered.

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Figure 4.3 - Energy changes during the course of an uncatalyzed reaction (solid green line) and a catalyzed reaction (dotted green
line). Image by Aleia Kim

Reversibility
The extent to which reactions will proceed forward is a function of the size of the energy difference between the product and
reactant states. The lower the energy of the products compared to the reactants, the larger the percentage of molecules that will be
present as products at equilibrium. It is worth noting that since an enzyme lowers the activation energy for a reaction that it can
speed the reversal of a reaction just as it speeds a reaction in the forward direction. At equilibrium, of course, no change in
concentration of reactants and products occurs. Thus, enzymes speed the time required to reach equilibrium, but do not affect the
balance of products and reactants at equilibrium.

Exceptions
The reversibility of enzymatic reactions is an important consideration for equilibrium, the measurement of enzyme kinetics, for
Gibbs free energy, for metabolic pathways, and for physiology. There are some minor exceptions to the reversibility of reactions,
though. They are related to the disappearance of a substrate or product of a reaction. Consider the first reaction below which is
catalyzed by the enzyme carbonic anhydrase:
− +
C O2 + H2 O ⇌ H2 C O3 ⇌ H C O +H (4.1.8)
3

In the forward direction, carbonic acid is produced from water and carbon dioxide. It can either remain intact in the solution or
ionize to produce bicarbonate ion and a proton. In the reverse direction, water and carbon dioxide are produced. Carbon dioxide, of
course, is a gas and can leave the solution and escape.
When reaction molecules are removed, as they would be if carbon dioxide escaped, the reaction is pulled in the direction of the
molecule being lost and reversal cannot occur unless the missing molecule is replaced. In the second reaction occurring on the
right, carbonic acid (H2CO3) is “removed” by ionization. This too would limit the reaction going back to carbon dioxide in water.
This last type of “removal” is what occurs in metabolic pathways. In this case, the product of one reaction (carbonic acid) is the
substrate for the next (formation of bicarbonate and a proton).
In the metabolic pathway of glycolysis, ten reactions are connected in this manner and reversing the process is much more
complicated than if just one reaction was being considered.

General mechanisms of action

As noted above, enzymatically catalyzed reactions are orders of magnitude faster than uncatalyzed and chemical-catalyzed
reactions. The secret of their success lies in a fundamental difference in their mechanisms of action.
Every chemistry student has been taught that a catalyst speeds a reaction without being consumed by it. In other words, the catalyst
ends up after a reaction just the way it started so it can catalyze other reactions, as well. Enzymes share this property, but in the

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middle, during the catalytic action, an enzyme is transiently changed. In fact, it is the ability of an enzyme to change that leads to
its incredible efficiency as a catalyst.

Changes
These changes may be subtle electronic ones, more significant covalent modifications, or structural changes arising from the
flexibility inherent in enzymes, but not present in chemical catalysts. Flexibility allows movement and movement facilitates
alteration of electronic environments necessary for catalysis. Enzymes are, thus, much more efficient than rigid chemical catalysts
as a result of their abilities to facilitate the changes necessary to optimize the catalytic process.

Substrate binding
Another important difference between the mechanism of action of an enzyme and a chemical catalyst is that an enzyme has binding
sites that not only ‘grab’ the substrate (molecule involved in the reaction being catalyzed), but also place it in a position to be
electronically induced to react, either within itself or with another substrate.
The enzyme itself may play a role in the electronic induction or the induction may occur as a result of substrates being placed in
very close proximity to each other. Chemical catalysts have no such ability to bind substrates and are dependent upon them
colliding in the right orientation at or near their surfaces.

Active site
Reactions in an enzyme are catalyzed at a specific location within it known as the ‘active site’. Substrates bind at the active site and
are oriented to provide access for the relevant portion of the molecule to the electronic environment of the enzyme where catalysis
occurs.

Enzyme flexibility
As mentioned earlier, a difference between an enzyme and a chemical catalyst is that an enzyme is flexible. Its slight changes in
shape (often arising from the binding of the substrate itself) help to optimally position substrates for reaction after they bind.

Figure 4.4 - Substrate binding by methylenetetrahydrofolate reductase

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 Figure 4.5 - Lysozyme with substrate binding site (blue), active site (red) and bound substrate (black). Wikipedia

Induced fit
These changes in shape are explained, in part, by Koshland’s Induced Fit Model of Catalysis (Figure 4.6), which illustrates that not
only do enzymes change substrates, but that substrates also transiently change enzyme structure. At the end of the catalysis, the
enzyme is returned to its original state. Koshland’s model is in contrast to the Fischer Lock and Key model, which says simply that
an enzyme has a fixed shape that is perfectly matched for binding its substrate(s). Enzyme flexibility also is important for control of
enzyme activity. Enzymes alternate between the T (tight) state, which is a lower activity state and the R (relaxed) state, which has
greater activity.

Figure 4.6 - Fischer’s lock and key model (left) Vs. Koshland’s induced fit model (right). Image by Aleia Kim

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Induced Fit
The Koshland Induced Fit model of catalysis postulates that enzymes are flexible and change shape on binding substrate. Changes
in shape help to 1) aid binding of additional substrates in reactions involving more than one substrate and/or 2) facilitate formation
of an electronic environment in the enzyme that favors catalysis. This model is in contrast to the Fischer Lock and Key Model of
catalysis which considers enzymes as having pre-formed substrate binding sites.

Ordered binding
The Koshland model is consistent with multi-substrate binding enzymes that exhibit ordered binding of substrates. For these
systems, binding of the first substrate induces structural changes in the enzyme necessary for binding the second substrate.
There is considerable experimental evidence supporting the Koshland model. Hexokinase, for example, is one of many enzymes
known to undergo significant structural alteration after binding of substrate. In this case, the two substrates are brought into very
close proximity by the induced fit and catalysis is made possible as a result.

Reaction types
Enzymes that catalyze reactions involving more than one substrate, such as

A+B ⇌ C +D (4.1.9)

can act in two different ways. Enzymatic reactions can be of several types, as shown in Figure 4.7. In one mechanism, called
sequential reactions, at some point in the reaction, both substrates will be bound to the enzyme. There are, in turn, two different
ways in which this can occur - random and ordered.
Figure 4.7 - Categories of enzymatic reactions
Types of Reactions

Single Substrate - Single Product A⇄B

Single Substrate - Multiple Products A⇄B+C

Multiple Substrates - Single Products A+B⇄C

Multiple Substrates - Multiple Products A+B⇄C+D

Consider lactate dehydrogenase, which catalyzes the reaction below:

+
N ADH + P yruvate → Lactate + N AD (4.1.10)

This enzyme requires that NADH must bind prior to the binding of pyruvate. As noted earlier, this is consistent with an induced fit
model of catalysis. In this case, binding of the NADH changes the enzyme shape/environment so that pyruvate can bind and
without binding of NADH, the substrate cannot access the pyruvate binding site. This type of multiple substrate reaction is called
sequential ordered binding, because the binding of substrates must occur in the right order for the reaction to proceed.

Random binding
A second mechanism of binding/catalysis is exhibited by creatine kinase which catalyzes the following reaction:
C reatine + AT P → Creatine phosphate + ADP (4.1.11)

For this enzyme, substrates can bind to it in any order. Creatine kinase displays sequential random binding. It is worth mentioning
that random binding is not inconsistent with Koshland’s induced fit model. Rather, random binding simply means that the enzyme’s
induced fit doesn’t affect substrate binding sites and involves other parts of the enzyme. In summary, sequential binding can occur
as ordered binding or as random binding.

Double displacement reactions

Not all enzymes that catalyze multi-substrate reactions, though, bind A and B by the sequential mechanisms above. This other
category of enzyme includes those that exhibit what are called “ping-pong” (or double displacement) mechanisms. In these
enzymes, the enzyme functions as both a catalyst and a carrier of a group between individually bound substrates. Examples of this

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type of enzyme include the class of enzymes known as transaminases. A general form of the reactions catalyzed by these enzymes
is shown in Figure 4.8.

Figure 4.8 - Double displacement reaction mechanism of a transaminase

In reversible transaminase reactions, an oxygen and an amine are swapping between the molecules. It can be represented as follows
(where N is the amine and O is the oxygen).
A=O + C=N ⇄ B-N + D=O
This reaction occurs not as one transfer reaction swapping the N and the O, but rather as a set of two half-reactions. In this case, the
enzyme is a both donor and a carrier of the group being swapped. The first half-reaction goes as follows
A=O + Enz-N ⇄ B-N + Enz=O
Next a second half-reaction goes as
C-N + Enz=O ⇄ D=O + Enz=N
The sum of these half-reactions then is
A=O + C=N ⇄ B-N + D=O
Note that at no time did the enzyme bind both A and C simultaneously. It is also important to recognize that the enzyme existed in
two states - Enz=O and Enz-N. The shuffling of the enzyme between these two states is what gives rise to the ping-pong name of
this mechanism - it literally goes back and forth like a ping-pong ball in a table tennis match.

Figure 4.9 - Binding of substrates (A+B) to free enzyme

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 Figure 4.10 ES complex formation

Figure 4.11 - ES* complex - The reaction occurs.

Figure 4.12 - EP complex - the reaction is complete.

Figure 4.13 - E + P - the products are released

Figure 4.14 - Summary

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Enzyme kinetics
To understand how an enzyme enhances the rate of a reaction, we must understand enzyme kinetics. We present a model here
proposed by Leonor Michaelis and Maud Menten. In order to understand the model, it is necessary to understand a few parameters.
First, we describe a reaction in simple terms proceeding as follows
E + S ⇄ ES -> E + P
where E is enzyme, S is substrate, and P is product. In this scheme, ES is the Enzyme-Substrate complex, which is simply the
enzyme bound to its substrate.
We could define the ES state a bit further with
E + S ⇄ ES -> ES* -> EP -> E + P
where ES* is the activated state and EP is the enzyme-product complex before release of the product.
The first consideration we have is velocity. The velocity of a reaction is the rate of creation of product over time, measured as the
concentration of product per time. The time is a critical consideration when measuring velocity. In a closed system (in which an
enzyme operates), all reactions will advance towards equilibrium. Enzymatically catalyzed reactions are no different in the end
result from non-enzymatic reactions, except that they get to equilibrium faster.

Equilibrium
At equilibrium, the ratio of product to reactant does not change. That is a property of equilibrium. Since the system is closed, the
concentration of product over time will not change. The velocity will thus be zero under these conditions and we will have learned
nothing about the reaction if we wait too long to study it.

Velocity

Figure 4.22 - Kcat/Km values for perfect enzymes. Image by Aleia Kim

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 Figure 4.23 - Triose phosphate isomerase - A perfect enzyme. Wikipedia
Consequently, in Michaelis-Menten kinetics, velocity is measured as initial velocity (V0). This is accomplished by measuring the
rate of formation of product early in the reaction before equilibrium is established and under these conditions, there is very little if
any of the reverse reaction occurring.
The other two assumptions are related. First, we use conditions where there is much more substrate than enzyme. This makes sense.
If the substrate is not in great excess, then the enzyme’s conversion of substrate to product will occur much faster than the enzyme
can bind substrate.

Waiting for substrate

Thus, the enzyme would “wait” for substrate to bind if there were not sufficient amounts of it to bind to the enzyme in a timely
fashion (when substrate concentration is low). This would not give an accurate measure of velocity, since the enzyme would be
inactive a good deal of the time. Because of this, we assume saturation of the enzyme with substrate will give a maximal velocity
of the reaction.

Steady state

Figure 4.15 - Concentration of product (P), substrate (S), enzyme (E), and enzyme-substrate complex (ES) versus time for an
enzymatic reaction

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 Figure 4.16 - Change in concentration of reaction materials over time

Figure 4.17 - Steady state versus non-steady state conditions

Last, the high concentration of substrate combined with measuring initial conditions results in studying reactions that are under so-
called steady state conditions (Figure 4.17). When steady state occurs, the concentration of the ES complex over time is not
significantly changing during the period of analysis.
Reiterating, the three assumptions for Michaelis-Menten kinetics are
1. Measurement of initial velocity of a reaction
2. Substrate in great excess compared to enzyme
3. Reaction conditions occurring under steady state

Experimental considerations
Now we turn our attention to how studies of the kinetic properties of an enzyme are conducted. To perform an analysis, one would
do the following experiment - 20 different tubes would be set up with enzyme buffer (to keep the enzyme stable), the same amount
of enzyme, and then a different amount of substrate in each tube, ranging from tiny amounts in the first tubes to very large amounts
in the last tubes. The reaction would be allowed to proceed for a fixed, short amount of time and then the reaction would be stopped
and the amount of product contained in each tube would be determined.
The initial velocity (V0) of the reaction then would be the concentration of product found in each tube divided by the time that the
reaction was allowed to run. Data from the experiment would be plotted on a graph using initial velocity (V0) on the Y-axis and the

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concentration of substrate on the X-axis, each tube, of course having a unique reaction velocity corresponding to a unique substrate
concentration.
For an enzyme following Michaelis-Menten kinetics, a curve like that shown in Figure 4.18 or 4.19 would result. At low
concentration of substrate, it is limiting and the enzyme converts it into product as soon as it can bind it. Consequently, at low
concentrations of substrate, the rate of increase of [P] is almost linear with [S] (Figure 4.19).

Figure 4.18 - Kinetics of an enzyme obeying Michaelis-Menten kinetics. Image by Aleia Kim

Figure 4.19 - Linear relationship between [P] and [S] at low [S]

Non-linear increase
As the substrate concentration increases, however, the velocity of the reaction in tubes with higher substrate concentration ceases to
increase linearly and instead begins to flatten out, indicating that as the substrate concentration gets higher and higher, the enzyme
has a harder time keeping up to convert the substrate to product.

Saturation
Not surprisingly, when the enzyme becomes completely saturated with substrate, it will not have to wait for substrate to diffuse to it
and will therefore be operating at maximum velocity.
For an enzyme following Michaelis-Menten kinetics will have its velocity (v) at any given substrate concentration given by the
following equation:

Vmax
Two terms in the equation above require explanation. The first is Vmax. It refers to the maximum velocity of an enzymatic reaction.
Maximum velocity for a reaction occurs when an enzyme is saturated with substrate. Saturation is important because it means (per
the assumption above) that none of the enzyme molecules are “waiting” for substrate after a product is released. Saturation ensures
that another substrate is always instantly available. The unit of Vmax is concentration of product per time = [P]/time.
On a plot of initial velocity versus substrate concentration (V0 vs. [S]), Vmax is the value on the Y axis that the curve
asymptotically approaches (dotted line in Figure 4.20). It should be noted that the value of Vmax depends on the amount of enzyme

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used in a reaction. If you double the amount of enzyme used, you will double the Vmax. If one wanted to compare the velocities of
two different enzymes, it would be necessary to use the same amounts of enzyme in the reaction each one catalyzes.

Figure 4.20 - Vmax, Vmax/2, and Km

Km
The second term is Km (also known as Ks). Referred to as the Michaelis constant, Km is the substrate concentration that causes the
enzyme to work at half of maximum velocity (Vmax/2). What it measures, in simple terms, is the affinity an enzyme has for its
substrate. The value of Km is inversely related to the affinity of the enzyme for its substrate. Enzymes with a high Km value will
have a lower affinity for their substrate (will take more substrate to get to Vmax/2) whereas those with a low Km will have high
affinity and take less substrate to get to Vmax/2. The unit of Km is concentration.
Affinities of enzymes for substrates vary considerably, so knowing Km helps us to understand how well an enzyme is suited to the
substrate being used. Measurement of Km depends on the measurement of Vmax.

Common mistake
A common mistake students make in describing Vmax is saying that Km = Vmax /2. This is, of course, not true. Km is a substrate
concentration and is the amount of substrate it takes for an enzyme to reach Vmax /2. On the other hand Vmax /2 is a velocity and
a velocity certainly cannot equal a concentration.

Kcat
It is desirable to have a measure of velocity that is independent of enzyme concentration. Remember that Vmax depended on the
amount of enzyme used. For this, we use the Kcat, also known as the turnover number. Kcat is a number that requires one to first
determine Vmax for an enzyme and then divide the Vmax by the concentration of enzyme used to determine Vmax. Thus,
Kcat = Vmax /[Enzyme]
Since Vmax has units of concentration per time and [Enzyme] has units of concentration, the units on Kcat are time-1. While that
might seem unintuitive, it means that the value of Kcat is the number of molecules of product made by each molecule of enzyme in
the time given. So, a Kcat value of 1000/sec means each enzyme molecule in the reaction at Vmax is producing 1000 molecules of
product per second. Note that since Kcat is a calculated value, it cannot be read from a V vs [S] graph as Vmax and Km can.

Figure 4.21 - Kcat (turnover number) values for several enzymes. Image by Aleia Kim

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Amazing Kcat values
A Kcat value of 1000 molecules of product per enzyme per second might seem like a high value, but there are enzymes known
(carbonic anhydrase, for example) that have a Kcat value of over 600,000/second (Figure 4.21). This astonishing value illustrates
clearly why enzymes seem almost magical in their action. In contrast to V , which varies with the amount of enzyme used, Kcat
max

is a constant for an enzyme under given conditions.

As seen earlier, enzymes that follow Michaelis-Menten kinetics produce hyperbolic plots of Velocity (V0) versus Substrate
Concentration [S] (Figure 4.18). Not all enzymes, though, follow Michaelis-Menten kinetics. Many enzymes have multiple protein
subunits and these sometimes interact differently upon binding of a substrate or an external molecule. See ATCase (HERE) for an
example.

Perfect enzymes
Now, if we think about what an ideal enzyme might be, it would be one that has a very high velocity and a very high affinity for its
substrate. That is, it wouldn’t take much substrate to get to Vmax/2 and the Kcat would be very high. Such enzymes would have
values of Kcat / Km that are maximum. Interestingly, there are several enzymes that have this property and their maximal Kcat /
Km values are all approximately the same. Such enzymes are referred to as being “perfect” because they have reached the
maximum possible value.

Figure 4.22 - Kcat/Km values for perfect enzymes. Image by Aleia Kim

Figure 4.23 - Triose phosphate isomerase - A perfect enzyme. Wikipedia

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 Figure 4.24 - Triose phosphate isomerase-catalyzed reaction

Diffusion limitation
Why should there be a maximum possible value of Kcat / Km? The answer is that movement of substrate to the enzyme becomes
the limiting factor for perfect enzymes. Movement of substrate by diffusion in water has a fixed rate at any temperature and that
limitation ultimately determines the maximum speed an enzyme can catalyze at. In a macroscopic world analogy, factories can’t
make products faster than suppliers can deliver materials. It is safe to say for a perfect enzyme that the only speed limit it has is the
rate of substrate diffusion in water.
Given the “magic” of enzymes alluded to earlier, it might seem that all enzymes should have evolved to be “perfect.” There are
very good reasons why most of them have not.

Speed
Speed is a dangerous thing. The faster a reaction proceeds in catalysis by an enzyme, the harder it is to control. As we all know
from learning to drive, speeding causes accidents. Just as drivers need to have speed limits for operating automobiles, so too must
cells exert some control on the ‘throttle’ of their enzymes. In view of this, one might wonder then why any cells have evolved any
enzymes to perfection. There is no single answer to the question, but a common one is illustrated by triose phosphate isomerase,
which catalyzes a reaction in glycolysis shown in Figure 4.24.
The enzyme appears to have evolved this ability because at lower velocities, there is breakdown of an unstable enediol intermediate
that then readily forms methyl glyoxal, a cytotoxic compound (Figure 4.25). Speeding up the reaction provides less opportunity for
the unstable intermediate to accumulate and fewer undesirable byproducts to be made.

Figure 4.25 - A high speed reaction avoids production of methyl glyoxal. Image by Aleia Kim

Dissociation constant
In studying proteins and ligands, it is important to understand the “tightness” with which a protein (P) “holds onto” a ligand (L).
This is measured with the dissociation constant (K ). The formation of a ligand-protein complex LP occurs as
d

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 L + P ⇌ LP (4.1.12)

The dissociation of the complex, therefore, is the reverse of this reaction, or

LP ⇌ L + P (4.1.13)

so the corresponding dissociation constant is defined as

[L][P ]
Kd = (4.1.14)
[LP ]

where [L], [P ], and [LP ] are the molar concentrations of the protein, ligand and the complex when they are joined together.
Smaller values of K indicate tight binding, whereas larger values indicate loose binding. The dissociation constant is the inverse
d

of the association constant.

1
Ka = (4.1.15)
Kd

Where multiple molecules bond together, such as

Jx Ky ⇌ xJ + yK (4.1.16)

The complex J x Ky is breaking down into x subunits of J and y subunits of K . The dissociation constant is then defined as
x y
[J ] [K ]
Kd = (4.1.17)
[ Jx Ky ]

where [J], [K], and [J x Ky ] are the concentrations of J, K, and the complex J x Ky , respectively.

Lineweaver-Burk plots
The study of enzyme kinetics is typically the most math intensive component of biochemistry and one of the most daunting aspects
of the subject for many students. Although attempts are made to simplify the mathematical considerations, sometimes they only
serve to confuse or frustrate students. Such is the case with modified enzyme plots, such as Lineweaver-Burk (Figure 4.26).
Indeed, when presented by professors as simply another thing to memorize, who can blame students? In reality, both of these plots
are aimed at simplifying the determination of parameters, such as K and V m. In making either of these modified plots, it is
max

important to recognize that the same data is used as in making a V0 vs. [S] plot. The data are simply manipulated to make the
plotting easier.

Figure 4.26 - A Lineweaver-Burk plot of 1/V vs 1/[S]. Image by Aleia Kim

0

Double reciprocal
For a LineWeaver-Burk plot, the manipulation is using the reciprocal of the values of both the velocity and the substrate
concentration. The inverted values are then plotted on a graph as 1/V0 vs. 1/[S]. Because of these inversions, Lineweaver-Burk
plots are commonly referred to as ‘double-reciprocal’ plots. As can be seen in Figure 4.26, the value of Km on a Lineweaver Burk

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plot is easily determined as the negative reciprocal of the x-intercept , whereas the Vmax is the inverse of the y-intercept. Other
related manipulation of kinetic data include Eadie-Hofstee diagrams, which plots V0 vs V0/[S] and gives Vmax as the Y-axis
intercept with the slope of the line being -Km.

Molecularity of reactions
The term molecularity refers to the number of molecules that must come together in order for a reaction to take place. Reactions of
the sort of A -> B (where ‘A’ is the reactant and ‘B’ is the product) are unimolecular, since A is directly changed into B. The rate of
the reaction is related only to the concentration of reactant A. For a bimolecular reaction where A + B ⇄ C the reaction depends on
the concentration of both A and B and its rate will be related to the product of the concentration of A and of B.

Coenzymes
Organic molecules that assist enzymes and facilitate catalysis are co-factors called coenzymes. The term co-factor is a broad
category usually subdivided into inorganic ions and coenzymes. If the coenzyme is very tightly or covalently bound to the enzyme,
it is referred to as a prosthetic group. Enzymes without their co-factors are inactive and referred to as apoenzymes. Enzymes
containing all of their co-factors are called holoenzymes.

Figure 4.27 - Enzyme cofactors. Image by Aleia Kim

Figure 4.28 - Coenzyme A (CoA)

Pre-steady state kinetic studies

In the study of kinetic rates of enzymatic reactions, time zero is a very critical point. It establishes when the mixing of substrate
with enzyme and measurement of formation of product begins. At time zero, there is no product. As shown in Figure 4.29, the
appearance of product (on a short time scale) goes through an early burst phase with a steep slope for [product]/time and then
changes.

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 Figure 4.29 - Burst phase of product formation
This change occurs during a critical period in an enzymatic reaction and gives information about the rate of reaction cycles. The
duration of the burst phase tells how long a single reaction turnover occurs, whereas the slow of the line post-burst phase tells the
amount of “functional” enzyme performing the reaction.
After the burst phase, the slope of the line of the amount of product versus time decreases. This is due to the reaction entering
conditions of steady state, used to study Michaelis-Menten kinetics. In steady state conditions, the amount of the enzyme-substrate
complex (ES) is relatively constant over time. In simple terms, this occurs when the rate of formation of the ES complex equals the
rate of conversion of the substrate to product by the enzyme with release of the product.

Earlier events
Events occurring prior to the conditions of steady state are referred to as pre-steady state. Depending on the enzyme, in as short as a
few milliseconds, steady state conditions can be present meaning that if one hopes to study formation of reaction intermediates in
pre-steady state, tools for this analysis must work very rapidly. One instrument commonly used for studying pre-steady state
kinetics is called a stopped flow instrument.
It loads an enzyme solution and a substrate into separate syringes whose output is pointed into a mixing chamber. The solutions are
rapidly mixed and measurements of product concentration begin. With a stopped flow instrument, dead times (time between mixing
and detection) can be achieved of as small as 0.3 msec.

Figure 4.30 - Stopped flow instrument for studying pre-steady state kinetics. Wikipedia

Ribozymes
Proteins do not have a monopoly on acting as biological catalysts. Some RNA molecules are also capable of speeding reactions.
The most famous of these molecules was discovered by Tom Cech in the early 1980s Studying excision of an intron in
Tetrahymena, Cech was puzzled at his inability to find any proteins catalyzing the process. Ultimately, the catalysis was recognized
as coming from the intron itself. It was a self-splicing RNA and since then, many other examples of catalytic RNAs have been
found. Catalytic RNA molecules are known as ribozymes.

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 359
Figure 4.31 - Cleavage of an RNA by a ribozyme. Wikipedia

Figure 4.32 - Hammerhead ribozyme. Wikipedia

Not unusual
Ribozymes, however, are not rarities of nature. The protein-making ribosomes of cells are essentially giant ribozymes. The 23S
rRNA of the prokaryotic ribosome and the 28S rRNA of the eukaryotic ribosome catalyze the formation of peptide bonds.
Ribozymes are also important in our understanding of the evolution of life on Earth. They have been shown to be capable via
selection to evolve self-replication. Indeed, ribozymes actually answer a chicken/egg dilemma - which came first, enzymes that do
the work of the cell or nucleic acids that carry the information required to produce the enzymes. As both carriers of genetic
information and catalysts, ribozymes are likely both the chicken and the egg in the origin of life.

This page titled 4.1: Basic Principles of Catalysis is shared under a CC BY-NC-SA license and was authored, remixed, and/or curated by Kevin
Ahern, Indira Rajagopal, & Taralyn Tan.

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4.2: Control of Enzymatic Activity
A printable version of this section is here: BiochemFFA_4_2.pdf. The entire textbook is available for free from the authors
at http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy

Regulation of enzyme activity

Apart from their ability to greatly speed the rates of chemical reactions in cells, enzymes have another property that makes them
valuable. This property is that their activity can be regulated, allowing them to be activated and inactivated, as necessary. This is
tremendously important in maintaining homeostasis, permitting cells to respond in controlled ways to changes in both internal and
external conditions.
Inhibition of specific enzymes by drugs can also be medically useful. Understanding the mechanisms that control enzyme activity
is, therefore, of considerable importance.

Inhibition
We will first discuss four types of enzyme inhibition – competitive, non-competitive, uncompetitive, and suicide inhibition. Of
these, the first three types are reversible. The last one, suicide inhibition, is not.

Competitive inhibition
Probably the easiest type of enzyme inhibition to understand is competitive inhibition and it is the one most commonly exploited
pharmaceutically. Molecules that are competitive inhibitors of enzymes resemble one of the normal substrates of an enzyme. An
example is methotrexate, which resembles the folate substrate of the enzyme dihydrofolate reductase (DHFR). This enzyme
normally catalyzes the reduction of folate, an important reaction in the metabolism of nucleotides.

Figure 4.33 - Competitive inhibitors resemble the normal substrate and compete for binding at the active site. Image by Aleia Kim
Inhibitor binding
When the drug methotrexate is present, some of the DHFR enzyme binds to it, instead of to folate, and during the time
methotrexate is bound, the enzyme is inactive and unable to bind folate. Thus, the enzyme is inhibited. Notably, the binding site on
DHFR for methotrexate is the active site, the same place that folate would normally bind. As a result, methotrexate ‘competes’ with
folate for binding to the enzyme. The more methotrexate there is, the more effectively it competes with folate for the enzyme’s
active site. Conversely, the more folate there is, the less of an effect methotrexate has on the enzyme because folate outcompetes it.

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 Figure 4.34 - Methotrexate and dihydrofolate. Image by Ben Carson
No effect on Vmax
How do we study competitive inhibition? It is typically done as follows. First, one performs a set of V0 vs. [S] reactions without
inhibitor (20 or so tubes, with buffer and constant amounts of enzyme, varying amounts of substrate, equal reaction times). V0 vs.
[S] is plotted (Figure 4.35 red line), as well as 1/V0 vs. 1/[S] (Figure 4.36 green line). Next, a second set of reactions is performed
in the same manner as before, except that a fixed amount of the methotrexate inhibitor is added to each tube. At low concentrations
of substrate, the methotrexate competes for the enzyme effectively, but at high concentrations of substrate, the inhibitor will have a
much reduced effect, since the substrate outcompetes it, due to its higher concentration (remember that the inhibitor is at fixed
concentration).

Figure 4.35 V0 vs [S] Plots for uninhibited reactions (red) and competitively inhibited reactions (blue). Both ultimately have same
Vmax
Graphically, the results of these inhibitor experiments are shown in Figure 4.35 (blue line) and Figure 4.36 (orange line). Notice
that at high substrate concentrations, the competitive inhibitor has essentially no effect, causing the V for the enzyme to remain
max

unchanged. To reiterate, this is due to the fact that at high substrate concentrations, the inhibitor doesn’t compete well. However, at
lower substrate concentrations, it does.

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 Figure 4.36 - Lineweaver-Burk plots - uninhibited reactions (green). Competitively inhibited reactions (orange). Lines cross on Y-
axis at 1/Vmax Since Vmax is the same for both reactions. Image by Aleia Kim
Increased K m

In competitively inhibited reactions, the apparent Km of the enzyme for the substrate increases (−1/K gets closer to zero - red
m

line in Figure 4.36) when the inhibitor is present compared to when the inhibitor is absent, thus illustrating the better competition of
the inhibitor at lower substrate concentrations. It may not be obvious why we call the changed Km the apparent Km of the enzyme.
The reason is that the inhibitor doesn’t actually change the enzyme’s affinity for the folate substrate. It only appears to do so. This
is because of the way that competitive inhibition works. When the competitive inhibitor binds the enzyme, it is effectively ‘taken
out of action.’ Inactive enzymes have NO affinity for substrate and no activity either. We can’t measure Km for an inactive enzyme.
The enzyme molecules that are not bound by methotrexate can, in fact, bind folate and are active. Methotrexate has no effect on
them and their Km values are unchanged. Why then, does Km appear higher in the presence of a competitive inhibitor? The reason
is that the competitive inhibitor is having a greater effect of reducing the amount of active enzyme at lower concentrations of
substrate than it does at higher concentrations of substrate. When the amount of enzyme is reduced, one must have more substrate
to supply the reduced amount of enzyme sufficiently to get to Vmax/2.
It is worth noting that in competitive inhibition, the percentage of inactive enzyme changes drastically over the range of [S] values
used. To start, at low [S] values, the greatest percentage of the enzyme is inhibited. At high [S], no significant percentage of
enzyme is inhibited. This is not always the case, as we shall see in non-competitive inhibition.

Non-competitive inhibition
A second type of inhibition employs inhibitors that do not resemble the substrate and bind not to the active site, but rather to a
separate site on the enzyme (Figure 4.37). The effect of binding a non-competitive inhibitor is significantly different from binding a
competitive inhibitor because there is no competition. In the case of competitive inhibition, the effect of the inhibitor could be
reduced and eventually overwhelmed with increasing amounts of substrate. This was because increasing substrate made increasing
percentages of the enzyme active. With non-competitive inhibition, increasing the amount of substrate has no effect on the
percentage of enzyme that is active. Indeed, in non-competitive inhibition, the percentage of enzyme inhibited remains the same
through all ranges of [S].

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 Figure 4.37 - Non-competitive inhibition - inhibitor does not resemble the substrate and binds to a site other than the active site.
Image by Aleia Kim
This means, then, that non-competitive inhibition effectively reduces the amount of enzyme by the same fixed amount in a typical
experiment at every substrate concentration used The effect of this inhibition is shown in Figure 4.38 & 4.39. As you can see, V max

is reduced in non-competitive inhibition compared to uninhibited reactions.

Figure 4.38 - V0 vs [S] plots of uninhibited reactions (red) and non-competitively inhibited reactions (blue). Vmax is reduced, but
Km values are unchanged in non-competitively inhibited reactions
This makes sense if we remember that Vmax is dependent on the amount of enzyme present. Reducing the amount of enzyme
present reduces V max . In competitive inhibition, this doesn’t occur detectably, because at high substrate concentrations, there is
essentially 100% of the enzyme active and the V appears not to change. Additionally, Km for non-competitively inhibited
max

reactions does not change from that of uninhibited reactions. This is because, as noted previously, one can only measure the K of m

active enzymes and K is a constant for a given enzyme.

m

Figure 4.39 - Lineweaver Burk plots of uninhibited reactions (green) and non-competitively inhibited reactions (purple). Image by
Aleia Kim

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Uncompetitive inhibition
A third type of enzymatic inhibition is that of uncompetitive inhibition, which has the odd property of a reduced Vmax as well as a
reduced Km. The explanation for these seemingly odd results is rooted in the fact that the uncompetitive inhibitor binds only to the
enzyme-substrate (ES) complex (Figure 4.40). The inhibitor-bound complex forms mostly under concentrations of high substrate
and the ES-I complex cannot release product while the inhibitor is bound, thus explaining the reduced V . max

Figure 4.40 - Uncompetitive inhibition. Image by Aleia Kim

The reduced Km is a bit harder to conceptualize. The reason is that the inhibitor-bound complex effectively reduces the
concentration of the ES complex. By Le Chatelier’s Principle, a shift occurs to form additional ES complex, resulting in less free
enzyme and more enzyme in the forms ES and ESI (ES with inhibitor). Decreases in free enzyme correspond to an enzyme with
greater affinity for its substrate. Thus, paradoxically, uncompetitive inhibition both decreases V and increases an enzyme’s
max

affinity for its substrate (K - Figures 4.41 & 4.42).

m

Figure 4.41 - V0 vs [S] plot for uncompetitive inhibition (blue) and uninhibited reactions (red)

Figure 4.42 - Uncompetitive inhibition (purple) and uninhibited reactions (green). Image by Aleia Kim

Suicide inhibition
In contrast to the first three types of inhibition, which involve reversible binding of the inhibitor to the enzyme, suicide inhibition is
irreversible, because the inhibitor becomes covalently bound to the enzyme during the inhibition. Suicide inhibition rather closely
resembles competitive inhibition because the inhibitor generally resembles the substrate and binds to the active site of the enzyme.
The primary difference is that the suicide inhibitor is chemically reactive in the active site and makes a bond with it that precludes

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its removal. Such a mechanism is that employed by penicillin (Figure 4.43), which covalently links to the bacterial enzyme, DD
transpeptidase and stops it from functioning. Since the normal function of the enzyme is to make a bond necessary for the
peptidoglycan complex of the bacterial cell wall, the cell wall cannot properly form and bacteria cannot reproduce.

Figure 4.43 - Action of penicillin. DD-transpeptidase builds peptidoglycan layer of bacterial cell wall (1-3). Binding of penicillin
by DD-transpeptidase stops peptidoglycan synthesis (4-5). Wikipedia

Control of enzymes
It is appropriate to talk at this point about mechanisms cells use to control enzymes. There are four general methods that are
employed:
1. allosterism,
2. covalent modification,
3. access to substrate, and
4. control of enzyme synthesis/breakdown.
Some enzymes are controlled by more than one of these methods.

Allosterism
The term allosterism refers to the fact that the activity of certain enzymes can be affected by the binding of small molecules.
Molecules causing allosteric effects come in two classifications. Ones that are substrates for the enzymes they affect are called
homotropic effectors and those that are not substrates are called heterotropic effectors.
The homotropic effectors usually are activators of the enzymes they bind to and the results of their action can be seen in the
conversion of the hyperbolic curve typical of a V0 vs. [S] plot for an enzyme (Figure 4.18), being converted to a sigmoidal plot
(Figure 4.44). This is due to the conversion of the enzyme from the T-state to the R-state on binding the substrate/homotropic
effector.

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 Figure 4.44 - Kinetic profile of an allosteric enzyme whose activity is controlled by a homotropic effector. Image by Aleia Kim
The V0 vs. [S] plot of allosteric enzyme reactions resembles the oxygen binding curve of hemoglobin (see Figure 2.83). Even
though hemoglobin is not an enzyme and is thus not catalyzing a reaction, the similarity of the plots is not coincidental. In both
cases, the binding of an external molecule is being measured – directly, in the hemoglobin plot, and indirectly by the V0 vs. [S]
plot, since substrate binding is a factor in enzyme reaction velocity.
Allosteric inhibition
Allosterically, regulation of these enzymes works by inducing different physical states (shapes, as it were) that affect their ability to
bind to substrate. When an enzyme is inhibited by binding an effector, it is converted to the T-state (T=tight), it has a reduced
affinity for substrate and it is through this means that the reaction is slowed.
Allosteric activation
On the other hand, when an enzyme is activated by effector binding, it converts to the R-state (R=relaxed) and binds substrate
much more readily. When no effector is present, the enzyme may be in a mixture of T- and R-states.
Feedback inhibition
An interesting kind of allosteric control is exhibited by HMG-CoA reductase, which catalyzes an important reaction in the pathway
leading to the synthesis of cholesterol. Binding of cholesterol to the enzyme reduces the enzyme’s activity significantly. Cholesterol
is not a substrate for the enzyme, so it is therefore a heterotropic effector.
Notably, though, cholesterol is the end-product of the pathway that HMG-CoA reductase catalyzes a reaction in. When enzymes
are inhibited by an end-product of the pathway in which they participate, they are said to exhibit feedback inhibition.
Feedback inhibition always operates by allosterism and further, provides important and efficient control of an entire pathway. By
inhibiting an early enzyme in a pathway, the flow of materials (and ATP hydrolysis required for their processing) for the entire
pathway is stopped or reduced, assuming there are not alternate supply methods.
Pathway control
In the cholesterol biosynthesis pathway, stopping this one enzyme has the effect of shutting off (or at least slowing down) the entire
pathway. This is significant because after catalysis by HMG-CoA reductase, there are over 20 further reactions necessary to make
cholesterol, many of them requiring ATP energy. Shutting down one reactions stops all of them. Another excellent example of
allosteric control and feedback inhibition is the enzyme ATCase, discussed below.
ATCase
Another interesting example of allosteric control and feedback inhibition is associated with the enzyme Aspartate
Transcarbamoylase (ATCase). This enzyme, which catalyzes a step in the synthesis of pyrimidine nucleotides, has 12 subunits.
These include six identical catalytic subunits and six identical regulatory subunits. The catalytic subunits bind to substrate and
catalyze a reaction. The regulatory subunits bind to either ATP or CTP. If they bind to ATP, the enzyme subunits arrange
themselves in the R-state.

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 Figure 4.45 - Schematic structure of ATCase. Regulatory Units = R, Catalytic Units = C. Image by Aleia Kim
R-state
The R-state of ATCase allows the substrate to have easier access to the six active sites and the reaction occurs more rapidly. For the
same amount of substrate, an enzyme in the R-state will have a higher velocity than the same enzyme that is not in the R-state. By
contrast, if the enzyme binds to CTP on one of its regulatory subunits, the subunits will arrange in the T-state and in this form, the
substrate will not have easy access to the active sites, resulting in a slower velocity for the same concentration of substrate
compared to the R-state. ATCase is interesting in that it can also flip into the R-state when one of the substrates (aspartate) binds to
an active site within one of the catalytic subunits.
Aspartate has the effect of activating the catalytic action of the enzyme by favoring the R-state. Thus, aspartate, which is a substrate
of the enzyme is a homotropic effector and ATP and CTP, which are not substrates of the enzyme are heterotropic effectors of
ATCase.

Figure 4.46 - Plots of V0 vs. [S] for ATCase. Left - Allosteric effect of aspartate. Right - Allosteric effects of ATP (activator) and
CTP (inhibitor). Image by Pehr Jacobson

Allosteric models
There are three models commonly used to explain how allosterism regulates multi-subunit enzyme activity. They are known as
the Monod-Wyman-Changeux (MWC) model (also known as the concerted model),
the sequential model (also known as KNF),
and the morpheein model.
All models describe a Tense (T) state that is less catalytically active and a Relaxed (R) state that is more catalytically active. The
models differ in how the states change.
Sequential model
In the sequential model, binding of an allosteric effector by one subunit causes it to change from T to R state (or vice versa) and
that change makes it easier for adjacent subunits to similarly change state. With this model , there is a cause/effect relationship

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between binding of an effector by one subunit and change of state by an adjacent subunit.
In hemoglobin, for example, binding of one oxygen by one unit of the complex may induce that unit to flip to the R-state and,
through interactions with other subunits, cause them to favor adopting the R configuration before they bind to oxygen. In this way,
binding of one subunit favors binding of others and cooperativity can be explained by the change in binding affinity as oxygen
concentration changes.

Figure 4.47 - Sequential model of allosteric regulation. Round = R-State. Square = T-state.. Image by Aleia Kim
MWC model
The MWC model is less intuitive. In it, the entire complex changes state from T to R (or vice versa) independently of the binding
of effectors. Flipping between T-states and R-states is postulated to be in an equilibrium of states in the absence of effector (for
example, a 50 to 1 ratio of T/R. This ratio is referred to as L, so L = T/R). Binding of effector by the enzyme complex has the
tendency of “locking” the complex in a state. Binding of inhibitors will increase the ratio of T/R whereas binding of activators will
increase R and thus decrease the ration of T/R.

Figure 4.48 - The MWC Model - The multisubunit complex flips as a whole and is in equilibrium. Wikipedia
Morpheein model
The morpheein model is similar to the MWC model, but with an added step of dissociation of the subunits. The MWC model
proposes that flipping between R and T states occurs by the complex as a whole and occurs on all units simultaneously. The
morpheein model instead proposes that the multi-subunit enzyme breaks down to individual units which can then flip in structure
and re-form the complex. In the morpheein model, only identically shaped units (all R, for example) can come together in the
complex, thus explaining the “all-R-” or “all-T-” state found in the MWC model.
A large number of enzymes, including prominent ones like citrate synthase, acetyl-CoA carboxylase, glutamate dehydrogenase,
ribonucleotide reductase and lactate dehydrogenase have behavior consistent with the morpheein model.

Figure 4.49 - Morpheein model of allosterism. States flip as monomers and aggregate only with same monomer. Wikipedia

Covalent control of enzymes

Some enzymes are synthesized in a completely inactive form and their activation requires covalent bonds in them to be cleaved.
Such inactive forms of enzymes are called zymogens. Examples include the proteins involved in blood clotting and proteolytic
enzymes of the digestive system, such as trypsin, chymotrypsin, pepsin, and others.
Synthesizing some enzymes in an inactive form makes very good sense when an enzyme’s activity might be harmful to the tissue
where it is being made. For example, the painful condition known as pancreatitis arises when digestive enzymes made in the
pancreas are activated too soon and end up attacking the pancreas.

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Cascades
For both the blood clotting enzymes and the digestive enzymes, the zymogens are activated in a protease cascade. This occurs
when activation of one enzyme activates others in a sort of chain reaction. In such a scheme the first enzyme activated
proteolytically cleaves the second zymogen, causing it to be activated, which in turn activates a third and this may proceed through
several levels of enzymatic action (Figure 4.50).
The advantage of cascades is that they allow a large amount of zymogens to become activated fairly quickly, since there is an
amplification of the signal at each level of catalysis.
Zymogens are also abundant in blood. Blood clotting involves polymerization of a protein known as fibrin. Since random
formation of fibrin is extremely hazardous because it can block the flow of blood, potentially causing heart attack/stroke, the body
synthesizes fibrin as a zymogen (fibrinogen) and its activation results from a “cascade” of activations of proteases that arise when a
signal is received from a wound. Similarly, the enzyme catalyzing removal of fibrin clots (plasmin) is also synthesized as a
zymogen (plasminogen), since random clot removal would also be hazardous (see below also).

Figure 4.50 - Protease activation scheme. Wikipedia

Phosphorylation/dephosphorylation
Another common mechanism for control of enzyme activity by covalent modification is phosphorylation. The phosphorylation of
enzymes (on the side chains of serine, threonine or tyrosine residues) is carried out by protein kinases. Enzymes activated by
phosphorylation can be regulated by the addition of phosphate groups by kinases or their removal by phosphatases. Thus, this type
of covalent modification is readily reversible, in contrast to proteolytic cleavage.

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 Figure 4.51 - Regulation by covalent modification of glycogen catabolism enzymes
Reduction/oxidation
An interesting covalent control of enzymes using reduction/oxidation is exhibited in photosynthetic plants. In the light phase of
photosynthesis, electrons are excited by light and flow through carriers to NADP+, forming NADPH. Thus, in the light, the
NADPH concentration is high. When NADPH concentration is high, the concentration of reduced ferredoxin (a molecule donating
electrons to NADP+) is also high.
Reduced ferredoxin can transfer electrons to thioredoxin, reducing it. Reduced thioredoxin can, in turn, transfer electrons to
proteins to reduce their disulfide bonds. Four enzymes related to the Calvin cycle can receive electrons from thioredoxin and
become activated, as a result.
These include sedoheptulose 1,7-bisphosphatase, ribulose-5-phosphate kinase, fructose 1,6-bisphosphatase, and glyceraldehyde 3-
phosphate dehydrogenase. Thus, in the light, electrons flow, causing NADPH to accumulate and ferredoxin to push electrons in the
direction of these enzymes above, activating them and favoring the Calvin cycle. In the dark, the concentration of reduced
NADPH, reduced ferredoxin, and reduced thioredoxin fall, resulting in loss of electrons by the Calvin cycle enzymes (oxidations
that re-form disulfide bonds) and the Calvin cycle inactivates.

Other enzyme control mechanisms

Other means of controlling enzymes relate to access to substrate (substrate-level control) and control of enzyme synthesis.
Hexokinase is an enzyme that is largely regulated by availability of its substrate, glucose. When glucose concentration is low, the
product of the enzyme’s catalysis, glucose-6-phosphate, inhibits the enzyme’s function.
Regulation of enzymes by controlling their synthesis is covered later in the book in the discussion relating to control of gene
expression.

This page titled 4.2: Control of Enzymatic Activity is shared under a CC BY-NC-SA license and was authored, remixed, and/or curated by Kevin
Ahern, Indira Rajagopal, & Taralyn Tan.

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4.3: Mechanisms of Catalysis
A printable version of this section is here: BiochemFFA_4_3.pdf. The entire textbook is available for free from the authors
at http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy
The magic of enzymes, as noted, is in their ability to create electronic environments conducive to initiation of a reaction. There are
more mechanisms of reaction than we could ever hope to cover in a book like this, and comprehensive discussion of these is not
our aim. Instead, we will cite some examples and go into detail on one of them - the mechanism of action of serine proteases.

Chymotrypsin
We will begin with mechanism of action of one enzyme - chymotrypsin. Found in our digestive system, chymotrypsin’s catalytic
activity is cleaving peptide bonds in proteins and it uses the side chain of a serine in its mechanism of catalysis. Many other
protein-cutting enzymes employ a very similar mechanism and they are known collectively as serine proteases (Figure 4.52).

Figure 4.52 - Substrate binding sites (S1 pockets) of three serine proteases. Image by Aleia Kim
These enzymes are found in prokaryotic and eukaryotic cells and all use a common set of three amino acids in the active site called
a catalytic triad (Figure 4.53). It consists of aspartic acid, histidine, and serine. The serine is activated in the reaction mechanism to
form a nucleophile in these enzymes and gives the class their name. With the exception of the recognition that occurs at the
substrate binding site, the mechanism shown here for chymotrypsin would be applicable to any of the serine proteases.

Figure 4.53 - 1. Active site of chymotrypsin showing the catalytic triad of serine - histidine-aspartic acid

Specificity
As a protease, chymotrypsin acts fairly specifically, cutting not all peptide bonds, but only those that are adjacent to relatively non-
polar amino acids in the protein. One of the amino acids it cuts adjacent to is phenylalanine. The enzyme’s action occurs in two
phases – a fast phase that occurs first and a slower phase that follows. The enzyme has a substrate binding site that includes a
region of the enzyme known as the S1 pocket. Let us step through the mechanism by which chymotrypsin cuts adjacent to
phenylalanine.

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Substrate binding
The process starts with the binding of the substrate in the S1 pocket (Figure 4.54). The S1 pocket in chymotrypsin has a
hydrophobic hole in which the substrate is bound. Preferred substrates will include amino acid side chains that are bulky and
hydrophobic, like phenylalanine. If an ionized side chain, like that of glutamic acid binds in the S1 pocket, it will quickly exit,
much like water would avoid an oily interior.

Figure 4.54 - 2. Binding of substrate to S1 pocket in the active site

Shape change on binding

When the proper substrate binds in the S1 pocket, its presence induces an ever so slight change in the shape of the enzyme. This
subtle shape change on the binding of the proper substrate starts the steps of the catalysis. Since the catalytic process only starts
when the proper substrate binds, this is the reason that the enzyme shows specificity for cutting at specific amino acids in the target
protein. Only amino acids with the side chains that interact well with the S1 pocket start the catalytic wheels turning.
The slight changes in shape involve changes in the positioning of three amino acids (aspartic acid, histidine, and serine) in the
active site known as the catalytic triad.

Figure 4.55 - 3. Formation of alkoxide ion

The shift of the negatively charged aspartic acid towards the electron rich histidine ring favors the abstraction of a proton by the
histidine from the hydroxyl group on the side chain of serine, resulting in production of a very reactive alkoxide ion in the active
site (Figure 4.55).

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 Figure 4.56 - 4. Nucleophilic attack

Since the active site at this point also contains the polypeptide chain positioned with the phenylalanine side chain embedded in the
S1 pocket, the alkoxide ion performs a nucleophilic attack on the peptide bond on the carboxyl side of phenylalanine sitting in the
S1 pocket (Figure 4.56). This reaction breaks the peptide bond (Figure 4.57) and causes two things to happen.

Figure 4.57 - 5. Stabilization by oxyanion hole. Breakage of peptide bond.

First, one end of the original polypeptide is freed and exits the active site (Figure 4.58). The second is that the end containing the
phenylalanine is covalently linked to the oxygen of the serine side chain. At this point we have completed the first (fast) phase of
the catalysis.

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 Figure 4.58 - 6. First peptide released. Other half bonded to serine.

Slower second phase

The second phase of the catalysis by chymotrypsin is slower. It requires that the covalent bond between phenylalanine and serine’s
oxygen be broken so the peptide can be released and the enzyme can return to its original state. The process starts with entry of
water into the active site. Water is attacked in a fashion similar to that of the serine side chain in the first phase, creating a reactive
hydroxyl group (Figure 4.59) that performs a nucleophilic attack on the phenylalanine-serine bond (Figure 4.60), releasing it and
replacing the proton on serine. The second peptide is released in the process and the reaction is complete with the enzyme back in
its original state (Figure 4.61).

Figure 4.59 - 7. Activation of water by histidine

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 Figure 4.60 - 8. Nucleophile attack by hydroxyl creates tetrahydryl intermediate stabilized by oxyanion hole. Bond to serine breaks.

Figure 4.61 - 9. Second half of peptide released. Enzyme active site restored.

Serine proteases
The list of serine proteases is quite long. They are grouped in two broad categories - 1) those that are chymotrypsin-like and 2)
those that are subtilisin-like. Though subtilisin-type and chymotrypsin-like enzymes use the same mechanism of action, including
the catalytic triad, the enzymes are otherwise not related to each other by sequence and appear to have evolved independently. They
are, thus, an example of convergent evolution - a process where evolution of different forms converge on a structure to provide a
common function.
The serine protease enzymes cut adjacent to specific amino acids and the specificity is determined by the size/shape/charge of
amino acid side chain that fits into the enzyme’s S1 binding pocket (Figure 4.62).

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 Figure 4.62 - Subtilisin - A serine protease
Examples of serine proteases include trypsin, chymotrypsin, elastase, subtilisin, signal peptidase I, and nucleoporin. Serine
proteases participate in many physiological processes, including blood coagulation, digestion, reproduction, and the immune
response.

Cysteine proteases
Cysteine proteases (also known as thiol proteases) catalyze the breakdown of proteins by cleaving peptide bonds using a
nucleophilic thiol from a cysteine (Figure 4.63). The cysteine is typically found in a catalytic dyad or triad also involving histidine
and (sometimes) aspartic acid (very much like serine proteases). The sulfhydryl group of cysteine proteases is more acidic than the
hydroxyl of serine proteases, so the aspartic acid of the triad is not always needed.
The mechanism of action is very similar to that of serine proteases. Binding of proper substrate results in activation of the thiol
(removal of the proton by the histidine group). The activated thiol acts as a nucleophile, attacking the peptide bond and causing it
break. One peptide is released and the other peptide becomes covalently linked to the sulfur. Hydrolysis by water releases the
second peptide and completes the cycle. Examples of cysteine proteases include papain, caspases, hedgehog protein, calpain, and
cathepsin K.

Figure 4.63 - Mechanism of action of proteases. In each case, a nucleophile is created - hydroxyl (aspartyl proteases), thiol
(cysteine proteases), and hydroxyl (metalloproteases). Image by Aleia Kim

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Caspases
Caspases (Cysteine-ASPartic ProteASEs) are a family of cysteine proteases that play important roles in the body. At the cellular
level they function in apoptosis and necrosis and in the body, they are involved in inflammation and the immune system.
Maturation of lymphocytes is one such role. They are best known, however, for their role in apoptosis, which has given rise to
descriptions of them as “executioner” proteins or “suicide proteases” that dismantle cells in programmed cell death.
There are 12 known human caspases. The enzymes are synthesized as pro-caspase zymogens with a prodomain and two other
subunits. The prodomain contains regions that allow it to interact with other molecules that regulate the enzyme’s activity. The
caspases come in two forms. The initiator caspases, when activated, activate the effector caspases. The effector caspases cleave
other proteins in the cell. Targets for effector caspase cleavage action include the nuclear lamins (fibrous proteins providing
structural integrity to the nucleus), ICAD/DFF45 (an inhibitor of DNAse), PARP (flags areas where DNA repair needed), and
PAK2 (apoptotic regulation).
The caspase activation cascade can itself be activated by granzyme B (a serine protease secreted by natural killer cells and
cytotoxic T-cells), cellular death receptors, and the apoptosome (large protein structure in apoptotic cells stimulated by release of
cytochrome C from the mitochondria). Each of these activators is responsible for activating different groups of caspases.

Metalloproteases
Metalloproteases (Figure 4.64) are enzymes whose catalytic mechanism for breaking peptide bonds involves a metal. Most
metalloproteases use zinc as their metal, but a few use cobalt, coordinated to the protein by three amino acid residues with a labile
water at the fourth position. A variety of side chains are used - histidine, aspartate, glutamate, arginine, and lysine. The water is the
target of action of the metal which, upon binding of the proper substrate, abstracts a proton to create a nucleophilic hydroxyl group
that attacks the peptide bond, cleaving it (Figure 4.64). Since the nucleophile here is not attached covalently to the enzyme, neither
of the cleaved peptides ends up attached to the enzyme during the catalytic process. Examples of metalloproteases include
carboxypeptidases, aminopeptidases, insulinases and thermolysin.

Figure 4.64 - Carboxypeptidase - A metalloprotease

Aspartyl proteases
As the name suggests, aspartyl proteases use aspartic acid in their catalytic mechanism (Figures 4.63 & 4.65). Like the
metalloproteases, aspartyl proteases activate a water to create a nucleophile for catalysis (Figure 4.65). The activated water attacks
the peptide bond of the bound substrate and releases the two pieces without the need to release a bound intermediate, since water is
not covalently attached to the enzyme. Common aspartyl proteases include pepsin, signal peptidase II, and HIV-1 protease.

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 Figure 4.65 - Activation of an aspartyl protease - aspartate side chain abstracts proton (top). Hydroxyl attacks peptide bond
(middle). Broken peptide pieces released (bottom). Image by Pehr Jacobson

Threonine proteases
Though threonine has an R-group with a hydroxyl like serine, the mechanism of action of this class of proteases differs somewhat
from the serine proteases. There are some similarities. First, the threonine’s hydroxyl plays a role in catalysis and that is to act as a
nucleophile. The nucleophile is created, however, not by a catalytic triad, but rather as a result of threonine’s own α-amine group
abstracting a proton.
Because of this, the nucleophilic threonine in a threonine protease must be at the n-terminus of the enzyme. Nucleophilic attack of
the peptide bond in the target protease results in breakage of the bond to release one peptide and the other is covalently attached to
serine, like the serine proteases. Also, as with the serine proteases, water must come in to release the covalently linked second
peptide to conclude the catalytic mechanism.

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Examples
Examples of threonine proteases include the catalytic subunits of the proteasome. Some acyl transferases (such as ornithine
acyltransferase) have evolved the same catalytic mechanism by convergent evolution. The latter enzymes use ornithine instead of
water to break the enzyme-substrate covalent bond, with the result that the acyl-group becomes attached to ornithine, instead of
water.

Protease inhibitors
Molecules which inhibit the catalytic action of proteases are known as protease inhibitors. These come in a variety of forms and
have biological and medicinal uses. Many biological inhibitors are proteins themselves. Protease inhibitors can act in several ways,
including as a suicide inhibitor, a transition state inhibitor, a denaturant, and as a chelating agent. Some work only on specific
classes of enzymes. For example, most known aspartyl proteases are inhibited by pepstatin. Metalloproteases are sensitive to
anything that removes the metal they require for catalysis. Zinc-containing metalloproteases, for example, are very sensitive to
EDTA, which chelates the zinc ion.
One category of proteinaceous protease inhibitors is known as the serpins. Serpins inhibit serine proteases that act like
chymotrypsin. 36 of them are known in humans.
Serpins are unusual in acting by binding to a target protease irreversibly and undergoing a conformational change to alter the active
site of its target. Other protease inhibitors act as competitive inhibitors that block the active site.
Serpins can be broad in their specificity. Some, for example, can block the activity of cysteine proteases. One of the best known
biological serpins is α-1-anti-trypsin (A1AT - Figure 4.66) because of its role in lungs, where it functions to inhibit the elastase
protease. Deficiency of A1AT leads to emphysema. This can arise as a result of genetic deficiency or by cigarette smoking.
Reactive oxygen species produced by cigarette smoking can oxidize a critical methionine residue (#358 of the processed form) in
A1AT, rendering it unable to inhibit elastase. Uninhibited, elastase can attack lung tissue and cause emphysema. Most serpins work
extracellularly. In blood, for example, serpins like antithrombin can help to regulate the clotting process.

Figure 4.66 - α-1-antitrypsin

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 Figure 4.67 - Incidence of α-1-antitrypsin (PiMZ) deficiency in Europe by percent. Wikipedia

Anti-viral Agents
Protease inhibitors are used as anti-viral agents to prohibit maturation of viral proteins - commonly viral coat proteins.
They are part of drug “cocktails” used to inhibit the spread of HIV in the body and are also used to treat other viral infections,
including hepatitis C. They have also been investigated for use in treatment of malaria and may have some application in anti-
cancer therapies as well.

This page titled 4.3: Mechanisms of Catalysis is shared under a CC BY-NC-SA license and was authored, remixed, and/or curated by Kevin
Ahern, Indira Rajagopal, & Taralyn Tan.

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4.4: Blood Clotting
A printable version of this section is here: BiochemFFA_4_4.pdf. The entire textbook is available for free from the authors
at http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy
Clotting is a process in which liquid blood is converted into a gelatinous substance that eventually hardens. The aim is to stop the
flow of blood from a vessel. The formation of a clot is the result of a series of enzymatic reactions that are triggered upon injury.
The process involves:
1. a step of activation (wounding) followed by
2. a cellular response (aggregation of blood platelets) and
3. a molecular response (polymerization of the protein called fibrin to create a meshwork that hardens).
Factors released in the cellular response help activate the molecular response. The process is highly conserved across species.

Cellular Response
Injury to the epithelial lining of a blood vessel begins the process of coagulation almost instantly. The cellular response has an
initial action followed by an amplification step. In the cellular response (Figure 4.68), the platelets bind directly to collagen using
Ia/IIa collagen-binding surface receptors and glycoprotein VI to form a plug. The signal to the platelets to take this action is
exposure of the underlying collagen, something that would not happen in the absence of a wound. Upon injury, platelet integrins
get activated and bind tightly to the extracellular matrix to anchor them to the site of the wound.
The von Willebrand factor (see below also) assists by forming additional links between the platelets’ glycoprotein Ib/IX/V and the
fibrils of the collagen.

Figure 4.68 - Blood coagulation scheme. The cellular response is shown in white on upper left. Everything else is part of the
molecular response. Wikipedia

Amplification
In the amplification part of the cellular response, the activated platelets release a large number of factors, including platelet factor 4
(a cytokine stimulating inflammation and moderating action of the heparin anticoagulant) and thromboxane A2, The latter has the
effect of increasing the “stickiness” of platelets, favoring their aggregation. In addition, a a Gq-protein linked receptor cascade is
activated, resulting in release of calcium from intracellular stores. This will play a role in the molecular response.

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Molecular response
The molecular response results in the creation of a web comprised of polymers of fibrin protein. Like the cellular pathway, the
molecular pathway begins with an initiation phase and continues with an amplification phase. Polymerization of fibrin results from
convergence of two cascading catalytic pathways. They are the intrinsic pathway (also called the contact activation pathway) and
the extrinsic pathway (also referred to as the tissue factor pathway). Of the two pathways, the tissue factor pathway has recently
been shown to be the more important.

Serine Protease Cascade

In both pathways, a series of zymogens of serine proteases are sequentially activated in rapid succession. The advantage of such a
cascading system is tremendous amplification of a small signal. At each step of the cascade, activation of a zymogen causes the
production of a considerable amount of an active serine protease, which is then able to activate the next zymogen which, in turn,
activates an even larger amount of the next zymogen in the system. This results in the ultimate activation of a tremendous amount
more fibrin than could be achieved if there were only a single step where an enzyme activated fibrinogen to fibrin.
Nomenclature
The zymogen factors in the molecular response are generally labeled with Roman numerals. A lowercase, subscripted ‘a’ is used
to designate an activated form.

The tissue factor pathway functions to create a thrombin burst, a process in which thrombin is activated very quickly. This is the
initiation phase. It is fairly straightforward because it has one focus - activation of thrombin. Thrombin, which converts fibrinogen
into the fibrin of the clot, is central also to the amplification phase, because it activates some of the factors that activate it, creating
an enormous increase in signal and making a lot of thrombin active at once.

Initiation phase
The initiation phase of the molecular response begins when Factor VII (the letter ‘F’ before the Roman numeral is often used as an
abbreviation for ‘factor’) gets activated to FVIIa after damage to the blood vessel (Figure 4.69 & 4.70). This happens as a result of
its interaction with Tissue Factor (TF, also called coagulation Factor III) to make a TF-FVIIa complex. The combined efforts of TF-
FVIIa, FIXa, and calcium (from the cellular response) inefficiently convert FX to FXa. FXa, FV, and calcium inefficiently convert
prothrombin (zymogen) to thrombin (active). A tiny amount of thrombin has been activated at the end of the initiation phase.

Figure 4.69 - Intrinsic and extrinsic pathways of blood coagulation. The aim is making a fibrin clot (lower right). Wikipedia

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 Figure 4.70 - Another view of the molecular response of the blood clotting pathway. Wikipedia

Amplification phase
To make sufficient thrombin to convert enough fibrinogen to fibrin to make a clot, thrombin activates other factors (FV, FXI,
FVIII) that help to make more thrombin. This is the amplification phase of the molecular process and is shown in the light blue
portion in the upper right part of Figure 4.68. The amplification phase includes factors in both the intrinsic and extrinsic pathways.
FVIII is normally bound in a complex with the von Willebrand factor and is inactive until it is released by action of thrombin.
Activation of FXI to FXIa helps favor production of more FIXa. FIXa plus FVIIIa stimulate production of a considerable amount
of FXa. FVa joins FXa and calcium to make a much larger amount of thrombin. Factors FVa and FVIIIa are critical to the
amplification process. FVIIIa stimulates FIXa’s production of FXa by 3-4 orders of magnitude. FVa helps to stimulate FXa’s
production of thrombin by about the same magnitude. Thus, thrombin stimulates activation of factors that, in turn, stimulate
activation of more thrombin.

Transglutaminase
In addition to helping to amplify product of itself and conversion of fibrinogen to fibrin, thrombin catalyzes the activation of FXIII
to FXIIIa. FXIIIa is a transglutaminase that helps to “harden” the clot (Figure 4.71 & 4.73). It accomplishes this by catalyzing
formation of a covalent bond between adjacent glutamine and lysine side chains in the fibrin polymers.

Figure 4.71 - Catalytic action of transglutaminase (top) and breaking of transglutamide bonds by hydrolysis (bottom)
Not all of the factors involved in the clotting process are activated by the pathway, nor are all factors serine proteases. This includes
FVIII and FV which are glycoproteins, and FXIII, which is the transglutaminase described above.
The blood clotting process must be tightly regulated. Formation of clots in places where no damage has occurred can lead to
internal clots (thrombosis) cutting off the flow of blood to critical regions of the body, such as heart or brain. Conversely, lack of

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clotting can lead to internal bleeding or, in severe cases, death due to unregulated external bleeding. Such is a danger for people
suffering from hemophilia.

Figure 4.72 - α-thrombin. Wikipedia

Diseases of Blood Clotting: Hemophilia
Hemophilia is a hereditary genetic disorder affecting the blood clotting process in afflicted individuals. The disease is X-linked
and thus occurs much more commonly in males. Deficiency of FVIII leads to Hemophilia A (about 1 in 5000 to 10,000 male
births) and deficiency of FIX produces Hemophilia B (about 1 in 20,000 to 35,000 male births).

Figure 4.73 - Product of transglutaminase action - cross links. Wikipedia

Figure 4.74 - Fibrin dimer - basic unit of a fibrin clot. Wikipedia

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 Figure 4.75 - Blood cells enmeshed in a fibrin clot
Hemophilia B spread through the royal families of Europe, beginning with Queen Victoria’s son, Leopold. Three of the queen’s
grandsons and six of her great-grandsons suffered from the disease. Hemophilia is treated by exogenous provision of missing
clotting factors and has improved life expectancy dramatically. In 1960, the life expectancy of a hemophiliac was about 11
years. Today, it is over 60.

Figure 4.76 - Queen Victoria, whose descendants suffered from hemophilia B

Diseases of Blood Clotting: von Willebrand’s disease

A related disease to hemophilia that is also genetically linked is von Willebrand’s Disease. The von Willebrand factor plays a
role in both the cellular and the molecular responses in blood clotting. First, the factor is a large multimeric glycoprotein present
in blood plasma and also is produced in the endothelium lining blood vessels.
The von Willebrand factor helps to anchor platelets near the site of the wound in the cellular response. It binds to several things.
First, it binds to platelets’ Ib glycoprotein. Second, it binds to heparin and helps moderate its action. Third, it binds to collagen
and fourth, the factor binds to FVIII in the molecular response, playing a protective role for it. In the absence of the von
Willebrand factor, FVIII is destroyed. Fifth, the von Willebrand factor binds to integrin of platelets, helping them to adhere
together and form a plug. Defects of the von Willebrand factor lead to various various bleeding disorders.

Blood “thinners”
The clotting of blood is essential for surviving wounds that cause blood loss. However, some people have conditions that
predispose them to the formation of clots that can lead to stroke, heart attack, or other problems, like pulmonary embolism. For
these people, anti-clotting agents (commonly called blood thinners) are used to reduce the likelihood of undesired clotting.
The first, and more common of these is aspirin. Aspirin is an inhibitor of the production of prostaglandins. Prostaglandins are
molecules with 20 carbons derived from arachidonic acid that have numerous physiological effects. Metabolically, the
prostaglandins are precursors of a class of molecules called the thromboxanes. Thromboxanes play roles in helping platelets to
stick together in the cellular response to clotting. By inhibiting the production of prostaglandins, aspirin reduces the production of
thromboxanes and reduces platelet stickiness and the likelihood of clotting.

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Vitamin K action
Another approach to preventing blood clotting is one that interferes with an important molecular action of Vitamin K. A pro-
clotting factor found in the blood, vitamin K is necessary for an important modification to prothrombin and other blood clotting
proteins. Vitamin K serves as an enzyme cofactor that helps to catalyze addition of an extra carboxyl group onto the side chain of
glutamic acid residues of several clotting enzymes (see HERE). This modification gives them the ability to bind to calcium (Figure
4.77), which is important for activating the serine protease cascade. During the reaction that adds carboxyl groups to glutamate, the
reduced form of vitamin K becomes oxidized. In order for vitamin K to stimulate additional carboxylation reactions to occur, the
oxidized form of vitamin K must be reduced by the enzyme vitamin K epoxide reductase.

Figure 4.77 - γ-carboxylglutamic acid (left) has a calcium binding Site. Unmodified glutamic acid (right) does not.

Warfarin blocks reduction

The compound known as warfarin (brand name = coumadin - Figure 4.78) interferes with the action of vitamin K epoxide
reductase and thus, blocks recycling of vitamin K. As a consequence, fewer prothrombins (and other blood clotting proteins) get
carboxylated, and less clotting occurs.

Figure 4.78 - Warfarin

Vitamin K-mediated carboxylation of glutamate occurs on the γ carbon of the amino acid’s side chain, for 16 different proteins, 7 of
which are involved in blood clotting, including prothrombin. When the carboxyl group is added as described, the side chain is able
to efficiently bind to calcium ions. In the absence of the carboxyl group, the side chain will not bind to calcium. Calcium released
near the site of the wound in the cellular response to clotting helps to stimulate activation of proteins in the serine protease cascade
of the molecular response.
Vitamin K comes in several forms. It is best described chemically as a group of 2-methyl-1,4-naphthoquinone derivatives. There
are five different forms recognized as vitamin Ks (K1, K2, K3, K4, and K5). Of these, vitamins K1 and K2 come from natural
sources and the others are synthetic. Vitamin K2, which is made from vitamin K1 by gut microorganisms, has several forms, with
differing lengths of of isoprenoid side-chains. The various forms are commonly named as MK-X, where X is a number, and MK
stands for menaquinone, which is the name given to this form of vitamin K. Figure 4.79 shows a common form known as MK-4
(menatetrenone).

Figure 4.79 - MK-4 (menatetrenone)

Hemorrhaging danger
It is very critical that the proper amount of warfarin be given to patients. Too much can result in hemorrhaging. Patients must have
their clotting times checked regularly to ensure that they are taking the right dose of anti-coagulant medication. Diet and the

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metabolism of Vitamin K in the body can affect the amount of warfarin needed. Vitamin K is synthesized in plants and plays a role
in photosynthesis. It can be found in the highest quantities in vegetables that are green and leafy. Patients whose diet is high in
these vegetables may require a different dose than those who rarely eat greens. Dietary vitamin K is also, as mentioned earlier,
metabolized by bacteria in the large intestine, where they convert vitamin K1 into vitamin K2.

Plasmin
Clots, once made in the body, do not remain there forever. Instead, a tightly regulated enzyme known as plasmin is activated, when
appropriate, to break down the fibrin-entangled clot. Like many of the enzymes in the blood clotting cascade, plasmin is a serine
protease. It is capable of cleaving a wide range of proteins. They include polymerized fibrin clots, fibronectin, thrombospondin,
laminin, and the von Willebrand factor.

Figure 4.81 - Plasmin

Plasmin plays a role in activating collagenases and in the process of ovulation by weakening the wall of the Graafian follicle in the
ovary. Plasmin is made in the liver as the zymogen known as plasminogen. Several different enzymes can activate it.
Tissue plasminogen activator (tPA), using fibrin as a co-factor, is one. Others include urokinase plasminogen activator (using
urokinase plasminogen activator receptor as a co-factor), kallikrein (plasma serine protease with many forms and blood functions),
and FXIa and FXIIa from the clotting cascade.

Plasmin inhibition
Plasmin’s activity can also be inhibited. Plasminogen activator inhibitor, for example, can inactivate tPA and urokinase. After
plasmin has been activated, it can also be inhibited by α2-antiplasmin and α2-macroglobulin (Figure 4.80). Thrombin also plays a
role in plasmin’s inactivation, stimulating activity of thrombin activatable fibrinolysis inhibitor. Angiostatin is a sub-domain of
plasmin produced by auto-proteolytic cleavage. It blocks the growth of new blood vessels and is being investigated for its anti-
cancer properties.

Figure 4.80 - Regulation of fibrin breakdown. Activators in blue. Inhibitors in red. Wikipedia

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Fibronectin
Fibronectin is a large (440 kDa) glycoprotein found in the extracellular matrix that binds to integral cellular proteins called
integrins and to extracellular proteins, including collagen, fibrin, and heparan sulfate. It comes in two forms. The soluble form is
found in blood plasma and is made by the liver. It is found in high concentration in the blood stream (300 µg/ml). The insoluble
form is found abundantly in the extracellular matrix.
The protein is assembled in the extracellular matrix and plays roles in cellular growth, adhesion, migration, and differentiation. It is
very important in wound healing.

Figure 4.82 - Fibronectin 1. Wikipedia

Assists in blood clot formation

Fibronectin from the blood plasma is localized to the site of the wound, assisting in formation of the blood clot to stop bleeding. In
the initial stages of wound healing, plasma fibronectin interacts with fibrin in clot formation. It also protects tissue surrounding the
wound. Later in the repair process, remodeling of the damaged area begins with the action of fibroblasts and endothelial cells at the
wound site. Their task is to degrade proteins of the blood clot matrix, replacing them with a new matrix like the undamaged,
surrounding tissue.
Fibroblasts act on the temporary fibronectin-fibrin matrix, remodeling it to replace the plasma fibronectin with cellular fibronectin.
This may cause the phenomenon of wound contraction, one of the steps in wound healing. Secretion of cellular fibronectin by
fibroblasts is followed by fibronectin assembly and integration with the extracellular matrix.

Embryogenesis
Fibronectin is essential for embryogenesis. Deleting the gene in mice causes lethality before birth. This is likely due to its role in
migration and guiding the attachment of cells as the embryo develops. Fibronectin also has a role in the mouth. It is found in saliva
and is thought to inhibit colonization of the mouth by pathogenic bacteria.

Platelet activating factor

Platelet Activating Factor (PAF) is a compound (Figure 4.83) produced primarily in cells involved in host defense. These include
platelets, macrophages, neutrophils, and monocytes, among others. It is produced in greater quantities in inflammatory cells upon
proper stimulation. The compound acts like a hormone and mediates platelet aggregation/degranulation, inflammation, and
anaphylaxis. It can transmit signals between cells to trigger and amplify inflammatory and clotting cascades.
When unregulated, signaling by PAF can cause severe inflammation resulting in sepsis and injury. Inflammation in allergic
reactions arises partly as a result of PAF and is an important factor in bronchoconstriction in asthma. In fact, at a concentration of
only 10 picomolar, PAF can cause asthmatic inflammation of the airways that is life threatening.

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 Figure 4.83 - Platelet Activating Factor. Wikipedia
I’m feeling so sad
‘Cuz I cut . . . . myself bad
Now I’m all worried ‘bout . . . . consequences
It’s starting to bleed
There’s some clo . . . . sure I need
So the body kicks . . . . in its defenses
It’s happened all so many times before
The blood flows out and then it shuts the door
Thank goodness my blood is clotting
Enmeshing the fibrin chains
Thank goodness my blood is clotting
The zymogens
Are activating and all is well
So I’ll stop bleeding again
The vitamin K’s
Help to . . . . bind to cee-ays
Adding C-O-. . . . O-H to amend things
Um-m-um-um-um-um
It hardens and stays
When a glu. . . . taminase
Creates co. . . . valent bonds . . . . for cementing
In just a moment, things are good to go
The clot’s in place and it has stopped the flow
But what about clot dissolving?
Untangling fibrin chains?
This calls for some problem solving
There is a way
Just activate up some t-PA
Get plasmin active in veins
Oh, oh, oh.
And thanks to the dis-enclotting’
As part of repairin’ veins
It’s part of my body’s plotting
The wound is gone
I’m back where I started and
Nothing’s wrong

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My blood flow is normal again.
Thank Goodness My Blood is Clotting
To the tune of "Don't Sleep in the Subway Darling"
Metabolic Melodies Website HERE
Recording by Liz Bacon and David Simmons
Lyrics by Kevin Ahern
Recording by Liz Bacon and David Simmons Lyrics by Kevin Ahern

This page titled 4.4: Blood Clotting is shared under a CC BY-NC-SA license and was authored, remixed, and/or curated by Kevin Ahern, Indira
Rajagopal, & Taralyn Tan.

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 CHAPTER OVERVIEW

5: Energy
5.1: Basics of Energy
5.3: Energy - Photophosphorylation
5.2: Electron Transport and Oxidative Phosphorylation

This page titled 5: Energy is shared under a CC BY-NC-SA license and was authored, remixed, and/or curated by Kevin Ahern, Indira Rajagopal,
& Taralyn Tan.

1
5.1: Basics of Energy
Source: BiochemFFA _5_1.pdf. The entire textbook is available for free from the authors at
http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy

Living organisms are made up of cells, and cells contain a horde of biochemical components. Living cells, though, are not random
collections of these molecules. They are extraordinarily organized or "ordered". By contrast, in the nonliving world, there is a
universal tendency to increasing disorder. Maintaining and creating order in cells takes the input of energy. Without energy, life is
not possible.

Oxidative Energy
The primary mechanism used by non-photosynthetic organisms to obtain energy is oxidation and carbon is the most commonly
oxidized energy source. The energy released during the oxidative steps is “captured” in ATP and can be used later for energy
coupling. The more reduced a carbon atom is, the more energy can be realized from its oxidation. Fatty acids are highly reduced,
whereas carbohydrates are moderately so. Complete oxidation of both leads to carbon dioxide, which has the lowest energy state.
Conversely, the more oxidized a carbon atom is, the more energy it takes to reduce it.

Figure 5.1.1 : Five oxidation states of carbon. Image by Pehr Jacobson

In the series shown in Figure 5.1.1, the most reduced form of carbon is on the left. The energy of oxidation of each form is shown
above it. The reduction states of fatty acids and carbohydrates can be seen by their formulas.
Palmitic acid: C H
16 34
O
2

Glucose: C H O
6 12 6

Palmitic acid only contains two oxygens per sixteen carbons, whereas glucose has six oxygen atoms per six carbons. Consequently,
when palmitic acid is fully oxidized, it generates more ATP per carbon (128/16) than glucose (38/6). It is because of this that we
use fat (contains fatty acids) as our primary energy storage material.

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 Figure 5.1.2 : Photosynthesis: The primary source of biological energy. Image by Aleia Kim

Oxidation vs. Reduction in Metabolism

Biochemical processes that break things down from larger to smaller are called catabolic processes. Catabolic processes are often
oxidative in nature and energy releasing. Some, but not all, of that energy is captured as ATP. If not all of the energy is captured as
ATP, what happens to the rest of it? The answer is simple. It is released as heat and it is for this reason we get hot when we
exercise.
By contrast, synthesizing large molecules from smaller ones (for example, making proteins from amino acids) is referred to as
anabolism. Anabolic processes are often reductive in nature (Figures 5.3 & 5.4) and require energy input. By themselves, they
would not occur, as they are reversing oxidation and decreasing entropy (making many small things into a larger one). To overcome
this energy barrier, cells must expend energy. For example, if one wishes to reduce CO to carbohydrate, energy must be used to
2

do so. Plants do this during the dark reactions of photosynthesis (Figure 5.1.3). The energy source for the reduction is ultimately
the sun. The electrons for the reduction come from water, and the CO is removed from the atmosphere and gets incorporated into
2

a sugar.

Figure 5.1.3 : Movement of biological energy. Image by Aleia Kim

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 Figure 5.1.4 : Photosynthesis as measured by chlorophyll concentration

Energy Coupling
The synthesis of the many molecules needed by cells needs the input of energy to occur. Cells overcome this energy obstacle by
using ATP to “drive” the reaction (Figure 5.1.6). The energy needed to drive reactions is harvested in very controlled conditions in
enzymes. This involves a process called ‘coupling’. Coupled reactions rely on linking an energetically favorable reaction (i.e., one
with a negative ∆G°’) with the reaction requiring an energy input, which has a positive ∆G°’. As long as the overall ∆G°’ of the
two reactions together is negative, the reaction can proceed. Hydrolysis of ATP is a very energetically favorable reaction that is
commonly linked to many energy requiring reactions in cells. Without the hydrolysis of ATP (or GTP, in some cases), the reaction
would not be feasible.

Figure 5.1.5 : Synthesis and breakdown pathways in metabolism. Image by Pehr Jacobson

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 Figure 5.1.6 : Cycling of biological energy via ADP and ATP. Image by Pehr Jacobson

Entropy and energy

Most students who have had some chemistry know about the Second Law of Thermodynamics with respect to increasing disorder
of a system. Cells are very organized or ordered structures, leading some to mistakenly conclude that life somehow violates the
second law. In fact, that notion is incorrect. The second law doesn’t say that entropy always increases, just that, left alone, it tends
to do so, in an isolated system. Cells are not isolated systems, though, in that they obtain energy, either from the sun, if they are
autotrophic, or food, if they are heterotrophic.
To counter the universal tendency towards disorder on a local scale requires energy. As an example, take a fresh deck of cards
which is neatly aligned with Ace-King-Queen . . . . 4,3,2 for each suit. Throw the deck into the air, letting the cards scatter. When
you pick them up, they will be more disordered than when they started. However, if you spend a few minutes (and expend a bit of
energy), you can reorganize the same deck back to its previous, organized state. If entropy always increased everywhere, you could
not do this. However, with the input of energy, you overcame the disorder. This illustrates an important concept: the cost of fighting
disorder is energy.

Biological energy
There are, of course, other reasons that organisms need energy. Muscular contraction, synthesis of molecules, neurotransmission,
signaling, thermoregulation, and subcellular movements are examples. Where does this energy come from? The currencies of
energy are generally high-energy phosphate-containing molecules. ATP is the best known and most abundant, but GTP is also an
important energy source (energy source for protein synthesis). CTP is involved in synthesis of glycerophospholipids and UTP is
used for synthesis of glycogen and other sugar compounds. In each of these cases, the energy is in the form of potential chemical
energy stored in the multi-phosphate bonds. Hydrolyzing those bonds releases the energy in them.
Of the triphosphates, ATP is the primary energy source, acting to facilitate the synthesis of the others by action of the enzyme
NDPK. ATP is made by three distinct types of phosphorylation – oxidative phosphorylation (in mitochondria),
photophosphorylation (in chloroplasts of plants), and substrate level phosphorylation (in enzymatically catalyzed reactions).

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 Figure 5.1.7 : Mitochondrion

Gibbs free energy in Biology

ATP is generally considered the “storage battery” of cells (See also ‘Molecular Battery Backups for Muscles HERE). In order to
understand how energy is captured, we must first understand Gibbs free energy and in doing so, we begin to see the role of energy
in determining the directions chemical reactions take.
Gibbs free energy may be thought of as the energy available to do work in a thermodynamic system at constant temperature and
pressure. Mathematically, the Gibbs free energy is given as:
G = H– T S (5.1.1)

where H is the enthalpy, T is the temperature in Kelvin, and S is the entropy. At standard temperature and pressure, every system
seeks to achieve a minimum of free energy. Thus, increasing entropy, S , will reduce Gibbs free energy. Similarly, if excess heat is
available (reducing the enthalpy, H ), the free energy can also be reduced.
Cells must work within the laws of thermodynamics, as noted, so all of their biochemical reactions, too, are ruled by these laws.
Now we shall consider energy in the cell. The change in Gibbs free energy (ΔG) for a reaction is crucial, for it, and it alone,
determines whether or not a reaction goes forward.

ΔG = ΔH – T Δ S. (5.1.2)

There are three cases

∆G < 0: the reaction proceeds as written
∆G = 0: the reaction is at equilibrium
∆G > 0: the reaction runs in reverse
For a reaction

aA −
↽⇀
− bB (5.1.3)

(where ‘a’ and ‘b’ are integers and A and B are molecules) at pH 7, ∆G can be determined by the following equation,
b
[B]
′
ΔG = ΔG° + RT ln( ) (5.1.4)
[A]a

For multiple substrate reactions, such as

aA + cC −
↽⇀
− bB + dD (5.1.5)

b d
[B] [D]
′
ΔG = ΔG° + RT ln( ) (5.1.6)
a c
[A] [C ]

The ∆G°’ term is called the change in Standard Gibbs Free energy, which is the change in energy that occurs when all of the
products and reactants are at standard conditions and the pH is 7.0. It is a constant for a given reaction.
In simple terms, we can collect all of the terms of the numerator together and call them {Products} and all of the terms of the
denominator together and call them {Reactants},

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 {Products}
′
ΔG = ΔG° + RT ln( ) (5.1.7)
{Reactants}

For most biological systems, the temperature, T, is a constant for a given reaction. Since ∆G°’ is also a constant for a given
reaction, the ∆G is changed almost exclusively as the ratio of {Products}/{Reactants} changes.

Importance of ∆G°’
If one starts out at standard conditions, where everything except protons is at 1M, the RTln({Products}/{Reactants}) term is zero,
so the ∆G°’ term equals the ∆G, and the ∆G°’ determines the direction the reaction will take (only under those conditions). This is
why people say that a negative ∆G°’ indicates an energetically favorable reaction, whereas a positive ∆G°’ corresponds to an
unfavorable one.
Increasing the ratio of {Products}/{Reactants} causes the value of the natural log (ln) term to become more positive (less negative),
thus making the value of ∆G more positive. Conversely, as the ratio of {Products}/{Reactants} decreases, the value of the natural
log term becomes less positive (more negative), thus making the value of ∆G more negative.

System response to stress

Intuitively, this makes sense and is consistent with Le Chatelier’s Principle – a system responds to stress by acting to alleviate the
stress. If we examine the ∆G for a reaction in a closed system, we see that it will always move to a value of zero (equilibrium), no
matter whether it starts with a positive or negative value.
Another type of free energy available to cells is that generated by electrical potential. For example, mitochondria and chloroplasts
partly use Coulombic energy (based on charge) from a proton gradient across their membranes to provide the necessary energy for
the synthesis of ATP. Similar energies drive the transmission of nerve signals (sodium and potassium gradients) and the movement
of some molecules in secondary active transport processes across membranes (e.g., H+ differential driving the movement of
lactose). From the Gibbs free energy change equation,
ΔG = ΔH – T Δ S (5.1.8)

it should be noted that an increase in entropy will help contribute to a decrease in ∆G. This happens, for example when a large
molecule is being broken into smaller pieces or when the rearrangement of a molecule increases the disorder of molecules around
it. The latter situation arises in the hydrophobic effect, which helps drive the folding of proteins.

Chemical and electrical potential

It is said that absence makes the heart grow fonder. We won’t tackle that philosophical issue here, but we will say that separation
provides potential energy that cells can and do harvest. The lipid bilayer of cell and (in eukaryotic cells) organelle membranes
provide the necessary barrier for separation.
Impermeable to most ions and polar compounds, biological membranes are essential for processes that generate cellular energy.
Consider Figure 5.8. A lipid bilayer separates two solutions with different concentrations of a solute. There is a greater
concentration of negative ions in the bottom and a greater concentration of positive ions on the top.
Whenever there is a difference in concentration of molecules across a membrane, there is said to be a concentration gradient across
it. A difference in concentration of ions across a membrane also creates a charge (or electrical) gradient. Because there is a
difference in both the chemical concentration of the ions and in the charge on the two sides of the membrane, this is described as an
electrochemical gradient (Figures 5.8 -5.10).

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 Figure 5.8: Differences in ion concentration across a membrane give rise to chemical and electrical gradients. Image by Pehr
Jacobson

Figure 5.1.9 : A chemical gradient. Image by Aleia Kim

Figure 5.1.10 : An electrochemical gradient of potassium ions. Image by Aleia Kim

Potential energy
Such gradients function like batteries and contain potential energy. When the potential energy is harvested by cells, they can create
ATP, transmit nerve signals, pump molecules across membranes, and more. It is important, therefore, to understand how to
calculate the potential energy of electrochemical gradients.
First, we consider chemical (solute) gradients. In Figure 5.9, two concentrations of glucose are separated by a lipid bilayer. Let’s
assume C2 be the concentration of glucose inside the cell (bottom) and C1 be the glucose concentration outside (top). The Gibbs
free energy associated with moving glucose in the direction of C2 (into the cell) is given by
∆G = RTln[C2/C1]
To move it in the direction of C1 (to the outside of the cell) the expression would be

ΔG = RT ln[ C1 / C2 ] (5.1.9)

Since C2 is smaller than C1 (i.e., there are fewer glucose molecules inside the cell) then the ∆G is negative and diffusion would be
favored into the cell, if the glucose could traverse the bilayer.
Conversely, if C2 was greater than C1 (more glucose was in the cell than outside) the ∆G would be positive, so movement in the
direction of C2 would not be favored and instead the glucose would tend to move towards C1 , that is, out of the cell.

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If C2 = C1, with the same concentration of glucose inside and outside, then the ∆G would be zero and there would be no net
movement, as the system would be at equilibrium.
In the example above, we considered glucose, which is an uncharged molecule. When ions are involved, their charges must also be
taken into consideration. Figure 5.1.10 depicts a similar situation across a lipid bilayer. In this case, a difference of concentration
and charge exists. There are more positive charges inside the cell than outside.
Using C2 to indicate the concentration of materials inside the cell and C1 for the concentration outside the cell (as before), then the
free energy for movement of an ion from top to bottom is given by the following equation
ΔG = RT ln[ C2 / C1 ] + ZF Δ ψ (5.1.10)

Note here that this equation must take into consideration both the concentration differences and the charge differences. Z refers to
the charge of the transported species, F is the Faraday constant (96,485 Coulombs/mol), and ∆ψ is the electrical potential difference
(voltage difference) across the membrane.
If we were to calculate the ∆G for movement of the potassium ion from top to bottom, it would be positive, since [C2/C1] is greater
than 1 (making for a positive ln term), and the ZF∆ψ is positive because positively charged ions (Z) are moving against a positive
charge gradient given by ∆ψ (greater concentration at the target (bottom) than the starting point (top)). If we were to calculate the
concentration of ions moving from bottom to top, then the ln term would be negative (C2<C1) and the ZF∆ψ would be negative as
well (Z=positive, but ∆ψ negative).

Reduction Potential
In discussing chemical potential, we must also consider reduction potential. Reduction potential measures the tendency of a
chemical to be reduced by electrons. It is also designated by several other names/variables. These include redox potential,
oxidation/reduction potential, ORP, pE, ε, E, and Eh.

Figure 5.1.1 1: Reference electrode for measuring reduction potentials. Image by Pehr Jacobson
Reduction potential is measured in volts, or millivolts. A substance with a higher reduction potential will have a greater tendency to
accept electrons and be reduced. If two substances are mixed in an aqueous solution, the one with the greater (more positive)
reduction potential will tend to take electrons away, thus being reduced, from the one with the lower reduction potential, which
becomes oxidized.
Relative measures
Absolute reduction potentials are difficult to measure, so reduction potentials are typically defined relative to a reference electrode.
In aqueous solutions, reduction potentials are measured as the potential difference between an inert sensing electrode (typically

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platinum) in contact with the test solution and a stable reference electrode (measured as a Standard Hydrogen Electrode: SHE) as
shown in Figure 5.1.11. The standard of reference for measurement is the half-reaction
H+ + e— → ½ H2
The electrode where this reaction occurs (referred to as a half-cell) is given the value of E° (Standard Reduction Potential) of 0.00
volts. The hydrogen electrode is connected via an external circuit to another half cell containing a mixture of the reduced and
oxidized species of another molecule (for example, Fe++ and Fe+++) at 1M each and standard conditions of temperature (25°C) and
pressure (1 atmosphere).
Direction and voltage measured
The direction and magnitude of electron movement is then measured. If the test mixture takes electrons from the hydrogen
electrode, the sign of the voltage is positive and if the direction is reversed, the voltage is negative.
Thus, compounds which have greater affinity for electrons than hydrogen will register a positive voltage and negative voltages
correspond to compounds with lesser affinity for electrons than hydrogen.
Movement of electrons
Under standard conditions, electrons will move from compounds generating lower voltages to ones generating higher (more
positive) voltages. Just as the standard Gibbs free energy change is the Gibbs free energy change under standard conditions, so, too,
is the standard reduction potential E° the reduction potential E under standard conditions.
The actual reduction potential of a half cell will vary with the concentration of each chemical species in the cell. The relationship
between the reduction potential E and the standard reduction potential E° is given by the following equation (also called the Nernst
equation)

where F is the Faraday constant (96,480 J/(Volts*moles), R is the gas constant (8.315 J/(moles*K), n is the number of moles of
electrons being transferred, and T is the absolute temperature in Kelvin.
At 25°C, this equation becomes

As for Gibbs free energy, it is useful to measure values at conditions found in cells. This means doing measurements at pH = 7,
which differs from having all species at 1M.
Adjustment
Because of this adjustment, a slightly different standard reduction potential is defined and we designate it by E°’, just as we defined
a special standard Gibbs free energy change at pH 7 as ΔG°’.
There is a relationship between the change in Gibbs free energy ΔG and the change in reduction potential (ΔE). It is

ΔG = −nF ΔE (5.1.11)

Similarly, the relation between the change in standard Gibbs free energy and the change in standard reduction potential is
\]ΔG°’ = -nFΔE°’\]

Energy Storage in Triphosphates

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 Movie 5.1: ATP: The fuel of the cell
Formation of triphosphates, like ATP, is essential to meeting the cell’s energy needs for synthesis, motion, and signaling. In a given
day, an average human body makes and breaks down more than its weight in triphosphates. This is especially remarkable
considering that there is only about 250 g of the molecule present in the body at any given time. Energy in ATP is released by
hydrolysis of a phosphate from the molecule.

Figure 5.1.1 2 ATP showing α, β and γ phosphates

The three phosphates, starting with the one closest to the sugar are referred to as α, β, and γ (Figure 5.1.12). It is the γ phosphate
that is cleaved in hydrolysis and the product is ADP. In a few reactions, the bond between the α and β is cleaved. When this
happens, a pyrophosphate (β linked to γ) is released and AMP is produced. This latter reaction to produce AMP releases more
energy (ΔG°’ = -45.6 kJ/mol) than the first reaction which produces ADP (ΔG°’ = -30.5 kJ/mol).
Since triphosphates are the “currency” that meet immediate needs of the cell, it is important to understand how triphosphates are
made. There are three phosphorylation mechanisms – 1) substrate level; 2) oxidative; and 3) photophosphorylation. We consider
them here individually.

Figure 5.1.1 3: Nucleotides, nucleosides, and bases

Substrate level phosphorylation

The easiest type of phosphorylation to understand is that which occurs at the substrate level. This type of phosphorylation involves
the direct synthesis of ATP from ADP and a high energy intermediate, typically a phosphate-containing molecule. Substrate level
phosphorylation is a relatively minor contributor to the total synthesis of triphosphates by cells. An example substrate
phosphorylation comes from glycolysis.
Phosphoenolpyruvate (PEP) + ADP ⇌ Pyruvate + ATP
This reaction has a very negative ∆G°’ (-31.4 kJ/mol), indicating that the PEP contains more energy than ATP, thus tending to
energetically favor ATP’s synthesis. Other triphosphates can be made by substrate level phosphorylation, as well. For example,
GTP can be synthesized by the following citric acid cycle reaction.
Succinyl-CoA + GDP + Pi ⇌ Succinate + GTP + CoA-SH

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Triphosphates can be interchanged readily in substrate level phosphorylations catalyzed by the enzyme Nucleoside Diphosphate
Kinase (NDPK). A generalized form of the reactions catalyzed by this enzyme is as follows:
XTP + YDP ⇌ XDP + YTP
where X = adenosine, cytidine, uridine, thymidine, or guanosine and Y can be any of these as well. Further, XTP and YDP can be
any of the deoxynucleotides as well.
Last, an unusual way of synthesizing ATP by substrate level phosphorylation is via the reaction catalyzed by adenylate kinase
2 ADP ⇌ ATP + AMP

ATP source
This reaction is an important means of generating ATP when the cell doesn’t have other sources of energy. Accumulation of AMP
resulting from this reaction activates enzymes, such as phosphofructokinase, of glycolysis, which will catalyze reactions to give the
cell additional, needed energy.
It is important to note that enzymes cannot make reactions happen that are energetically unfavorable. Enzymes speed reactions, but
do not change their direction. Cells are thus bound by the rules of Gibbs free energy. So, how do energetically unfavorable
reactions happen in a cell?

Reaction coupling
Reactions that are energetically unfavorable, can be made favorable by coupling them with the hydrolysis of ATP, a very
energetically favorable reaction. There are numerous parallels in the “real world.” Movement of automobiles is energetically
unfavorable, but coupling movement of the automobile to oxidation of gasoline makes an unfavorable process favorable. Another
approach to making an unfavorable reaction favorable is to manipulate the concentration of reactants and products. Consider the
reaction below, which occurs in pyrimidine nucleotide metabolism:
orotate + PRPP ⇌ OMP + PPi
The ΔG°’ for this reaction is -0.8 kJ/mol, meaning that if one starts with equal concentrations of reactants and products, at
equilibrium, there will be a small excess of products. In the cell, however, this reaction moves strongly to the right (ΔG = very
negative). Given that the ΔG°’ is very close to zero, a very negative ΔG can only occur if the concentrations of reactants and
products are altered, since

′
[OMP][PPi ]
ΔG = ΔG° + RT ln( ) (5.1.12)
[Orotate][PRPP]

Manipulation is exactly what happens here. The key item whose concentration is adjusted in this reaction is the pyrophosphate
(PPi). This is possible because cells contain an enzyme called pyrophosphorylase that catalyzes the following reaction
PPi + H2O ⇌ 2 Pi
Hydrolysis of pyrophosphate is very energetically favored, causing the PPi produced in the reaction to be quickly hydrolyzed. As a
result, the concentration of PPi in the cell is kept very low. A low concentration of a product (PPi) causes the natural log (ln) term
of the orotate equation to become more negative, driving the ΔG term for the overall reaction to become much more negative.

Pushing and pulling

Reactions that yield pyrophosphate as a product are produced in the synthesis of DNA and RNA, as well as many other molecules.
As shown in the previous example, this pyrophosphate is rapidly hydrolyzed, causing the overall reaction to move in the direction
of pyrophosphate production. When reactants are removed/reduced in a metabolic reaction to decrease the concentration of a
product, we say that the reaction is “pulled”, to represent the increase in the forward reaction as a result of product depletion.
Pushing happens when reactants in a reaction are added/increased. This too has the effect of reducing the ΔG of a reaction and
making it more favorable because the ratio of [Products]/[Reactants] is decreased with increasing [Reactants]. Pushing and pulling
of reactions are additional tools for cells to overcome energy barriers, just like coupling energetically favorable processes to
energetically unfavorable ones.

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This page titled 5.1: Basics of Energy is shared under a CC BY-NC-SA license and was authored, remixed, and/or curated by Kevin Ahern, Indira
Rajagopal, & Taralyn Tan.

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5.3: Energy - Photophosphorylation
Source: BiochemFFA_5_3.pdf. The entire textbook is available for free from the authors at
http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy

Photophosphorylation
The third type of phosphorylation to make ATP is found only in cells that carry out photosynthesis. This process is similar to
oxidative phosphorylation in several ways. A primary difference is the ultimate source of the energy for ATP synthesis. In oxidative
phosphorylation, the energy comes from electrons produced by oxidation of biological molecules. In photosynthesis, the energy
comes from the light of the sun. Photons from the sun interact with chlorophyll molecules in reaction centers in the chloroplasts
(Figures 5.3.1 and 5.3.2) of plants or membranes of photosynthetic bacteria.
The similarities of photophosphorylation to oxidative phosphorylation include:
a membrane associated electron transport chain
creation of a proton gradient
harvesting energy of the proton gradient by making ATP with the help of an ATP synthase.
Some of the differences include :
the source of the electrons – H2O for photosynthesis versus NADH/FADH2 for oxidative phosphorylation
direction of proton pumping – into the thylakoid space of the chloroplasts versus outside the matrix of the mitochondrion
movement of protons during ATP synthesis – out of the thylakoid space in photosynthesis versus into the mitochondrial matrix
in oxidative phosphorylation
nature of the terminal electron acceptor – NADP+ in photosynthesis versus O2 in oxidative phosphorylation.

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 Figure 5.3.2 : The chloroplast. Image by Aleia Kim

Electron transport: chloroplasts vs mitochondria

In some ways, the movement of electrons in chloroplasts during photosynthesis is opposite that of electron transport in
mitochondria. In photosynthesis, water is the source of electrons and their final destination is NADP+ to make NADPH. In
mitochondria, NADH/FADH2 are electron sources and H2O is their final destination. How do biological systems get electrons to
go both ways? It would seem to be the equivalent of going to and from a particular place while always going downhill, since
electrons will move according to potential.

Solar power

Figure 5.3.3 : Overview of photosynthesis. Wikipedia

The answer is the captured energy of the photons from the sun (Figure 5.59), which elevates electrons to an energy where they
move “downhill” to their NADPH destination in a Z-shaped scheme. The movement of electrons through this scheme in plants

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requires energy from photons in two places to “lift” the energy of the electrons sufficiently.
Last, it should be noted that photosynthesis actually has two phases, referred to as the light cycle (described above) and the dark
cycle, which is a set of chemical reactions that captures CO2 from the atmosphere and “fixes” it, ultimately into glucose. The dark
cycle is also referred to as the Calvin Cycle and is discussed HERE.

Figure 5.3.4 : Chloroplast anatomy. Wikipedia

Photosynthesis
Photosynthesis is an energy capture process found in plants and other organisms to harvest light energy and convert it into chemical
energy. This photochemical energy is stored ultimately in carbohydrates which are made using ATP (from the energy harvesting),
carbon dioxide and water. In most cases, a byproduct of the process is oxygen, which is released from water in the capture process.
Photosynthesis is responsible for most of the oxygen in the atmosphere and it supplies the organic materials and most of the energy
used by life on Earth.

Steps
The steps in the photosynthesis process varies slightly between organisms. In a broad overview, it always starts with energy capture
from light by protein complexes, containing chlorophyll pigments, called reaction centers. Plants sequester these proteins in
chloroplasts, but bacteria, which don’t have organelles, embed them in their plasma membranes.
Energy from the light is used to strip electrons away from electron donors (usually water) and leave a byproduct (oxygen, if water
was used). Electrons are donated to a carrier and ultimately are accepted by NADP+, to become NADPH. As electrons travel
towards NADP+, they generate a proton gradient across the thylakoid membrane, which is used to drive synthesis of ATP. Thus
NADPH, ATP, and oxygen are the products of the first phase of photosynthesis called the light reactions. Energy from ATP and
electrons from NADPH are used to reduce CO2 and build sugars, which are the ultimate energy storage directly arising from
photosynthesis.

Chloroplasts
Chloroplasts are found in almost all aboveground plant cells, but are primarily concentrated in leaves. The interior of a leaf, below
the epidermis is made up of photosynthesis tissue called mesophyll, which can contain up to 800,000 chloroplasts per square
millimeter.
The chloroplast’s membrane has a phospholipid inner membrane, a phospholipid outer membrane, and a region between them
called the intermembrane space (Figure 5.61). Within the inner chloroplast membrane is the stroma, in which the chloroplast DNA
and the enzymes of the Calvin cycle are located. Also within the stroma are stacked, flattened disks known as thylakoids which are
defined by their thylakoid membranes. The space within the thylakoid membranes are termed the thylakoid spaces or thylakoid
lumen. The protein complexes containing the light-absorbing pigments, known as photosystems, are located on the thylakoid
membrane. Besides chlorophylls, carotenes and xanthophylls are also present, allowing for absorption of light energy over a wider
range. The same pigments are used by green algae and land plants.
Brown algae and diatoms add fucoxanthin (a xanthophyll) and red algae add phycoerythrin to the mix. In plants and algae, the
pigments are held in a very organized fashion complexes called antenna proteins that help funnel energy, through resonance energy
transfer, to the reaction center chlorophylls. A system so organized is called a light harvesting complex. The electron transport
complexes of photosynthesis are also located on the thylakoid membranes.

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 Figure 5.3.5 : Side view of thylakoids. Wikipedia

Figure 5.3.6 : Complexes in the thylakoid membrane. Image by Aleia Kim

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Light reactions of photosynthesis
In chloroplasts, the light reactions of photosynthesis involving electron transfer occur in the thylakoid membranes (Figure 5.3.6).
Separate biochemical reactions involving the assimilation of carbon dioxide to make glucose are referred to as the Calvin cycle,
also sometimes referred to as the “dark reactions”. This will be discussed elsewhere in the section on metabolism (HERE).
The chloroplasts are where the energy of light is captured, electrons are stripped from water, oxygen is liberated, electron transport
occurs, NADPH is formed, and ATP is generated. The thylakoid membrane corresponds to the inner membrane of the
mitochondrion for transport of electrons and proton pumping (Figure 5.3.4).
The thylakoid membrane does its magic using four major protein complexes. These include Photosystem II (PS II), Cytochrome
b6f complex (Cb6f), Photosystem I (PS I), and ATP synthase. The roles of these complexes, respectively, are to capture light
energy, create a proton gradient from electron movement, capture light energy (again), and use proton gradient energy from the
overall process to synthesize ATP.

Figure 5.3.7 : Movement of electrons through photosystems. Cyclic photophosphorylation shown by blue dashed line. Image by
Aleia Kim and Pehr Jacobson

Light harvesting
Harvesting the energy of light begins in PS II with the absorption of a photon of light at a reaction center. PS II performs this duty
best with light at a wavelength of 680 nm and it readily loses an electron to excitation when this occurs, leaving PS II with a
positive charge. This electron must be replaced. The ultimate replacement source of electrons is water, but water must lose four
electrons and PS II can only accept one at a time.

Manganese centers
An intermediate Oxygen Evolving Complex (OEC) contains four manganese centers that provide the immediate replacement
electron that PSII requires. After four electrons have been donated by the OEC to PS II, the OEC extracts four electrons from two
water molecules, liberating oxygen and dumping four protons into the thylakoid space, thus contributing to the proton gradient. The
excited electron from PS II must be passed to another carrier very quickly, lest it decay back to its original state. It does this, giving
its electron within picoseconds to pheophytin (Figure 5.3.8).
Pheophytin passes the electron on to protein-bound plastoquinones . The first is known as PQA. PQA hands the electron off to a
second plastoquinone (PQB), which waits for a second electron and collects two protons to become PQH2, also known as
plastoquinol (Figure 5.3.9). PQH2 passes these to the Cytochrome b6f complex (Cb6f) which uses passage of electrons through it
to pump protons into the thylakoid space. ATP synthase makes ATP from the proton gradient created in this way. Cb6f drops the

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electron off at plastocyanin, which holds it until the next excitation process begins with absorption of another photon of light at 700
nm by PS I.

Absorption of light at PS I
With absorption of a photon of light by PS I, a process begins, that is similar to the process in PS II. PS I gains a positive charge as
a result of the loss of an excited electron and pulls the electron in plastocyanin away from it. Meanwhile, the excited electron from
PS I passes through an iron-sulfur protein, which gives the electron to ferredoxin (another iron sulfur protein). Ferredoxin then
passes the electron off to the last protein in the system known as Ferredoxin:NADP+ oxidoreductase, which gives the electron and
a proton to NADP+, creating NADPH.
Note that reduction of NADP+ to NADPH requires two electrons and one proton, so the four electrons and two protons from
oxidation of water will result in production of two molecules of NADPH. At this point, the light cycle is complete - water has been
oxidized, ATP has been created, and NADPH has been made. The electrons have made their way from water to NADPH via
carriers in the thylakoid membrane and their movement has released sufficient energy to make ATP. Energy for the entire process
came from four photons of light.
The two photosystems performing all of this magic are protein complexes that are similar in structure and means of operation. They
absorb photons with high efficiency so that whenever a pigment in the photosynthetic reaction center absorbs a photon, an electron
from the pigment is excited and transferred to another molecule almost instantaneously. This reaction is called photo-induced
charge separation and it is a unique means of transforming light energy into chemical forms.

Cyclic photophosphorylation
Besides the path described above for movement of electrons through PS I, plants have an alternative route that electrons can take.
Instead of electrons going through ferredoxin to form NADPH, they instead take a backwards path through the the proton-pumping
b6f complex. This system, called cyclic photophosphorylation (Figure 5.3.8) which generates more ATP and no NADPH, is similar
to a system found in green sulfur bacteria. The ability of plants to switch between non-cyclic and cyclic photosystems allows them
to make the proper ratio of ATP and NADPH they need for assimilation of carbon in the dark phase of photosynthesis. This ratio
turns out to be 3 ATPs to 2 NADPHs.

Figure 5.3.8 - Movement of electrons and protons through the thylakoid membrane. Image by Aleia Kim

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 Figure 5.3.9 - Photosystem II of cyanobacteria. Wikipedia

Photosynthetic energy
The output of the photophosphorylation part of photosynthesis (O2, NADPH, and ATP), of course, is not the end of the process of
photosynthesis. For the growing plant, the NADPH and ATP are used to capture carbon dioxide from the atmosphere and convert it
(ultimately) into glucose and other important carbon compounds. This, as noted previously, occurs in the Calvin Cycle (see HERE)
in what is called the dark phase of the process. The oxygen liberated in the process is a necessary for respiration of all aerobic life
forms on Earth. Indeed, it is believed that essentially all of the oxygen in the atmosphere today is the result the splitting of water in
photosynthesis over the many eons that the process has existed.

This page titled 5.3: Energy - Photophosphorylation is shared under a CC BY-NC-SA license and was authored, remixed, and/or curated by Kevin
Ahern, Indira Rajagopal, & Taralyn Tan.

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5.2: Electron Transport and Oxidative Phosphorylation
Source: BiochemFFA_5_2.pdf. The entire textbook is available for free from the authors at
http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy

In eukaryotic cells, the vast majority of ATP synthesis occurs in the mitochondria in a process called oxidative phosphorylation.
Even plants, which generate ATP by photophosphorylation in chloroplasts, contain mitochondria for the synthesis of ATP through
oxidative phosphorylation.
Oxidative phosphorylation is linked to a process known as electron transport (Figure 5.14). The electron transport system, located
in the inner mitochondrial membrane, transfers electrons donated by the reduced electron carriers NADH and FADH2 (obtained
from glycolysis, the citric acid cycle or fatty acid oxidation) through a series of electrons acceptors, to oxygen. As we shall see,
movement of electrons through complexes of the electron transport system essentially “charges” a battery that is used to make ATP
in oxidative phosphorylation. In this way, the oxidation of sugars and fatty acids is coupled to the synthesis of ATP, effectively
extracting energy from food.

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Chemiosmotic model
Dr. Peter Mitchell introduced a radical proposal in 1961 to explain the mechanism by which mitochondria make ATP. It is known
as the chemiosmotic hypothesis and has been shown over the years to be correct. Mitchell proposed that synthesis of ATP in
mitochondria depends on an electrochemical gradient, across the mitochondrial inner membrane, that arises ultimately from the
energy of reduced electron carriers, NADH and FADH2.

Electron transport
Further, the proposal states that the gradient is created when NADH and FADH2 transfer their electrons to an electron transport
system (ETS) located in the inner mitochondrial membrane. Movement of electrons through a series of of electron carriers is
coupled to the pumping of protons out of the mitochondrial matrix across the inner mitochondrial membrane into the space
between the inner and outer membranes. The result is creation of a gradient of protons whose potential energy can be used to make
ATP. Electrons combine with oxygen and protons at the end of the ETS to make water.

ATP synthase
In oxidative phosphorylation, ATP synthesis is accomplished as a result of protons re-entering the mitochondrial matrix via the
transmembrane ATP synthase complex, which combines ADP with inorganic phosphate to make ATP. Central to the proper
functioning of mitochondria through this process is the presence of an intact mitochondrial inner membrane impermeable to
protons.

Tight coupling
When this is the case, tight coupling is said to exist between electron transport and the synthesis of ATP (called oxidative
phosphorylation). Chemicals which permeabilize the inner mitochondrial membrane to protons cause uncoupling, that is, they
allow the protons to leak back into the mitochondrial matrix, rather than through the ATP synthase, so that the movement of
electrons through the ETS is no longer linked to the synthesis of ATP.

Power plants
Mitochondria are called the power plants of the cell because most of a cell’s ATP is produced there in the process of oxidative
phosphorylation. The mechanism by which ATP is made in oxidative phosphorylation is one of the most interesting in all of
biology.

Considerations
The process has three primary considerations. The first is electrical – electrons from reduced electron carriers, such as NADH and
FADH2, enter the electron transport system via Complex I and II, respectively. As seen in Figure 5.16 and Figure 5.17, electrons
move from one complex to the next, not unlike the way they move through an electrical circuit. Such movement occurs a a result of
a set of reduction-oxidation (redox) reactions with electrons moving from a more negative reduction potential to a more positive
one.

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One can think of this occurring as a process where carriers “take” electrons away from complexes with lower reduction potential,
much the way a bully takes lunch money from a smaller child. In this scheme, the biggest “bully” is oxygen in Complex IV.
Electrons gained by a carrier cause it to be reduced, whereas the carrier giving up the electrons is oxidized.

Entry of electrons to system

Movement of electrons through the chain begins either by 1) transfer from NADH to Complex I (Figure 5.16) or 2) movement of
electrons through a covalently bound FADH2 (Figure 5.17) in the membrane-bound succinate dehydrogenase (Complex II). (An
alternate entry point for electrons from FADH2 is the Electron Transferring Flavoprotein via the electron-transferring-flavoprotein
dehydrogenase, not shown).

Traffic cop
Both Complex I and II pass electrons to the inner membrane’s coenzyme Q (CoQ - Figures 5.18 & 5.19). In each case, coenzyme Q
accepts electrons in pairs and passes them off to Complex III (CoQH2-cytochrome c reductase) singly. Coenzyme Q thus acts as a
traffic cop, regulating the flow of electrons through the ETS.

Docking station
Complex III is a docking station or interchange for the incoming electron carrier (coenzyme Q) and the outgoing carrier
(cytochrome c). Movement of electrons from Coenzyme Q to Complex III and then to cytochrome C occurs as a result of what is
referred to as the Q-cycle (see below).
Complex III acts to ferry electrons from CoQ to cytochrome c. Cytochrome c takes one electron from Complex III and passes it to
Complex IV (cytochrome oxidase). Complex IV is the final protein recipient of the electrons. It passes them to molecular oxygen

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(O2) to make two molecules of water. Making two water molecules requires four electrons, so Complex IV must accept, handle,
and pass to molecular oxygen four separate electrons, causing the oxidation state of oxygen to be sequentially changed with
addition of each electron.

Proton pumping
As electrons pass through complexes I, III, and IV, there is a release of a small amount of energy at each step, which is used to
pump protons from the mitochondrial matrix (inside of mitochondrion) and deposit them in the intermembrane space (between the
inner and outer membranes of the mitochondrion). The effect of this redistribution is to increase the electrical and chemical
potential across the membrane.

Potential energy
As discussed earlier, electrochemical gradients have potential energy. Students may think of the process as “charging the battery.”
Just like a charged battery, the potential arising from the proton differential across the membrane can be used to do things. In the
mitochondrion, what the proton gradient does is facilitate the production of ATP from ADP and Pi. This process is known as
oxidative phosphorylation, because the phosphorylation of ADP to ATP is dependent on the oxidative reactions occurring in the
mitochondria.

Having understood the overall picture of the synthesis of ATP linked to the movement of electrons through the ETS, we will take a
closer look at the individual components of the ETS.

Complex I
Complex I (also called NADH:ubiquinone oxidoreductase or NADH dehydrogenase (ubiquinone)) is the electron acceptor from
NADH in the electron transport chain and the largest complex found in it.
Complex I contains 44 individual polypeptide chains, numerous iron-sulfur centers, a molecule of flavin mononucleotide (FMN)
and has an L shape with about 60 transmembrane domains. In the process of electron transport through it, four protons are pumped
across the inner membrane into the intermembrane space and electrons move from NADH to coenzyme Q, converting it from
ubiquinone (no electrons) to ubiquinol (gain of two electrons). An intermediate form, ubisemiquinone (gain of one electron), is
found in the Q-cycle.
Electrons travel through the complex via seven primary iron sulfur centers. The best known inhibitor of the complex, rotenone,
works by binding to the CoQ binding site. Other inhibitors include ADP-ribose (binds to the NADH site) and piericidin A
(rotenone analog). The process of electron transfer through complex I is reversible and when this occurs, superoxide (a reactive
oxygen species) may be readily generated.

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Complex II
Complex II (also called succinate dehydrogenase or succinate-coenzyme Q reductase ) is a membrane bound enzyme of the citric
acid cycle that plays a role in the electron transport process, transferring electrons from its covalently bound FADH2 to coenzyme
Q. The process occurs, as shown in Figure 5.20 and Figure 5.21, with transfer of electrons from succinate to FAD to form FADH2
and fumarate. FADH2, in turn, donates electrons to a relay system of iron-sulfur groups and they ultimately reduce ubiquinone
(CoQ) along with two protons from the matrix to ubiquinol. The role of the heme group in the process is not clear. Inhibitors of the
process include carboxin, malonate, malate, and oxaloacetate. The role of citric acid cycle intermediates as inhibitors is thought to
be due to inhibition of the reversal of the transfer process which can produce superoxide.

Coenzyme Q
Coenzyme Q (Figure 5.23) is a 1,4 benzoquinone whose name is often given as Coenzyme Q10, CoQ, or Q10. The 10 in the name
refers to the number of isoprenyl units it contains that anchor it to the mitochondrial inner membrane. CoQ is a vitamin-like lipid
substance found in most eukaryotic cells as a component of the electron transport system. The requirement for CoQ increases with
increasing energy needs of cells, so the highest concentrations of CoQ in the body are found in tissues that are the most
metabolically active - heart, liver, and kidney.
Three forms
CoQ is useful because of its ability to carry and donate electrons and particularly because it can exist in forms with two extra
electrons (fully reduced - ubiquinol), one extra electron (semi-reduced - ubisemiquinone), or no extra electrons (fully oxidized -
ubiquinone). This ability allows CoQ to provide transition between the first part of the electron transport system that moves
electrons in pairs and the last part of the system that moves electrons one at a time.

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Complex III
Complex III (also known as coenzyme Q : cytochrome c — oxidoreductase or the cytochrome bc1 complex - Figure 5.24) is the
third electron accepting complex of the electron transport system. It is a transmembrane protein with multiple subunits present in
the mitochondria of all aerobic eukaryotic organisms and and the cell membrane of almost all bacteria. The complex contains 11
subunits, a 2-iron ferredoxin, cytochromes b and c1 and belongs to the family of oxidoreductase enzymes.
It accepts electrons from coenzyme Q in electron transport and passes them off to cytochrome c. In this cycle, known as the Q
cycle, electrons arrive from CoQ in pairs, but get passed to cytochrome c individually. In the overall process, two protons are
consumed from the matrix and four protons are pumped into the intermembrane space. Movement of electrons through the complex
can be inhibited by antimycin A, myxothiazol, and stigmatellin. Complex III is also implicated in creation of superoxide (a reactive
oxygen species) when electrons from it leak out of the chain of transfer. The phenomenon is more pronounced when antimycin A is
present.

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Q-cycle
In the Q-cycle, electrons are passed from ubiquinol (QH2) to cytochrome c using Complex III as an intermediary docking station
for the transfer. Two pair of electrons enter from QH2 and one pair is returned to another CoQ to re-make QH2. The other pair is
donated singly to two different cytochrome c molecules.

Step one
The Q-cycle happens in a two step process. First, a ubiquinol (CoQH2) and a ubiquinone (CoQ) dock at Complex III. Ubiquinol
transfers two electrons to Complex III. One electron goes to a docked cytochrome c, reducing it and it exits (replaced by an
oxidized cytochrome c). The other goes to the docked uniquinone to create the semi-reduced semiubiquinone (CoQ.-) and leaving
behind a ubiquinone, which exits. This is the end of step 1.

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Step two
The gap left behind by the ubiquinone (Q) that departed is replaced by another ubiquinol (QH2). It too donates two electrons to
Complex III, which splits them. One goes to the newly docked oxidized cytochrome c, which is reduced and exits. The other goes
to the ubisemiquinone. Two protons from the matrix combine with it to make another ubiquinol. It and the ubiquinone created by
the electron donation exit Complex III and the process starts again. In the overall process, two protons are consumed from the
matrix and four protons are pumped into the intermembrane space.

Cytochrome c
Cytochrome c (Figure 5.26) is a small (12,000 Daltons), highly conserved protein, from unicellular species to animals, that is
loosely associated with the inner mitochondrial membrane where it functions in electron transport. It contains a heme group which
is used to carry a single electron from Complex III to Complex IV. Cytochrome c also plays an important role in apoptosis in higher
organisms. Damage to the mitochondrion that results in release of cytochrome c can stimulate assembly of the apoptosome and
activation of the caspase cascade that leads to programmed cell death.

Complex IV
Complex IV, also known as cytochrome c oxidase is a 14 subunit integral membrane protein at the end of the electron transport
chain (Figure 5.27). It is responsible for accepting one electron each from four cytochrome c proteins and adding them to molecular
oxygen (O2) along with four protons from the mitochondrial matrix to make two molecules of water. Four protons from the matrix
are also pumped into the intermembrane space in the process. The complex has two molecules of heme, two cytochromes (a and
a3), and two copper centers (called CuA ad CuB). Cytochrome c docks near the CuA and donates an electron to it. The reduced
CuA passes the electron to cytochrome a, which turns it over to the a3-CuB center where the oxygen is reduced. The four electrons
are thought to pass through the complex rapidly resulting in complete reduction of the oxygen-oxygen molecule without formation
of a peroxide intermediate or superoxide, in contrast to previous predictions.

Respirasome
There has been speculation for many years that a supercomplex of electron carriers in the inner membrane of the mitochondrion
may exist in cells with individual carriers making physical contact with each other. This would make for more efficient transfer
reactions, minimize the production of reactive oxygen species and be similar to metabolons of metabolic pathway enzymes, for

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which there is some evidence. Now, evidence appears to be accumulating that complexes I, III, and IV form a supercomplex, which
has been dubbed the respirasome1.

Oxidative phosphorylation
The process of oxidative phosphorylation uses the energy of the proton gradient established by the electron transport system as a
means of phosphorylating ADP to make ATP. The establishment of the proton gradient is dependent upon electron transport. If
electron transport stops or if the inner mitochondrial membrane’s impermeability to protons is compromised, oxidative
phosphorylation will not occur because without the proton gradient to drive the ATP synthase, there will be no synthesis of ATP.

ATP synthase
The protein complex harvesting energy from the proton gradient and using it to make ATP from ADP is an enzyme that has several
names - Complex V, PTAS (Proton Translocating ATP Synthase), and ATP synthase (Figure 5.29). Central to its function is the
movement of protons through it (from the intermembrane space back into the matrix). Protons will only provide energy to make
ATP if their concentration is greater in the intermembrane space than in the matrix and if ADP is available.
It is possible, in some cases, for the concentration of protons to be greater inside the matrix than outside of it. When this happens,
the ATP synthase can run backwards, with protons moving from inside to out, accompanied by conversion of ATP to ADP + Pi.
This is usually not a desirable circumstance and there are some controls to reduce its occurrence.
Normally, ATP concentration will be higher inside of the mitochondrion and ADP concentration be higher outside the
mitochondrion. However, when the rate of ATP synthesis exceeds the rate of ATP usage, then ATP concentrations rise outside the
mitochondrion and ADP concentrations fall everywhere.
This may happen, for example, during periods of rest. It has the overall effect of reducing transport and thus lowering the
concentration of ADP inside the matrix. Reducing ADP concentration in the matrix reduces oxidative phosphorylation and has
effects on respiratory control (see HERE).
Another important consideration is that when ATP is made in oxidative phosphorylation, it is released into the mitochondrial
matrix, but must be transported into the cytosol to meet the energy needs of the rest of the cell. This is accomplished by action of
the adenine nucleotide translocase, an antiport that moves ATP out of the matrix in exchange for ADP moving into the matrix. This
transport system is driven by the concentrations of ADP and ATP and ensures that levels of ADP are maintained within the
mitochondrion, permitting continued ATP synthesis.
One last requirement for synthesis of ATP from ADP is that phosphate must also be imported into the matrix. This is accomplished
by action of the phosphate translocase, which is a symport that moves phosphate into the mitochondrial matrix along with a proton.
There is evidence that the two translocases and ATP synthase may exist in a complex, which has been dubbed the ATP synthasome.

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In summary, the electron transport system charges the battery for oxidative phosphorylation by pumping protons out of the
mitochondrion. The intact inner membrane of the mitochondrion keeps the protons out, except for those that re-enter through ATP
Synthase. The ATP Synthase allows protons to re-enter the mitochondrial matrix and harvests their energy to make ATP.

ATP synthase mechanism

Figure 5.32 illustrate the multi-subunit nature of this membrane protein, which acts like a turbine at a hydroelectric dam. The
movement of protons through the ATP Synthase c-ring causes it and the γ-ε stalk attached to it to turn. It is this action that is
necessary for making ATP.
In ATP Synthase, the spinning components, or rotor, are the membrane portion (c ring) of the F0 base and the γ-ε stalk, which is
connected to it. The γ-ε stalk projects into the F1 head of the mushroom structure. The F1 head contains the catalytic ability to
make ATP. The F1 head is hexameric in structure with paired α and β proteins arranged in a trimer of dimers. ATP synthesis occurs
within the β subunits.

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Rotation of γ unit
Turning of the γ shaft (caused by proton flow) inside the α-β trimer of the F1 head causes each set of β proteins to change structure
slightly into three different forms called Loose, Tight, and Open (L,T,O - Figure 5.31). Each of these forms has a function.
The Loose form binds ADP + Pi. The Tight form “squeezes” them together to form the ATP. The Open form releases the ATP into
the mitochondrial matrix. Thus, as a result of the proton flow through the ATP synthase, from the intermembrane space into the
matrix, ATP is made from ADP and Pi.

Respiratory control
When a mitochondrion has an intact inner membrane and protons can only return to the matrix by passing through the ATP
synthase, the processes of electron transport and oxidative phosphorylation are said to be tightly coupled.
Interdependence
In simple terms, tight coupling means that the processes of electron transport and oxidative phosphorylation are interdependent.
Without electron transport going on in the cell, oxidative phosphorylation will soon stop.
The reverse is also true, because if oxidative phosphorylation stops, the proton gradient will not be dissipated as it is being built by
the electron transport system and will grow larger and larger. The greater the gradient, the greater the energy needed to pump
protons out of the mitochondrion. Eventually, if nothing relieves the gradient, it becomes too large and the energy of electron
transport is insufficient to perform the pumping. When pumping stops, so too does electron transport.
ADP dependence
Another relevant point is that ATP synthase is totally dependent upon a supply of ADP. In the absence of ADP, the ATP synthase
stops functioning and when it stops, so too does movement of protons back into the mitochondrion. With this information, it is
possible to understand the link between energy usage and metabolism. The root of this, as noted, is respiratory control.
At rest
To illustrate these links, let us first consider a person, initially at rest, who then suddenly jumps up and runs away. At first, the
person’s ATP levels are high and ADP levels are low (no exercise to burn ATP), so little oxidative phosphorylation is occurring and
thus the proton gradient is high. Electron transport is moving slowly, if at all, so it is not using oxygen and the person’s breathing is
slow, as a result.
Exercise
When running starts, muscular contraction, which uses energy, causes ATP to be converted to ADP. Increasing ADP in muscle cells
favors oxidative phosphorylation to attempt to make up for the ATP being burned. ATP synthase begins working and protons begin
to come back into the mitochondrial matrix. The proton gradient decreases, so electron transport re-starts.
Electron transport needs an electron acceptor, so oxygen use increases and when oxygen use increases, the person starts breathing
more heavily to supply it. When the person stops running, ATP concentrations get rebuilt by ATP synthase. Eventually, when ATP
levels are completely restored, ADP levels fall and ATP synthase stops or slows considerably. With little or no proton movement,
electron transport stops because the proton gradient is too large. When electron transport stops, oxygen use decreases and the rate
of breathing slows down.
Electron transport critical
The really interesting links to metabolism occur relative to whether or not electron transport is occurring. From the examples, we
can see that electron transport will be relatively slowed when not exercising and more rapid when exercise (or other ATP usage) is

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occurring. Remember that electron transport is the way in which reduced electron carriers, NADH and FADH2, donate their
electrons to the ETS , becoming oxidized to NAD+ and FAD, respectively.
Oxidized carriers, such as NAD+ and FAD are needed by catabolic pathways, like glycolysis, the citric acid cycle, and fatty acid
oxidation. Anabolic pathways, such as fatty acid/fat synthesis and gluconeogenesis rely on reduced electron carriers, such as
FADH2, NADH, and the related carrier, NADPH.

Links to exercise
High levels of NADH and FADH2 prevent catabolic pathways from operating, since NAD+ and FAD levels will be low and these
are needed to accept the electrons released during catabolism by the oxidative processes.
Thanks to respiratory control, when one is exercising, NAD+ and FAD levels increase (because electron transport is running), so
catabolic pathways that need NAD+ and FAD can function. The electrons lost in the oxidation reactions of catabolism are captured
by NAD+ and FAD to yield NADH and FADH2, which then supply electrons to the electron transport system and oxidative
phosphorylation to make more needed ATP.
Thus, during exercise, cells move to a mode of quickly cycling between reduced electron carriers (NADH/FADH2) and oxidized
electron carriers (NAD+/FAD). This allows rapidly metabolizing tissues to transfer electrons to NAD+/FAD and it allows the
reduced electron carriers to rapidly become oxidized, allowing the cell to produce ATP.
Rest
When exercise stops, NADH and FADH2 levels rise (because electron transport is slowing) causing catabolic pathways to
slow/stop. If one does not have the proper amount of exercise, reduced carriers remain high in concentration for long periods of
time. This means we have an excess of energy and then anabolic pathways, particularly fatty acid synthesis, are favored, so we get
fatter.

Altering respiratory control

One might suspect that altering respiratory control could have some very dire consequences and that would be correct. Alterations
can take the form of either inhibiting electron transport/oxidative phosphorylation or uncoupling the two . These alterations can be
achieved using compounds with specific effects on particular components of the system.
All of the chemicals described here are laboratory tools and should never be used by people. The first group for discussion are the
inhibitors. In tightly coupled mitochondria, inhibiting either electron transport or oxidative phosphorylation has the effect of
inhibiting the other one as well.
Electron transport inhibitors

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Common inhibitors of electron transport include rotenone and amytal, which stop movement of electrons past Complex I,
malonate, malate, and oxaloacetate, which inhibit movement of electrons through Complex II, antimycin A which stops movement
of electrons past Complex III, and cyanide, carbon monoxide, azide, and hydrogen sulfide, which inhibit electron movement
through Complex IV (Figure 5.33). All of these compounds can stop electron transport directly (no movement of electrons) and
oxidative phosphorylation indirectly (proton gradient will dissipate). While some of these compounds are not commonly known,
almost everyone is aware of the hazards of carbon monoxide and cyanide, both of which can be lethal.
ATP synthase inhibitor
It is also possible to use an inhibitor of ATP synthase to stop oxidative phosphorylation directly (no ATP production) and electron
transport indirectly (proton gradient not relieved so it becomes increasingly difficult to pump protons out of matrix). Oligomycin A
(Figure 5.34) is an inhibitor of ATP synthase.

Rotenone
Rotenone, which is a plant product, is used as a natural insecticide that is permitted for organic farming. When mitochondria are
treated with this, electron transport will stop at Complex I and so, too, will the pumping of protons out of the matrix. When this
occurs, the proton gradient rapidly dissipates, stopping oxidative phosphorylation as a consequence. There are other entry points for
electrons than Complex I, so this type of inhibition is not as serious as using inhibitors of Complex IV, since no alternative route for
electrons is available. It is for this reason that cyanide, for example, is so poisonous.
2,4-DNP

Figure 5.35). Treatment of mitochondria with 2,4 DNP makes the mitochondrial inner membrane “leaky” to protons. This has the
effect of providing an alternate route for protons to reenter the matrix besides going through ATP synthase, and uncouples oxidative
phosphorylation from electron transport.
Imagine a dam holding back water with a turbine generating electricity through which water must flow. When all water flows
through the turbine, the maximum amount of electricity can be generated. If one pokes a hole in the dam, though, water will flow
through the hole and less electricity will be created. The generation of electricity will thus be uncoupled from the flow of water. If
the hole is big enough, the water will all drain out through the hole and no electricity will be made.
Bypassing ATP synthase
Imagine, now, that the proton gradient is the equivalent of the water, the inner membrane is the equivalent of the dam and the ATP
synthase is the turbine. When protons have an alternate route, little or no ATP will be made because protons will pass through the
membrane’s holes instead of spinning the turbine of ATP synthase.
It is important to recognize, though, that uncoupling by 2,4 DNP works differently from the electron transport inhibitors or the ATP
synthase inhibitor. In those situations, stopping oxidative phosphorylation resulted in indirectly stopping electron transport, since
the two processes were coupled and the inhibitors did not uncouple them. Similarly, stopping electron transport indirectly stopped
oxidative phosphorylation for the same reason.

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Such is not the case with 2,4 DNP. Stopping oxidative phosphorylation by destroying the proton gradient allows electron transport
to continue unabated (it actually stimulates it), since the proton gradient cannot build no matter how much electron transport runs.
Consequently, electron transport runs like crazy but oxidative phosphorylation stops. When that happens, NAD+ and FAD levels
rise, and catabolic pathways run unabated with abundant supplies of these electron acceptors. The reason such a scenario is
dangerous is because the body is using all of its nutrient resources, but no ATP is being made. Lack of ATP leads to cellular (and
organismal) death. In addition, the large amounts of heat generated can raise the temperature of the body to unsafe levels.

Thermogenin
One of the byproducts of uncoupling electron transport is the production of heat. The faster metabolic pathways run, the more heat
is generated as a byproduct. Since 2,4 DNP causes metabolism to speed up, a considerable amount of heat can be produced.
Controlled uncoupling is actually used by the body in special tissues called brown fat. In this case, brown fat cells use the heat
created to help thermoregulate the temperature of newborn children.
Permeabilization of the inner membrane is accomplished in brown fat by the synthesis of a protein called thermogenin (also known
as uncoupling protein). Thermogenin binds to the inner membrane and allows protons to pass through it, thus bypassing the ATP
synthase. As noted for 2,4 DNP, this results in activation of catabolic pathways and the more catabolism occurs, the more heat is
generated.
Dangerous drug
In uncoupling, whether through the action of an endogenous uncoupling protein or DNP, the energy that would have normally been
captured in ATP is lost as heat. In the case of uncoupling by thermogenin, this serves the important purpose of keeping newborn
infants warm. But in adults, uncoupling merely wastes the energy that would have been harvested as ATP. In other words, it mimics
starvation, even though there is plenty of food, because the energy is dissipated as heat.
This fact, and the associated increase in metabolic rate, led to DNP being used as a weight loss drug in the 1930s. Touted as an
effortless way to lose weight without having to eat less or exercise more, it was hailed as a magic weight loss pill. It quickly
became apparent, however, that this was very dangerous. Many people died from using this drug before laws were passed to ban
the use of DNP as a weight loss aid.
Alternative oxidase
Another approach to generating heat that doesn’t involve breaking respiratory control is taken by some fungi, plants, and protozoa.
They use an alternative electron transport. In these organisms, there is an enzyme called alternative oxidase (Figure 5.36).
Alternative oxidase is able to accept electrons from CoQ and pass them directly to oxygen.

The process occurs in coupled mitochondria. Its mechanism of action is to reduce the yield of ATP, since fewer protons are being
pumped per reduced electron carrier. Thus NAD+ concentrations increase, oxygen consumption increases, and the efficiency of
ATP production decreases.
Organisms using this method must activate catabolic pathways by the increase in NAD+ concentration. This, in turn produces
quantities of NADH and FADH2 necessary to make sufficient amounts of ATP. The byproduct of this increased catabolism is more
heat. Not surprisingly, the alternative oxidase pathway can be activated by cold temperatures.

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Energy efficiency
Cells are not 100% efficient in energy use. Nothing we know is. Consequently, cells do not get as much energy out of catabolic
processes as they put into anabolic processes. A good example is the synthesis and breakdown of glucose, something liver cells are
frequently doing. The complete conversion of glucose to pyruvate in glycolysis (catabolism) yields two pyruvates plus 2 NADH
plus 2 ATPs. Conversely, the complete conversion of two pyruvates into glucose by gluconeogenesis (anabolism) requires 4 ATPs,
2 NADH, and 2 GTPs. Since the energy of GTP is essentially equal to that of ATP, gluconeogenesis requires a net of 4 ATPs more
than glycolysis yields. This difference must be made up in order for the organism to meet its energy needs. It is for this reason that
we eat. In addition, the inefficiency of our capture of energy in reactions results in the production of heat and helps to keep us
warm, as noted. You can read more about glycolysis (HERE) and gluconeogenesis (HERE).
Metabolic controls of energy
It is also noteworthy that cells do not usually have both catabolic and anabolic processes for the same molecules occurring
simultaneously inside of them (for example, breakdown of glucose and synthesis of glucose) because the cell would see no net
production of anything but heat and a loss of ATPs with each turn of the cycle. Such cycles are called futile cycles and cells have
controls in place to limit the extent to which they occur. Since futile cycles can, in fact, yield heat, they are used as sources of heat
in some types of tissue. Brown adipose tissue of mammals uses this strategy, as described earlier. See also HERE for more on heat
generation with a futile cycle.

Reactive oxygen species

Figure 5.37) are oxygen containing molecules, such as peroxides, hydroxyl radical, superoxide, peroxynitrite, and others that are
very chemically reactive. Though some ROS, such as peroxide and nitric oxide have important biological functions in signaling,
increases in reactive oxygen species in times of stress can cause significant damage in the cell. Exogenous sources of ROS, such as
pollution, tobacco, smoke, radiation or drugs can also cause significant problems.
Endogenous production of ROS is directed towards intracellular signaling (H2O2 and nitric oxide, for example) and defense. Many
cells, for example, have NADPH oxidase (Figure 5.38) embedded in the exterior portion of the plasma membranes, in peroxisomes,
and endoplasmic reticulum. It produces superoxides in the reaction below to kill bacteria .

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In the immune system, cells called phagocytes engulf foreign cells and then use ROS to kill them. ROS can serve as signals for
action. In zebrafish, damaged tissues have increased levels of H2O2 and this is thought to be a signal for white blood cells to
converge on the site. In fish lacking the genes to produce hydrogen peroxide, white blood cells do not converge at the damage site.
Sources of hydrogen peroxide include peroxisomes, which generate it as a byproduct of oxidation of long chain fatty acids.

Aging
Reactive oxygen species are at the heart of the free radical theory of aging, which states that organisms age due to the accumulation
of damage from free radicals in their cells. In yeast and Drosophila, there is evidence that reducing oxidative damage can increase
lifespan. In mice, increasing oxidative damage decreases life span, though in Caenorhabditis, blocking production of superoxide
dismutase actually increases lifespan, so the role of ROS in aging is not completely clear.
It is clear, though, that accumulation of mitochondrial damage is problematic for individual cells. Bcl-2 proteins on the surface of
mitochondria monitor damage and if they detect it, will activate proteins called Bax to stimulate the release of cytochrome c from
the mitochondrial membrane, stimulating apoptosis (programmed cell death). Eventually the dead cell will be phagocytosed.
A common endogenous source of superoxide is the electron transport chain. Superoxide can be produced when movement of
electrons into and out of the chain don’t match well. Under these circumstances, semi-reduced CoQ can donate an electron to O2 to
form superoxide (O2-). Superoxide can react with many molecules, including DNA where it can cause damage leading to mutation.
If it reacts with the aconitase enzyme, ferrous iron (Fe++) can be released which, in turn, can react in the Fenton reaction to
produce another reactive oxygen species, the hydroxyl radical (Figure 5.39) .
Countering the effects of ROS are enzymes, such as catalase, superoxide dismutase, and anti-oxidants, such as glutathione and
vitamins C and E.
Glutathione protects against oxidative damage by being a substrate for the enzyme glutathione peroxidase. Glutathione peroxidase
catalyzes the conversion of hydrogen peroxide to water (next page).

Catalase

Figure 5.40) is an important enzyme for cells of all types that live in an oxygen environment. A first line of defense against reactive
oxygen species, catalase catalyzes the breakdown of hydrogen peroxide into water and oxygen.
2 H2O2 <=> 2 H2O + O2
The enzyme, which employs four heme groups in its catalysis, works extremely rapidly, converting up to 40,000,000 molecules of
hydrogen peroxide to water and oxygen per enzyme per second. It is abundantly found in peroxisomes.
In addition to catalase’s ability to break down hydrogen peroxide, the enzyme can also use hydrogen peroxide to oxidize a wide
variety organic compounds, including phenols, formic acid, formaldehyde, acetaldehyde, and alcohols, but with much lower
efficiency.
Health
The importance of catalase for health is uncertain. Mice deficient in the enzyme appear healthy and humans with low levels of the
enzyme display few problems. On the other hand, mice engineered to produce higher levels of catalase, in at least one study, lived
longer. The ability of organisms to live with lower levels or no catalase may arise from another group of enzymes, the
peroxiredoxins, which also act on hydrogen peroxide and may make up for lower quantities of catalase. Last, there is evidence that

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reduced levels of catalase with aging may be responsible for the graying of hair. Higher levels of H2O2 with reduced catalase
results in a bleaching of hair follicles.

Superoxide dismutase

Another important enzyme for protection against reactive oxygen species is superoxide dismutase (SOD), which is found, like
catalase, in virtually all organisms living in an oxygen environment. Superoxide dismutase, also like catalase, has a very high Kcat
value and, in fact, has the highest Kcat/Km known for any known enzyme. It catalyzes the reactions at the top of the next column
(superoxides shown in red):
The enzyme thus works by a ping-pong (double displacement) mechanism (see HERE), being converted between two different
forms.
The hydrogen peroxide produced in the second reaction is easily handled by catalase and is also less harmful than superoxide,
which can react with nitric oxide (NO) to form very toxic peroxynitrite ions (Figure 5.43). Peroxynitrite has negative effects on
cells, as shown in Figure 5.45.

In addition to copper, an ion of Zn++ is also bound by the enzyme and likely plays a role in the catalysis. Forms of superoxide
dismutase that use manganese, nickel, or iron are also known and are mostly found in prokaryotes and protists, though a manganese
SOD is found in most mitochondria. Copper/zinc enzymes are common in eukaryotes.
Three forms of superoxide dismutase are found in humans and localized to the cytoplasm (SOD1 - Figure 5.45), mitochondria
(SOD2 - Figure 5.46), and extracellular areas (SOD3 - Figure 5.47). Mice lacking any of the three forms of the enzyme are more
sensitive to drugs, such as paraquat. Deficiency of SOD1 in mice leads to hepatocellular carcinoma and early loss of muscle tissue
related to aging. Drosophila lacking SOD2 die before birth and those lacking SOD1 prematurely age.
In humans, superoxide dismutase mutations are associated with the genetically-linked form of Amyotrophic Lateral Sclerosis
(ALS) and over-expression of the gene is linked to neural disorders associated with Down syndrome.

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Mixed function oxidases
Other enzymes catalyzing reactions involving oxygen include the mixed function oxidases. These enzymes use molecular oxygen
for two different purposes in one reaction. The mixed function part of the name is used to indicate reactions in which two different
substrates are being oxidized simultaneously. Monooxygenases are examples of mixed function oxidases. An example of a mixed
function oxidase reaction is shown below.
AH + BH2 + O2 <=> AOH + B + H2O
In this case, the oxygen molecule has one atom serve as an electron acceptor and the other atom is added to the AH, creating an
alcohol.

Cytochrome P450 enzymes

Cytochrome P450 enzymes (called CYPs) are family of heme-containing mixed function oxidase enzymes found in all domains of
life. Over 21,000 CYP enzymes are known. The most characteristic reaction catalyzed by these enzymes follows
Monooxygenase reactions such as this are relatively rare in the cell due to their use of molecular oxygen. CYPs require an electron
donor for reactions like the one shown here and frequently require a protein to assist in transferring electrons to reduce the heme
iron. There are six different classes of P450 enzymes based on how they get electrons
1. Bacterial P450 - electrons from ferredoxin reductase and ferredoxin
2. Mitochondrial P450 - electrons from adrenodoxin reductase and adrenodoxin
3. CYB5R/cyb5 - electrons come from cytochrome b5
4. FMN/Fd - use a fused FMN reductase
5. Microsomal P450 - NADPH electrons come via cytochrome P450 reductase or from cytochrome b5 and cytochrome b5
reductase
6. P450 only systems - do not require external reducing power
The CYP genes are abundant in humans and catalyze thousand of reactions on both cellular and extracellular chemicals. There are
57 human genes categorized into 18 different families of enzymes. Some CYPs are specific for one or a few substrates, but others
can act on many different substrates.
CYP enzymes are found in most body tissues and perform important functions in synthesis of steroids (cholesterol, estrogen,
testoterone, Vitamin D, e.g.), breakdown of endogenous compounds (bilirubin), and in detoxification of toxic compounds including
drugs. Because they act on many drugs, changes in CYP activity can produce unexpected results and cause problems with drug
interactions.
Bioactive compounds, for example, in grapefruit juice, can inhibit CYP3A4 activity, leading to increased circulating concentrations
of drugs that would normally have been acted upon by CYP3A4. This is the reason that patients prescribed drugs that are known to
be CYP3A4 substrates are advised to avoid drinking grapefruit juice while under treatment. St. Johns Wort, an herbal treatment, on
the other hand, induces CYP3A4 activity, but inhibits CYP1A1, CYP1B1, and CYP2D6. Tobacco smoke induces CYP1A2 and
watercress inhibits CYP2E1.

Cytochromes
Cytochromes are heme-containing proteins that play major roles in the process of electron transport in the mitochondrion and in
photosynthesis in the chloroplast. They exist either as monomers (cytochrome c) or as subunits within large redox complexes
(Complex III and Complex IV of electron transport. An atom of iron at the center of the heme group plays a central role in the
process, flipping between the ferrous (Fe++) and ferric (Fe+++) states as a result of the movement of electrons through it.
There are several different cytochromes. Cytochrome c (Figure 5.47) is a soluble protein loosely associated with the
mitochondrion. Cytochromes a and a3 are found in Complex IV. Complex III has cytochromes b and c1 and the plastoquinol-
plastocyanin reductase of the chloroplast contains cytochromes b6 and f. Another important class of enzymes containing
cytochromes is the cytochrome P450 oxidase group (see above). They get their name from the fact that they absorb light at 450 nm
when their heme iron is reduced.

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Iron-Sulfur Proteins
Iron-sulfur proteins contain iron-sulfur clusters in a variety of formats, including sulfide-linked di-, tri-, and tetrairon centers
existing in different oxidation states (Figures 5.48 & 5.49). The clusters play a variety of roles, but the best known ones are in
electron transport where they function in the redox reactions involved in the movement of electrons.
Complexes I and Complex II contain multiple Fe-S centers. Iron-sulfur proteins, though, have many other roles in cells. Aconitase
uses an iron-sulfur center in its catalytic action and the ability of the enzyme to bind iron allows it to function as a barometer of
iron concentration in cells. Iron-sulfur centers help to generate radicals in enzymes using S-Adenosyl Methionine (SAM) and can
serve as a source of sulfur in the synthesis of biotin and lipoic acid. Some iron-sulfur proteins even help to regulate gene
expression.

Ferredoxin
Ferredoxins are iron-sulfur containing proteins performing electron transfer in a wide variety of biological systems and processes.
They include roles in photosynthesis in chloroplasts. Ferredoxins are classified structurally by the iron-sulfur clustered centers they
contain. Fe2S2 clusters (Figure 5.50) are found in chloroplast membranes and can donate electrons to glutamate synthase, nitrate
reductase, and sulfite reductase and serve as electron carriers between reductase flavoproteins and bacterial dioxygenase systems.
Adrenodoxin is a soluble human Fe2S2 ferredoxin (also called ferredoxin 1) serving as an electron carrier (to cytochrome P450) in
mitochondrial monooxygenase systems. Fe4S4 ferredoxins are subdivided as low and high potential ferredoxins, with the latter
ones functioning in anaerobic electron transport chains.

Ferritin
Ferritin is an intracellular iron-storage protein found in almost all living organisms, from bacteria to higher plants and animals. It is
a globular protein complex with 24 subunits and is the primary intracellular iron-storage protein in eukaryotes and prokaryotes.
Ferritin functions to keep iron in a soluble and non-toxic form. Its ability to safely store iron and release it in a controlled fashion
allow it to act like the prime iron buffer and solubilizer in cells - keeping the concentration of free iron from going to high or falling
too low. Ferritin is located in the cytoplasm in most tissues, but it is also found in the serum acting as an iron carrier. Ferritin that
doesn’t contain any iron is known as apoferritin.

Monoamine oxidases
Monoamine oxidases are enzymes that catalyze the oxidative deamination of monoamines, such as serotonin, epinephrine, and
dopamine. Removal of the amine with oxygen results in the production of an aldehyde and ammonia. The enzymes are found inside
and outside of the central nervous system.
There are two types of monoamine oxidase enzymes - MAO-A and MAO-B. MAO-A is particularly important for oxidizing
monoamines consumed in the diet. Both MAO-A and MAO-B play important roles in inactivating monoaminergic
neurotransmitters. Both enzymes act on dopamine, tyramine (Figure 5.50), and tryptamine. MAO-A is the primary enzyme for
metabolizing melatonin, serotonin, norepinephrine, and epinephrine, while MAO-B is the primary enzyme for phenethylamine
(Figure 5.51) and benzylamine. MAO-B levels have been reported to be considerably reduced with tobacco usage.
Actions of monoamine oxidases thus affects levels of neurotransmitters and consequently are thought to play roles in neurological
and/or psychiatric disorders. Aberrant levels of MAOs have been linked to numerous psychological problems, including
depression, attention deficit disorder (ADD), migraines, schizophrenia, and substance abuse. Medications targeting MAOs are
sometimes used to treat depression as a last resort - due to potential side effects. Excess levels of catecholamines, such as
epinephrine, norepinephrine, and dopamine, can result in dangerous hypertension events.

DNA damage theory of aging

The DNA Damage Theory of Aging is based on the observation that, over time, cells are subject to extensive oxidative events. As
already noted, these afford opportunities for the formation of ROS that can damage cellular molecules, and it follows that
accumulation of such damage, especially to the DNA would be deleterious to the cell. The build-up of DNA damage could, thus, be
responsible for the changes in gene expression that we associate with aging.
Numerous damage events

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The amount of DNA damage that can occur is considerable. In mice, for example, it is estimated that each cell experiences 40,000
to 150,000 damage events per day. The damage, which happens to nuclear as well as to mitochondrial DNA, can result in apoptosis
and/or cellular senescence. DNA repair systems, of course, protect against damage to DNA, but over time, unrepairable damage
may accumulate.
Oxidative damage
DNA damage can occur in several ways. Oxidation can damage nucleotides and alter their base-pairing tendencies. Oxidation of
guanine by reactive oxygen species, for example, can produce 8-oxo-guanine (Figures 5.52 and 5.53). This oxidized nucleobase
commonly produced lesion in DNA arising from action of reactive oxygen species like superoxides. 8-oxoguanine is capable of
forming a stable base pairing interaction within a DNA duplex with adenine, potentially giving rise to a mutation when DNA
replication proceeds. 8-oxoguanine can be repaired if recognized in time by a DNA glycosylase, which acts to clip out the damaged
base and it can then be replaced by the proper one. Polycyclic aromatic hydrocarbons from cigarette smoke, diesel exhaust, or
overcooked meat can covalently bind to DNA and, if unrepaired, lead to mutation. Chemical damage to DNA can result in broken
or cross-linked DNAs.
Diseases of DNA repair
The importance of DNA repair in the aging process is made clear by diseases affecting DNA repair that lead to premature aging.
These include Werner syndrome, for whom the life expectancy is 47 years. It arises as a result of loss of two enzymes in base
excision repair. People suffering from Cockayne syndrome have a life expectancy of 13 years due to mutations that alter
transcription-coupled nucleotide excision repair, which is an important system for fixing oxidative damage.
Further, the life expectancies of 13 species of mammalian organisms correlates with the level of expression of the PARP DNA
repair-inducing protein. Interestingly, people who lived past the age of 100 had a higher level of PARP than younger people in the
population.

Antioxidants
There is a growing interest in the subject of antioxidants because of health concerns raised by our knowledge of problems created
as a result of spontaneous oxidation of biomolecules by Reactive Oxygen Species (ROS), such as superoxide. Antioxidants have
the chemical property of protecting against oxidative damage by being readily oxidized themselves, preferentially to other
biomolecules.
Biologically, cells have several lines of antioxidant defense. They include molecules, such as vitamins C, A, and E, glutathione, and
enzymes that destroy ROS such as superoxide dismutase, catalase, and peroxidases.

Health effects

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Oxidation by ROS is mutagenic and has been linked to atherosclerosis. Nonetheless, randomized studies of oral supplementation of
various vitamin combinations have shown no protective effect against cancer and supplementation of Vitamin E and selenium has
revealed no decrease in the risk of cardiovascular disease. Further, no reduction in mortality rates as a result of supplementation
with these materials has been found, so the protective effects, if any, of antioxidants on ROS in human health remain poorly
understood.
Glutathione

Figures 5.55 & 5.56). The three amino acids in glutathione (glutamate, cysteine, and glycine) are joined in an unusual fashion. The
glutamate is joined to the center cysteine by a peptide bond between the R-group carboxyl of glutamate and the α-amine of
cysteine. The bond between cysteine and glycine is a normal peptide bond between the α-carboxyl of cysteine and the α-amine of
glycine.
The thiol group of cysteine is a reducing agent that reduces disulfide bonds to sulfhydryls in cytoplasmic proteins. This, in turn, is
the bridge when two glutathiones get oxidized and form a disulfide bond with each other (Figure 5.56). Glutathione’s two oxidative
states are abbreviated as follows: GSH (reduced) and GSSG (oxidized).
Disulfide-joined glutathiones can be separated by reduction of their bonds with glutathione reductase, using electrons from
NADPH for the reduction.
Non-ribosomal synthesis
Glutathione is not made by ribosomes. Rather, two enzymes catalyze its synthesis. The enzyme γ-glutamylcysteine synthetase
catalyzes the joining of the glutamate to the cysteine and then glutathione synthetase catalyzes the peptide bond formation between
the cysteine and the glycine. Each step requires energy from ATP.
Essential for life
Glutathione is important for life. Mice lacking the first enzyme involved in its synthesis in the liver die in the first month after
birth. In healthy cells, 90% of glutathione is in the GSH state. Higher levels of GSSG correspond to cells that are oxidatively
stressed.
Besides reducing disulfide bonds in cells, glutathione is also important for the following:
• Neutralization of free radicals and reactive oxygen species.
• Maintenance of exogenous antioxidants such as vitamins C and E in their reduced forms.
Regulation of the nitric oxide cycle
Resveratrol

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 Figure 5.57) is a phenolic compound produced in the skin of plants such as grapes, raspberries, and blueberries, in response to
injury or when they are being attacked by pathogens. Numerous health benefits are claimed for the compound, though evidence of
such benefits is in short supply. Resveratrol is metabolized rapidly in the body, so it is difficult to maintain levels of it.
Some data indicates resveratrol may improve the functioning of mitochondria. It also acts as an antioxidant and causes
concentration of another anti-oxidant, glutathione, to increase. The compound appears to induce expression of manganese
superoxide dismutase (protects against reactive oxygen species) and inhibits several phosphodiesterases. This causes an increase in
cAMP which results in increases in oxidation of fatty acids, mitochondria formation, gluconeogenesis, and glycogen breakdown. It
has been claimed to be the cause of the French Paradox in which drinking of red wine is supposed to give protection for the
cardiovascular system. Research data is lacking in support of the claim, however. Resveratrol is known to activate Sirtuin proteins,
which play roles in gene inactivation.

Summary
In summary, energy is needed for cells to perform the functions that they must carry out in order to stay alive. At its most basic
level, this means fighting a continual battle with entropy, but it is not the only need for energy that cells have.
References
1. Winge, D.R., Mol Cell Biol. 2012 Jul; 32(14): 2647–2652. doi: 10.1128/MCB.00573-12
Energy: Electron Transport & Oxidative Phosphorylation
429
YouTube Lectures
by Kevin
HERE & HERE
YouTube Lectures
by Kevin
HERE & HERE
430
Figure 5.14 - Overview of electron transport (bottom left and top right) and oxidative phosphorylation (top left - yellow box) in the
mitochondrion
431
Figure 5.15 - Loss of electrons by NADH to form NAD+. Relevant reactions occur in the top ring of the molecule.
432
Figure 5.16 - Flow of electrons from NADH into the electron transport system. Entry is through complex I
Image by Aleia Kim
Figure 5.17 - Flow of electrons from FADH2 into the electron transport chain. Entry is through complex II.
Image by Aleia Kim
Interactive Learning

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Module
HERE
433
Figure 5.18 - Complex I embedded in the inner mitochondrial membrane. The mitochondrial matrix at at the top
Wikipedia
434
Figure 5.19 - Complex II embedded in inner mitochondrial membrane. Matrix is up.
Wikipedia
YouTube Lectures
by Kevin
HERE & HERE
435
Figure 5.20 - Movement of electrons through complex I from NADH to coenzyme Q. The mitochondrial matrix is at the bottom
Image by Aleia Kim
Figure 5.21 - Movement of electrons from succinate through complex II (A->B->C->D->Q). Mitochondrial matrix on bottom.
Image by Aleia Kim
436
Figure 5.22 - Complex II in inner mitochondrial membrane showing electron flow. Matrix is up.
Wikipedia
Figure 5.23 - Coenzyme Q
437
Movie 5.2 - The Q-cycle
Wikipedia
Figure 5.24 - The Q-Cycle Image by Aleia Kim
Figure 5.24 - Complex III
Wikipedia
438
YouTube Lectures
by Kevin
HERE & HERE
Figure 5.25 - The Q-cycle. Matrix is down.
Image by Aleia Kim
439
Figure 5.26 - Movement of electrons and protons through complex IV. Matrix is down
Image by Aleia Kim
Figure 5.25 - Cytochrome c with bound heme Group
Wikipedia
440

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Figure 5.27 - Mitochondrial anatomy. Electron transport complexes and ATP synthase are embedded in the inner mitochondrial
membrane
Image by Aleia Kim
441
Figure 5.28 - ATP synthase. Protons pass from intermembrane space (top) through the complex and exit in the matrix (bottom).
Image by Aleia Kim
Interactive Learning
Module
HERE
442
Movie 5.3 - ATP Synthase - ADP + Pi (pink) and ATP (red). The view is end-on from the cytoplasmic side viewing the β subunits
Movie 5.3 - ATP Synthase - ADP + Pi (pink) and ATP (red). The view is end-on from the cytoplasmic side viewing the β subunits
443
Figure 5.29 - Important structural features of the ATP synthase
Image by Aleia Kim
444
Figure 5.30 - Loose (L), Tight (T), and Open (O) structures of the F1 head of ATP synthase. Change of structure occurs by rotation
of γ-protein (purple) in center as a result of proton movement. Individual α and β units do not rotate
Image by Aleia Kim
445
Figure 5.31 - Respiration overview in eukaryotic cells
Wikipedia
YouTube Lectures
by Kevin
HERE & HERE
446
Rest
ATP High / ADP Low
Oxidative Phosphorylation Low
Electron Transport Low
Oxygen Use Low
NADH High / NAD+ Low
Citric Acid Cycle Slow
Exercise
ATP Low / ADP High
Oxidative Phosphorylation High
Electron Transport High
Oxygen Use High
NADH Low / NAD+ High

5.2.24 https://bio.libretexts.org/@go/page/7830
Citric Acid Cycle Fast
Interactive Learning
Module
HERE
447
Figure 5.32 - Three inhibitors of electron transport
Image by Aleia Kim
448
Figure 5.33 - Oligomycin A - An inhibitor of ATP synthase
Figure 5.34 - 2,4 DNP - an uncoupler of respiratory control
449
In Cells With Tight Coupling
O2 use depends on metabolism
NAD+ levels vary with exercise
Proton gradient high with no exercise
Catabolism depends on energy needs
ETS runs when OxPhos runs and vice versa
In Cells That Are Uncoupled
O2 use high
NAD+ Levels high
Little or no proton gradient
Catabolism high
OxPhos does not run, but ETS runs rapidly
YouTube Lectures
by Kevin
HERE & HERE
450
451
Figure 5.35 - Alternative oxidase (AOX) of fungi, plants, and protozoa bypasses part of electron transport by taking electrons from
CoQ and passing them to oxygen.
452
Figure 5.36 - Structure of an oxygen free radical
Wikipedia
NADPH + 2O2
NADP+ + 2O2− + H+
Figure 5.37 - Three sources of reactive oxygen species (ROS) in cells
Wikipedia
453
454
YouTube Lectures
by Kevin

5.2.25 https://bio.libretexts.org/@go/page/7830
HERE & HERE
Figure 5.38 A hydroxyl radical
Wikipedia
455
Reduced Glutathione (GSH) + H2O2
Oxidized Glutathione (GSSG) + H2O
Figure 5.40 - Detoxifying reactive oxygen species
Figure 5.39 - Catalase
456
1. O2- + Enzyme-Cu++
O2 + Enzyme-Cu+
2. O2- + Enzyme-Cu+ + 2H+
H2O2 + Enzyme-Cu++
Figure 5.41 - SOD2 of humans
Figure 5.42 3 - Peroxynitrite Ion
Figure 5.44 - SOD1 of humans
Wikipedia
Figure 5.45 - SOD3 of humans
457
Figure 5.43 - Peroxynitrite’s effects on cells lead to necrosis or apoptosis
Wikipedia
458
RH + O2 + NADPH + H+
ROH + H2O + NADP+
459
Figure 5.46 - Cytochrome c with its heme group
460
YouTube Lectures
by Kevin
HERE & HERE
Figure 5.47 - Fe2S2 Cluster
Figure 5.48 - Redox reactions for Fe4S4 clusters
461
Figure 5.49 - Tyramine
Figure 5.50 - Phenethylamine
462
Figure 5.51 - Guanine and 8-oxo-guanine
Figure 5.52 - Adenine-8-oxo-guanine base pair. dR = deoxyribose

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463
Figure 5.53 - Good antioxidant sources
464
Figure 5.55 - Oxidized glutathiones (GSSG) joined by a disulfide bond
Wikipedia
Figure 5.54 - Structure of reduced glutathione (GSH)
465
Figure 5.56 - Resveratrol
YouTube Lectures
by Kevin
HERE & HERE
466
Graphic images in this book were products of the work of several talented students. Links to their Web pages are below
Click HERE for
Martha Baker’s
Web Page
Click HERE for
Pehr Jacobson’s
Web Page
Click HERE for
Aleia Kim’s
Web Page
Click HERE for
Penelope Irving’s
Web Page
Problem set related to this section HERE
Point by Point summary of this section HERE
To get a certificate for mastering this section of the book, click HERE
Kevin Ahern’s free iTunes U Courses - Basic / Med School / Advanced
Biochemistry Free & Easy (our other book) HERE / Facebook Page
Kevin and Indira’s Guide to Getting into Medical School - iTunes U Course / Book
To see Kevin Ahern’s OSU ecampus courses - BB 350 / BB 450 / BB 451
To register for Kevin Ahern’s OSU ecampus courses - BB 350 / BB 450 / BB 451
Biochemistry Free For All Facebook Page (please like us)
Kevin Ahern’s Web Page / Facebook Page / Taralyn Tan’s Web Page
Kevin Ahern’s free downloads HERE
OSU’s Biochemistry/Biophysics program HERE
OSU’s College of Science HERE

5.2.27 https://bio.libretexts.org/@go/page/7830
Oregon State University HERE
Email Kevin Ahern / Indira Rajagopal / Taralyn Tan
I'm a little mitochondrion
Who gives you energy
I use my proton gradient
To make the ATPs

He's a little mitochondrion

Who gives us energy
He uses proton gradients
To make some ATPs

Electrons flow through Complex II

To traffic cop Co-Q
Whenever they arrive there in
An FADH-two

Electrons flow through Complex II

To traffic cop Co-Q
Whenever they arrive there in
An FADH-two
Tightly coupled is my state
Unless I get a hole
Created in my membrane by
Some di-ni-tro-phe-nol

Yes tightly coupled is his state

Unless he gets a hole
Created in his membrane by
Some di-ni-tro-phenol

Both rotenone and cyanide

Stop my electron flow
And halt the calculation of
My "P" to "O" ratio

Recording by Tim Karplus

Lyrics by Kevin Ahern
Recording by Tim Karplus Lyrics by Kevin Ahern
I’m a Little Mitochondrion
To the tune of “I’m a Lumberjack”
Metabolic Melodies Website HERE
In the catabolic pathways that our cells employ
Oxidations help create the ATP
While they lower Gibbs free energy
Thanks to enthalpy
If a substrate is converted from an alcohol
To an aldehyde or ketone it is clear
Those electrons do not disappear

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They just rearrange – very strange

N-A-D is in my ears and in my eyes

Help-ing mol-e-cules get oxidized
Making N-A-D-H then
And the latter is a problem anaerobically
‘Cuz accumulations of it muscles hate
They respond by using pyruvate
To produce lactate

Catalyzing is essential for the cells to live

So the enzymes grab their substrates eagerly
If they bind with high affinity
Low Km you see, just as me

N-A-D is in my ears and in my eyes

Help-ing mol-e-cules get oxidized
Making N-A-D-H then
N-A-D
To the tune of “Penny Lane”
Metabolic Melodies Website HERE
Recorded by Tim Karplus
Lyrics by Kevin Ahern
Recorded by Tim Karplus Lyrics by Kevin Ahern
When oxygen’s electrons all are in the balanced state
There’s twelve of them for oh-two. The molecule is great
But problems sometimes happen on the route to complex IV
Making reactive species that the cell cannot ignore
Oh superoxide dismutase is super catalytic
Keeping cells from getting very peroxynitritic
Faster than a radical, its actions are terrific
Superoxide dismutase is super catalytic
Enzyme, enzyme deep inside
Blocking all the bad oxides
The enzyme’s main advantage is it doesn’t have to wait
By binding superoxide in a near-transition state
It turns it to an oxygen in mechanism one
Producing “h two oh two” when the cycle is all done
Oh superoxide dismutase you’re faster than all them
You’ve got the highest ratio of kcat over KM
This means that superoxide cannot cause too much mayhem
Superoxide dismutase is faster than all them
Superoxide dismutase

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Stopping superoxide’s ways
The enzyme’s like a ping-pong ball that mechanistic-ly
Bounces between two copper states, plus one and two you see
So S-O-D behaves just like an anti-oxidant
Giving as much protection as a cell could ever want
Oh superoxide dismutase, the cell’s in love with you
Because you let electron transport do what it must do
Without accumulation of a radical oh two
Superoxide dismutase - that’s why a cell loves you
Superoxide Dismutase
To the tune of “Supercalifragilistiexpialidocious”
Metabolic Melodies Website HERE
Lyrics by Kevin Ahern
No Recording Yet For This Song

This page titled 5.2: Electron Transport and Oxidative Phosphorylation is shared under a CC BY-NC-SA license and was authored, remixed,
and/or curated by Kevin Ahern, Indira Rajagopal, & Taralyn Tan.

5.2.30 https://bio.libretexts.org/@go/page/7830
 CHAPTER OVERVIEW

6: Metabolism
6.1: Metabolism - Sugars
6.2: Citric Acid Cycle & Related Pathways
6.3: Fats and Fatty Acids
6.4: Other Lipids
6.5: Amino Acids and the Urea Cycle
6.6: Nucleotides

Thumbnail: Metabolic Metro Map. Image used with permission (CC BY-SA 4.0; Chakazul).

This page titled 6: Metabolism is shared under a CC BY-NC-SA license and was authored, remixed, and/or curated by Kevin Ahern, Indira
Rajagopal, & Taralyn Tan.

1
6.1: Metabolism - Sugars
Glycolysis
Carbohydrates, whether synthesized by photosynthetic organisms, stored in cells as glycogen, or ingested by heterotrophs, must be
broken down to obtain energy for the cell’s activities as well as to synthesize other molecules required by the cell. Starch and
glycogen, polymers of glucose, are the main energy storage forms of carbohydrates in plants and animals, respectively. To use these
sources of energy, cells must first break down the polymers to yield glucose. The glucose is then taken up by cells through
transporters in cell membranes. The metabolism of glucose, as well as other six carbon sugars (hexoses) begins with the catabolic
pathway called glycolysis. In this pathway, sugars are oxidized and broken down into pyruvate molecules. The corresponding
anabolic pathway by which glucose is synthesized is termed gluconeogenesis. Neither glycolysis nor gluconeogenesis is a major
oxidative/reductive process, with one step in each one involving loss/gain of electrons, but the product of glycolysis, pyruvate, can
be completely oxidized to carbon dioxide (Figure 6.2). Indeed, without production of pyruvate from glucose in glycolysis, a major
energy source for the cell would not be available.
Glucose is the most abundant hexose in nature and is traditionally used to illustrate the reactions of glycolysis, but fructose (in the
form of fructose-6- phosphate) is also readily metabolized, while galactose can easily be converted into glucose for catabolism in
the pathway as well. The end metabolic products of glycolysis are two molecules of ATP, two molecules of NADH and two
molecules of pyruvate (Figure 6.3), which, in turn, can be oxidized further in the citric acid cycle.
Entry points for glycolysis
Glucose and fructose are the sugar ‘funnels’ serving as entry points to the glycolytic pathway. Other sugars must be converted to
either of these forms to be metabolized in glycolysis. Some pathways, including the Calvin
Figure 6.2 - Metabolic fates of glucose Image by Aleia Kim Your cells may have a mounting crisis Should they not go through
glyco-lye-sis No glucose energy releases Until it’s fractured into pieces
Figure 6.3 - Glycolysis and its Regulators Image by Ben Carson Cycle and the Pentose Phosphate Pathway (PPP) contain
intermediates in common with glycolysis, so in that sense, almost any cellular sugar can be metabolized here.
Other pathways
Intermediates of glycolysis and gluconeogenesis that are common to other pathways include glucose-6-phosphate (PPP, glycogen
metabolism), Fructose-6-phosphate (Calvin Cycle, PPP), Glyceraldehyde-3- phosphate (Calvin Cycle, PPP), dihydroxyacetone
phosphate (PPP, glycerol metabolism, Calvin Cycle), 3- phosphoglycerate (Calvin Cycle, PPP), phosphoenolpyruvate (C4 plant
metabolism, Calvin Cycle), and pyruvate (fermentation, acetyl-CoA genesis, amino acid metabolism). It is worth noting that
glycerol from the breakdown of fat can readily be metabolized to dihydroxyacetone phosphate (DHAP) and thus enter the
glycolysis pathway. It is the only part of a fat that is used in these pathways.
Reaction 1
Glucose gets a phosphate from ATP to make glucose-6-phosphate (G6P) in a reaction catalyzed by the enzyme hexokinase, a
transferase enzyme.
Glucose + ATP ⇄ G6P + ADP + H+
Hexokinase is one of three regulated enzymes in glycolysis and is inhibited by one of the products of its action - G6P. Hexokinase
has flexibility in its substrate binding and is able to phosphorylate a variety of hexoses, including fructose, mannose, and galactose.
Why phosphorylate glucose?
Phosphorylation of glucose serves two important purposes. First, the addition of a phosphate group to glucose effectively traps it in
the cell, as G6P cannot diffuse across the lipid bilayer. Second, the reaction decreases the concentration of free glucose, favoring
additional import of the molecule. G6P is a substrate for the pentose phosphate pathway and can also be converted to glucose-1-
phosphate (G1P) for use in glycogen synthesis and galactose metabolism (Figure 6.5).
It is worth noting that the liver has an enzyme like hexokinase called glucokinase, which Figure 6.4 - Reaction #1 -
Phosphorylation of glucose - catalyzed by hexokinase has a much higher Km (lower affinity) for glucose. This is important,
because the liver is a site of glucose synthesis (gluconeogenesis) where cellular concentrations of glucose can be relatively high.

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With a lower affinity glucose phosphorylating enzyme, glucose is not converted to G6P unless glucose concentrations get high, so
the liver is able to release the glucose it makes into the bloodstream for the rest of the body to use.
Reaction 2
Next, G6P is converted to fructose-6-phosphate (F6P), in a reaction catalyzed by the enzyme
G P ⇄ F P (6.1.1)
6 6

The reaction has a low ΔG°’ , so it is readily favorable in either direction with Figure 6.6 - Mechanism of conversion of G6P to
F6P in reaction #2 Figure 6.5 - The centrality of glucose-6-phosphate in metabolism Image by Aleia Kim only slight changes in
concentration of reactants.
Reaction 3
ceF 6P + AT P ⇄ F 1, 6BP + ADP + H + (6.1.2)

The second input of energy occurs when F6P gets another phosphate from ATP in a reaction catalyzed by the enzyme
phosphofructokinase-1 (PFK-1 - another transferase) to make fructose-1,6- bisphosphate (F1,6BP). PFK-1 is a very important
enzyme regulating glycolysis, with several allosteric activators and inhibitors (see HERE).
Like the hexokinase reaction the energy from ATP is needed to make the reaction energetically favorable. PFK-1 is the most
important regulatory enzyme in the pathway and this reaction is the ratelimiting step. It is also one of three essentially irreversible
reactions in glycolysis.
A variant enzyme found in plants and some bacterial uses pyrophosphate rather than ATP as the energy source and due to the lower
energy input from hydrolysis of the pyrophosphate, that reaction is reversible.
Reaction 4
F , 6 BP ⇄ D−GLYAL P + DHAP (6.1.3)
1 3

With the glycolysis pump thus primed, the pathway proceeds to split the F1,6BP into two 3-carbon intermediates. This reaction
catalyzed by the lyase known as aldolase is energetically a “hump” to overcome in the glycolysis direction (∆G°’ = +24 kJ/mol
Figure 6.7 - Reaction #3 - Conversion of F6P to F1,6BP by PFK Wikipedia Figure 6.8 - Reaction #4 - Breakdown of F1,6BP into
GLYAL3P (left) and DHAP (right) by aldolase °K) so to get over the energy hump, cells must increase the concentration the
reactant (F1,6BP) and decrease the concentration of the products, which are D-glyceraldehyde- 3-phosphate (D-GLYAL3P) and
dihydroxyacetone phosphate (DHAP).
A novel scheme facilitates decreasing concentration of the products (see below). Aldolases cut the ketose ring by two different
mechanisms and these enzymes are grouped as Class I (in animals and plants) and Class II (in fungi and bacteria).
Reaction 5
DHAP ⇄ D−GLYAL P (6.1.4)
3

In the next step, DHAP is converted to DGLYAL3P in a reaction catalyzed by the enzyme triosephosphate isomerase. At this point,
the six carbon glucose molecule has been broken down to two units of three carbons each - D-GLYAL3P. From this point forward
each reaction of glycolysis contains two of each molecule. Reaction #5 is fairly readily reversible in cells.
The enzyme is of note because it is one example of a “perfect enzyme.” Enzymes in this category have very high ratios of Kcat/Km
that approach a theoretical maximum limited only by the diffusion of substrate into the active site of the enzyme. The apparent
reason for the enzyme evolving in this way is that the mechanism of the reaction produces an unstable, toxic intermediate (Figure
6.9). With the reaction proceeding as rapidly as it does, there is less chance of the intermediate escaping and causing damage in the
cell.
Reaction 6
+ +
D−GLYAL P + NAD + PiD−1 , 3 BPG + NADH + H (6.1.5)
3

Figure 6.9 - Reaction #5 - Triose phosphate isomerase with unstable, toxic intermediate (methyl glyoxal) Image by Ben Carson
In this reaction, D-GLYAL3P is oxidized in the only oxidation step of glycolysis catalyzed by the enzyme glyceraldehyde-3-
phosphate dehydrogenase, an oxidoreductase. The aldehyde in this reaction is oxidized, then linked to a phosphate to make an ester

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- D-1,3-bisphospho-glycerate (D- 1,3BPG). Electrons from the oxidation are donated to NAD+, creating NADH.
NAD+ is a critical constituent in this reaction and is the reason that cells need a fermentation option at the end of the pathway (see
below).
Note here that ATP energy was not required to put the phosphate onto the oxidized D-GLYAL3P. The reason for this is because the
energy provided by the oxidation reaction is sufficient for adding the phosphate.
Reaction 7

D−1 , 3 BPG + ADP ⇄ 3 PG + ATP (6.1.6)

The two phosphates in the tiny 1,3BPG molecule repel each other and give the molecule high potential energy. This energy is
utilized by the enzyme phosphoglycerate kinase (another transferase) to phosphorylate ADP and make ATP, as well as the product,
3-phosphoglycerate (3-PG). This is an example of a substrate-level phosphorylation. Such mechanisms for making ATP require an
intermediate with a high enough energy to phosphorylate ADP to make ATP. Figure 6.10 - Reaction #6 - Oxidation of GLYAL3P,
catalyzed by glyceraldehyde-3-phosphate dehydrogenase Figure 6.11 - Reaction #7 - Substrate-level Phosphorylation by 1,3-BPG
Though there are a few substrate level phosphorylations in cells (including another one at the end of glycolysis), the vast major of
ATP is made by oxidative phosphorylation in the mitochondria (in animals). In addition to oxidative phosphorylation, plants also
make ATP by photophosphorylation in their chloroplasts. Since there are two 1,3 BPGs produced for every glucose, the two ATPs
produced in this reaction replenish the two ATPs used to start the cycle and the net ATP count at this point of the pathway is zero.
Reaction 8
3 −PG ⇄ 2 −PG (6.1.7)

Conversion of the 3-PG intermediate to 2-PG (2- phosphoglycerate) occurs by an important mechanism. An intermediate in this
readily reversible reaction (catalyzed by phosphoglycerate mutase - a mutase enzyme) is 2,3-BPG. This intermediate, which is
stable, is released with low frequency by the enzyme instead of being con- Figure 6.13 - Two routes to formation of 2,3-BPG
Figure 6.14 - 2,3- Bisphosphoglycerate (2,3-BPG) Figure 6.12 - Reaction #8 - Conversion of 3-PG to 2-PG verted to 2-PG. 2,3BPG
is important because it binds to hemoglobin and stimulates release of oxygen. The molecule can also be made from 1,3-BPG as a
product of a reaction catalyzed by bisphophglycerate mutase (Figure 6.13). Cells which are metabolizing glucose rapidly release
more 2,3-BPG and, as a result, get more oxygen, supporting their needs. Notably, cells which are metabolizing rapidly are using
oxygen more rapidly and are more likely to be deficient in it.
Reaction 9
2 −PG ⇄ PEP + H O (6.1.8)
2

2-PG is converted by enolase (a lyase) to phosphoenolpyruvate (PEP) by removal of water, creating a very high energy
intermediate. The reaction is readily reversible, but with PEP, the cell has one of its highest energy molecules and that is important
for the next reaction.
Reaction 10
+
PEP + ADP + H ⇄ PYR + ATP (6.1.9)

Conversion of PEP to pyruvate by pyruvate kinase is the second substrate level phosphorylation of glycolysis, creating ATP. This
reaction is what some refer to as the “Big Bang” of glycolysis because there is almost enough energy in PEP to stimulate
production of a second ATP (ΔG°’ = 31.6 kJ/ mol), but it is not used. Consequently, this energy is lost as heat. If you wonder why
you get hot when you exercise, the heat produced in the breakdown of glucose is a prime contributor and the pyruvate kinase
reaction is a major source. Figure 6.16 - Reaction #10 - The big bang - PEP phosphorylates ADP with a lot of energy to spare
Wikipedia Figure 6.15 - Reaction #9 - Enolase-catalyzed removal of water Wikipedia
Pyruvate kinase is the third and last enzyme of glycolysis that is regulated (see below). The primary reason this is the case is to be
able to prevent this reaction from occurring when cells are making PEP while going through gluconeogenesis (see more HERE).
Catabolism of other sugars
Though glycolysis is a pathway focused on the metabolism of glucose and fructose, the fact that other sugars can be readily
metabolized into glucose means that glycolysis can be used for extracting energy from them as well. Galactose is a good example.

6.1.3 https://bio.libretexts.org/@go/page/7836
It is commonly produced in the produced in the body as a result of hydrolysis of lactose, catalyzed by the enzyme known as lactase
(Figure 6.17). Deficiency of lactase is the cause of lactose intolerance.
Galactose begins preparation for entry into glycolysis by being converted to galactose-1- phosphate (catalyzed by galactokinase -
Figure 6.18). Galactose-1-phosphate swaps with glucose-1-phosphate from UDP-glucose to make UDP-galactose (Figure 6.19). An
epimerase converts UDPgalactose back to UDP-glucose and the cycle is complete. Each turn of the cycle thus takes in one
galactose-1-phosphate and releases one glucose-1-phosphate.
Deficiency of galactose conversion enzymes results in accumulation of galactose (from breakdown of lactose). Excess galactose is
converted to galactitol, a sugar alcohol. Galactitol in the human eye lens causes it to absorb water and this may be a factor in
formation of cataracts.Figure 6.17 - Breakdown of lactose to glucose and galactose by lactase Image by Pehr Jacobson Figure 6.18
- Galactokinase Reaction Image by Penelope Irving Free fructose can also enter glycolysis by two mechanisms. First, it can be
phosphorylated to fructose-6-phosphate by hexokinase. A more interesting alternate entry point is that shown in Figure 6.20.
Phosphorylation of fructose by fructokinase produces fructose-1-phosphate and cleavage of that by fructose-1- phosphate aldolase
yields DHAP and glyceraldehyde.
Phosphorylation of glyceraldehyde by triose kinase yields GLYAL3P. This alternative entry means for fructose may have important
implications because DHAP and GLYAL3P are introduced into the glycolysis pathway while bypassing PFK-1 regulation. Some
have proposed this may be important when considering metabolism of high fructose corn syrup, since it forces production of
pyruvate, a precursor of acetyl-CoA, which is itself a precursor of fatty acids when ATP levels are high.
Mannose metabolism
Mannose can also be metabolized in glycolysis. In this case, it enters via fructose by the following two-step process - 1)
phosphoryla- Figure 6.19 - Conversion of galactose-1-phosphate into glucose-6-phosphate Image by Aleia Kim tion by hexokinase
to make mannose-6- phosphate followed by its conversion to fructose-6-phosphate, catalyzed by phosphomannoisomerase (Figure
6.21).
Glycerol metabolism
Glycerol is an important molecule for the synthesis of fats, glycerophospholipids, and other membrane lipids. Most commonly it is
made into glycerol-3- phosphate (Figure 6.22) and the glycolysis/gluconeogenesis pathways are important both for producing the
compound and for metabolizing it. The relevant intermediate in these pathways both for producing and for using glycerol-3-
phosphate is DHAP. The enzyme glycerol-3-phosphate dehydrogenase reversibly converts glycerol-3- phosphate into DHAP
(Figure 6.22).
This reaction, which is an oxidation, transfers electrons to NAD+ to produce NADH. In the reverse reaction, production of
glycerol-3- phosphate from DHAP, of course, requires electrons from NADH for the reduction. Both glycolysis and
gluconeogenesis are sources DHAP, meaning when the cell needs glycerol- 3-phosphate that it can use sugars (glucose, fructose,
mannose, or galactose) as sources in glycolysis. For gluconeogenesis, sources include pyruvate, alanine and Figure 6.20 - Entry of
fructose into glycolysis, bypassing PFK-1 Image by Penelope Irving Figure 6.21 - Entry of other sugars into glycolysis Image by
Penelope Irving lactate (both can easily be made into pyruvate), oxaloacetate, aspartic acid (which can be made into oxaloacetate
by transamination), and others. All of the intermediates of the citric acid cycle (and glyoxylate cycle) can be converted ultimately
to oxaloacetate, which is a gluconeogenesis intermediate, as well.
It is worth noting that animals are unable to use fatty acids as materials for gluconeogenesis in net amounts, but they can, in fact,
use glycerol in both glycolysis and gluconeogenesis. It is the only part of the fat molecule that can be so used.
Pyruvate metabolism
As noted, pyruvate produced in glycolysis can be oxidized to acetyl-CoA, which is itself oxidized in the citric acid cycle to carbon
dioxide. That is not the only metabolic fate of pyruvate, though (Figure 6.23).
Pyruvate is a “starting” point for gluconeogenesis, being converted to oxaloacetate in the mitochondrion in the first step. Pyruvate
in animals can also be reduced to lactate by adding electrons from NADH (Figure 6.24). This reaction produces NAD+ and is
critical for generating the latter molecule to keep the glyceraldehyde-3-phosphate dehydrogenase reaction of glycolysis (reaction
#6) going under conditions when there is no oxygen.
This is because oxygen is necessary for the electron transport system (ETS) to operate and it performs the important function of
converting NADH back to NAD+. When the ETS is running, NADH donates electrons to Complex I and is oxidized to NAD+ in

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the process, generating the intermediate needed for oxidizing GLYAL-3P. In the absence of oxygen, however, NADH cannot be
converted to Figure 6.22 - Reactions in glycerol metabolism Image by Penelope Irving NAD+ by the ETS, so an alternative means
of making NAD+ is necessary for keeping glycolysis running under low oxygen conditions (fermentation).
Bacteria and yeast generate NAD+ under oxygen deprived conditions by doing fermentation in a different way (Figure 6.25). They
use NADH-requiring reactions that regenerate NAD+ while producing ethanol from pyruvate instead of making lactate. Thus,
fermentation of pyruvate is essential to keep glycolysis operating when oxygen is limiting. It is also for these reasons that brewing
of beer (using yeast) involves depletion of oxygen and muscles low in oxygen produce lactic acid (animals).
Pyruvate is a precursor of alanine which can be easily synthesized by transfer of a nitrogen from an amine donor, such as glutamic
acid. Pyruvate can also be converted into oxaloacetate by carboxylation in the process of gluconeogenesis (see below).
The enzymes involved in pyruvate metabolism include pyruvate dehydrogenase (makes acetyl-CoA), lactate dehydrogenase (makes
lactate), transaminases (make alanine), pyruvate carboxylase (makes ox- Figure 6.23 - Pyruvate metabolism. When oxygen is
absent, pyruvate is converted to lactate (animals) or ethanol (bacteria and yeast). When oxygen is present, pyruvate is converted to
acetyl-CoA. Not shown - Pyruvate transamination to alanine or carboxylation to form oxaloacetate. aloacetate), and pyruvate
decarboxylase (a part of pyruvate dehydrogenase that makes acetaldehyde in bacteria and yeast).
Catalytic action and regulation of the pyruvate dehydrogenase complex is discussed in the section on the citric acid cycle (HERE).

Gluconeogenesis
The anabolic counterpart to glycolysis is gluconeogenesis (Figure 6.26), which occurs mostly in the cells of the liver and kidney
and virtually no other cells in the body. In seven of the eleven reactions of gluconeogenesis (starting from pyruvate), the same
enzymes are used as in glycolysis, but the reaction directions are reversed. Notably, the ∆G values of these reactions in the cell are
typically near zero, meaning their direction can be readily controlled by changing substrate and product concentrations by small
amounts.
The three regulated enzymes of glycolysis all catalyze reactions whose cellular ∆G values are not close to zero, making
manipulation of reaction direction for their reac- Figure 6.24 - Formation of lactate in animal fermentation produces NAD+ for
G3PDH Image by Ben Carson Figure 6.25 - Formation of ethanol in microbial fermentation produces NAD+ for G3PDH Image by
Ben Carson tions non-trivial. Consequently, cells employ “work-around” reactions catalyzed by four different enzymes to favor
gluconeogenesis, when appropriate.
Bypassing pyruvate kinase
Two of the enzymes (pyruvate carboxylase and PEP carboxykinase - PEPCK) catalyze reactions that bypass pyruvate kinase.
F1,6BPase bypasses PFK-1 and G6Pase bypasses hexokinase. Notably, pyruvate carboxylase and G6Pase are found in the
mitochondria and endoplasmic reticulum, respectively, whereas the other two are found in the cytoplasm along with all of the
enzymes of glycolysis.
Biotin An important coenzyme used by pyruvate carboxylase is biotin (Figure 6.27). Biotin is commonly used by carboxylases to
carry CO2 to incorporate into the substrate.
Also known as vitamin H, biotin is a water soluble B vitamin (B7) needed for many metabolic processes, including fatty acid
synthesis, gluconeogenesis, and amino acid metabolism. Deficiency of the vitamin is rare, since it is readily produced by gut
Gluconeogenesis and glycolysis. Only the enzymes differing in gluconeogenesis are shown Image by Aleia Kim teria. There are
many claims of advantages of taking biotin supplements, but there is no strong indication of benefits in most cases. Deficiencies are
associated with inborn genetic errors, alcoholism, burn patients, and people who have had a gastrectomy. Some pregnant and
lactating women may have reduced levels due to increased biotin catabolism.
Reciprocal regulation
All of the enzymes of glycolysis and nine of the eleven enzymes of gluconeogenesis are all in the cytoplasm, necessitating a
coordinated means of controlling them. Cells generally need to minimize the extent to which paired anabolic and catabolic
pathways are occurring simultaneously, lest they produce a futile cycle, resulting in wasted energy with no tangible product except
heat. The mechanisms of controlling these pathways have opposite effects on catabolic and anabolic processes. This method of
control is called reciprocal regulation (see above).
Reciprocal regulation is a coordinated means of simultaneously controlling metabolic pathways that do opposite things. In
reciprocal regulation, a single molecule (allosteric regulation) or a single covalent modification

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(phosphorylation/dephosphorylation,
Allosteric Regulation of Glycolysis & Gluconeogenesis
Reciprocal Regulation
AMP - Activates PFK-1, Inhibits F1,6BPase
F2,6BP - Activates PFK-1, Inhibits F1,6BPase
Citrate - Activates PFK-1, Inhibits F1,6BPase
Glycolysis Only
ATP - Inhibits PFK-1 and Pyruvate Kinase
Alanine - Inhibits Pyruvate Kinase
Gluconeogenesis Only
ADP - Inhibits Pyruvate Carboxylase and PEPCK
Acetyl-CoA - Activates Pyruvate Carboxylase
Figure 6.27 - Biotin carrying carbon dioxide (red) Wikipedia for example) has opposite effects on the different pathways.
Reciprocal allosteric effects For example, in glycolysis, the enzyme known as phosphofructokinase (PFK-1) is allosterically
activated by AMP and a molecule known as F2,6BP (Figure 6.28). The corresponding enzyme from gluconeogenesis catalyzing a
reversal of the glycolysis reaction is known as F1,6BPase. F1,6BPase is inhibited by both AMP and F2,6BP.
Reciprocal covalent effects
In glycogen metabolism, the enzymes phosphorylase kinase and glycogen phosphorylase catalyze reactions important for the
breakdown of glycogen. The enzyme glycogen synthase catalyzes the synthesis of glyco- Directional velocity Inverts with
reciprocity If glycolysis is flowing Glucose synthesis awaits But when the latter is a-going Sugar breakdown then abates Figure
6.28 - Regulation of glycolysis (orange path) and gluconeogenesis (black path) Image by Aleia Kim gen. Each of these enzymes is,
at least partly, regulated by attachment and removal of phosphate.
Phosphorylation of phosphorylase kinase and glycogen phosphorylase has the effect of making them more active, whereas
phosphorylation of glycogen synthase makes it less active. Conversely, dephosphorylation has the reverse effects on these enzymes
- phosphorylase kinase and glycogen phosphorylase become less active and glycogen synthase becomes more active.
Simple and efficient
The advantage of reciprocal regulation schemes is that they are very efficient. It doesn’t take separate molecules or separate
treatments to control two pathways simultaneously. Further, its simplicity ensures that when one pathway is turned on, the other is
turned off.
This is especially important with catabolic/ anabolic regulation, because having both pathways going on simultaneously in a cell is
not very productive, leading only to production of heat in a futile cycle. A simple futile cycle is shown on Figure 6.29. If
unregulated, the cyclic pathway in the figure (shown in black) will make ATP in creating pyruvate from PEP and will use ATP to
make oxaloacetate from pyruvate.
It will also use GTP to make PEP from oxaloacetate. Thus, each turn of the cycle will make one ATP, use one ATP and use one
GTP for a net loss of energy. The process will start with pyruvate and end with pyruvate, so there is no net production of molecules.
(see HERE for one physiological use of a futile cycle).
Specific gluconeogenesis controls
Besides reciprocal regulation, other mechanisms help control gluconeogenesis. First, PEPCK is controlled largely at the level of
synthesis. Overexpression of PEPCK (stimulated by glucagon, glucocorticoid hormones, and cAMP and inhibited by insulin)
produces symptoms of diabetes.
Pyruvate carboxylase is sequestered in the mitochondrion (one means of regulation) Figure 6.29 - A simple futile cycle - follow the
black lines Image by Aleia Kim Interactive Learning Module HERE and is sensitive to acetyl-CoA, which is an allosteric activator.
Acetyl-CoA concentrations increase as the citric acid cycle activity decreases. Glucose-6- phosphatase is present in low

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concentrations in many tissues, but is found most abundantly and importantly in the major gluconeogenic organs – the liver and
kidney cortex.
Specific glycolysis controls
Control of glycolysis and gluconeogenesis is unusual for metabolic pathways, in that regulation occurs at multiple points. For
glycolysis, this involves three enzymes:
1. Hexokinase (Glucose ⇄ G6P)
2. Phosphofructokinase-1 (F6P ⇄ F1,6BP)
3. Pyruvate kinase (PEP ⇄ Pyruvate).
Regulation of hexokinase is the simplest of these. The enzyme is unusual in being inhibited by its product, glucose-6-phosphate.
This ensures when glycolysis is slowing down hexokinase is also slowing down to reduce feeding the pathway.
Pyruvate kinase
It might also seem odd that pyruvate kinase, the last enzyme in the pathway, is regulated (Figure 6.30), but the reason is simple.
Pyruvate kinase catalyzes the most energetically rich reaction of glycolysis. The reaction is favored so strongly in the forward
direction that cells must do a ‘two-step’ around it in the reverse direction when making glucose in the gluconeogenesis pathway. In
other words, it takes two enzymes, two reactions, and two triphosphates (ATP and GTP) to go from one pyruvate back to one PEP
in gluconeogenesis. When cells are needing to make glu- igure 6.30 - Regulation of pyruvate kinase For cells a glucose cycling’s
cost Is energy in reams Four ATPs each time is lost From breaking/making schemes So use for metabolic heat To make it warm
inside your feet Else it’s of no utility To practice such futility cose, they can’t be sidetracked by having the PEP they have made in
gluconeogenesis be converted directly back to pyruvate by pyruvate kinase. Consequently, pyruvate kinase must be inhibited
during gluconeogenesis or a futile cycle will occur and no glucose will be made.
Another interesting control mechanism called feedforward activation involves pyruvate kinase. Pyruvate kinase is activated
allosterically by the glycolysis intermediate, F1,6BP. This molecule is a product of the PFK-1 reaction and a substrate for the
aldolase reaction.
Reactions pulled
As noted above, the aldolase reaction is energetically unfavorable (high positive ∆G°’), thus allowing F1,6BP to accumulate. When
this happens, some of the excess F1,6BP binds to pyruvate kinase, which activates and jump- Figure 6.31 - Regulation of Synthesis
and Breakdown of F2,6BP Image by Penelope Irving starts the conversion of PEP to pyruvate. The resulting drop in PEP levels has
the effect of “pulling” on the reactions preceding pyruvate kinase. As a consequence, the concentrations of GLYAL3P and DHAP
fall, helping to pull the aldolase reaction forward.
PFK-1 regulation
PFK-1 has a complex regulation scheme. First, it is reciprocally regulated (relative to F1,6BPase) by three molecules. F2,6BP
activates PFK-1 and inhibits F1,6BPase. PFK-1 is also allosterically activated by AMP, whereas F1,6BPase is inhibited. On the
other hand, citrate inhibits PFK-1, but activates F1,6BPase.
PFK-1 is also inhibited by ATP and is exquisitely sensitive to proton concentration, easily losing activity when the pH drops only
slightly. PFK- 1’s inhibition by ATP is noteworthy and odd at first glance because ATP is also a substrate whose increasing
concentration should favor the reaction instead of inhibit it. The root of this conundrum is that PFK-1 has two ATP binding sites -
one at an allosteric site that binds ATP relatively inefficiently and one that the active site that binds ATP with high affinity. Thus,
only when ATP concentration is high is binding at the allosteric site favored and only then can ATP turn off the enzyme.
F2,6BP regulation
Regulation of PFK-1 by F2,6BP is simple at the PFK-1 level, but more complicated at the level of synthesis of F2,6BP. Despite
having a name sounding like a glycolysis/ gluconeogenesis intermediate (F1,6BP), F2,6BP is not an intermediate in either pathway.
Instead, it is made from fructose-6-phosphate and ATP by the enzyme known as phosphofructokinase-2 (PFK- 2 - Figure 6.31).
Cori cycle
With respect to energy, the liver and muscles act complementarily. The liver is the major or- Figure 6.32 - The Cori cycle Image by
Aleia Kim gan in the body for the synthesis of glucose. Muscles are major users of glucose to make ATP. Actively exercising
muscles use oxygen faster than the blood can deliver it. As a consequence, the muscles go anaerobic and produce lactate. This

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lactate is of no use to muscle cells, so they dump it into the blood. Lactate travels in the blood to the liver, which takes it up and
reoxidizes it back to pyruvate, catalyzed by the enzyme lactate dehydrogenase (Figure 6.32).
Pyruvate in the liver is then converted to glucose by gluconeogenesis. The glucose thus made by the liver is dumped into the
bloodstream where it is taken up by muscles and used for energy, completing the important intercellular pathway known as the Cori
cycle.
Glucose alanine cycle
The glucose alanine cycle (also known as the Cahill Cycle), has been described as the amine equivalent of the Cori cycle (Figure
6.33). The Cori cycle, of course, exports lac- Figure 6.33 - Overlap between the Cori cycle and the glucose alanine cycle tate from
muscles (when oxygen is limiting) to the liver via the bloodstream. The liver, in turn, converts lactate to glucose, which it ships
back to the muscles via the bloodstream. The Cori Cycle is an essential source of glucose energy for muscles during periods of
exercise when oxygen is used faster than it can be delivered.
In the glucose-alanine cycle, cells are generating toxic amines and must export them. This is accomplished by transaminating
pyruvate (the product of glycolysis) to produce the amino acid alanine.
The glucose-alanine process requires the enzyme alanine aminotransferase, which is found in muscles, liver, and intestines. Alanine
is exported in the process to the blood and picked up by the liver, which deaminates it to release the amine for synthesis of urea and
excretion. The pyruvate left over after the transamination is a substrate for gluconeogenesis. Glucose produced in the liver is then
exported to the blood for use by cells, thus completing the cycle.
Polysaccharide metabolism
Sugars are metabolized rapidly in the body and that is one of the primary reasons they are used. Managing levels of glucose in the
body is very important - too much leads to complications related to diabetes and too little gives rise to hypoglycemia (low blood
sugar). Sugars in the body are maintained by three processes - 1) diet; 2) synthesis (gluconeogenesis); and 3) storage. The storage
forms of sugars are, of course, the polysaccharides and their metabolism is our next topic of discussion.
Amylose and amylopectin
The energy needs of a plant are much less dynamic than those of animals. Muscular contraction, nervous systems, and information
processing in the brain require large amounts of quick energy. Because of this, the polysaccharides stored in plants are somewhat
less complicated than those of animals. Plants store glucose for energy in the form of amylose (Figure 6.34 and see HERE) and
amylopectin and for structural integrity in the form of cellulose (see HERE). These structures differ in that cellulose contains
glucose units solely joined by β-1,4 bonds, whereas amylose has only α-1,4 bonds and amylopectin has α-1,4 and α-1,6 bonds.
Figure 6.34 Amylose, a polymer of glucose in plants
Glycogen
Animals store glucose primarily in liver and muscle in the form of a compound related to amylopectin known as glycogen. The
structural differences between glycogen and amylopectin are solely due to the frequency of the α-1,6 branches of glucoses. In
glycogen they occur about every 10 residues instead of every 30-50, as in amylopectin (Figure 6.35).
Glycogen provides an additional source of glucose besides that produced via gluconeogenesis. Because glycogen contains so many
glucoses, it acts like a battery backup for the body, providing a quick source of glucose when needed and providing a place to store
excess glucose when glucose concentrations in the blood rise.
The branching of glycogen is an important feature of the molecule metabolically as well. Since glycogen is broken down from the
"ends" of the molecule, more branches translate to more ends, and more glucose that can be released at once.
Just as in gluconeogenesis, the cell has a separate mechanism for glycogen synthesis that is distinct from glycogen breakdown. As
noted previously, this allows the cell to separately control the reactions, avoiding futile cycles, and enabling a process to occur
efficiently (synthesis of glycogen) that would not occur if Figure 6.35 - Glycogen Structure - α-1,4 links with α-1,6 branches every
7-10 residues it were simply the reversal of glycogen breakdown.
Glycogen breakdown
Breakdown of glycogen involves 1) release of glucose-1-phosphate (G1P), 2) rearranging the remaining glycogen (as necessary) to
permit continued breakdown, and 3) conversion of G1P to G6P for further metabolism. G6P can be 1) used in glycolysis, 2)
converted to glucose by gluconeogenesis, or 3) oxidized in the pentose phosphate pathway.

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Glycogen phosphorylase (sometimes simply called phosphorylase) catalyzes breakdown of glycogen into glucose-1- Phosphate
(G1P - Figure 6.36). The reaction that produces G1P from glycogen is a phosphorolysis, not a hydrolysis reaction. The distinction
is that hydrolysis reactions use water to cleave bigger molecules into smaller ones, but phosphorolysis reactions use phosphate
instead for the same purpose. Note that the phosphate is just that - it does NOT come from ATP. Since ATP is not used to put
phosphate on G1P, the reaction saves the cell energy.
Glycogen debranching enzyme
Glycogen phosphorylase will only act on nonreducing ends of a glycogen chain that are at least 5 glucoses away from a branch
point. A second enzyme, Glycogen Debranching Enzyme (GDE) (also called debranching enzyme), is therefore needed to convert
α (1-6) branches to α (1-4) branches. GDE acts on glycogen branches that have reached their limit of phosphorylysis with glycogen
phosphorylase. Figure 6.36 - Breaking of α-1,4 bonds of glycogen by glycogen phosphorylase Image by Aleia Kim Interactive
Learning Module HERE
GDE acts to transfer a trisaccharide from an α-1,6 branch onto an adjacent α-1,4 branch, leaving a single glucose at the 1,6 branch.
Note that the enzyme also catalyzes the hydrolysis of the remaining glucose at the 1,6 branch point (Figure 6.37). Thus, the
breakdown products from glycogen are G1P and glucose (mostly G1P). Glucose can, of course, be converted to Glucose-6-
Phosphate (G6P) as the first step in glycolysis by either hexokinase or glucokinase.
G1P can be converted to G6P by action of an enzyme called phosphoglucomutase. This reaction is readily reversible, allowing G6P
and G1P to be interconverted as the concentration of one or the other increases. This is important, because phosphoglucomutase is
needed to form G1P for glycogen synthesis.
Regulation of glycogen metabolism
Regulation of glycogen metabolism is complex, occurring both allosterically and via hormone-receptor controlled events that result
in protein phosphorylation or dephosphorylation. In order to avoid a futile cycle of glycogen synthesis and breakdown
simultaneously, cells have evolved an elaborate set of controls that ensure only one pathway is primarily active at a time.
Regulation of glycogen metabolism is managed by the enzymes glycogen phosphorylase and glycogen synthase. Glycogen
phosphorylase is regulated by both allosteric factors (ATP, G6P, AMP, and glucose) and by covalent modification (phosphorylation
/ dephosphorylation). Its regulation is consistent with the energy needs of the cell. High energy molecules (ATP, G6P, glucose) al-
Figure 6.37 - Catalytic activity of debranching enzyme losterically inhibit glycogen phosphorylase, while the low energy molecule
AMP allosterically activates it.
GPa/GPb allosteric regulation
Glycogen phosphorylase exists in two different covalent forms – one form with phosphate (called GPa here) and one form lacking
phosphate (GPb here). GPb is converted to GPa by phosphorylation by an enzyme known as phosphorylase kinase. GPa and GPb
can each exist in an 'R' state and a 'T' state (Figure 6.38). For both GPa and GPb, the R state is the more active form of the enzyme.
GPa's negative allosteric effector (glucose) is usually not abundant in cells, so GPa does not flip into the T state often. There is no
positive allosteric effector of GPa. When glucose is absent, GPa automatically flips into the R (more active) state (Figure 6.39). It
is for this reason that people tend to think of GPa as being the more active covalent form of the enzyme.
GPb can convert from the GPb T state to the GPb R state by binding AMP. Unless a cell is low in energy, AMP concentration is
low. Thus GPb is not converted Figure 6.38 - Glycogen phosphorylase regulation - covalent (horizontal) and allosteric (vertical)
Image by Aleia Kim to the R state very often. This is why people think of the GPb form as less active than GPa. On the other hand,
ATP and/or G6P are usually present at high enough concentration in cells that GPb is readily flipped into the T state (Figure 6.40).
GPa/GPb covalent regulation
The relative amounts of GPa and GPb largely govern the overall process of glycogen breakdown, since GPa tends to be active more
often than GPb. It is i
Phosphorylase kinase itself has two covalent forms – phosphorylated (active) and dephosphorylated (inactive). It is phosphorylated
by the enzyme Protein Kinase A (PKA - ). Another way to activate the enzyme is allosterically with calcium (Figure 6.41).
Phosphory- Figure 6.39 - Allosteric regulation of GPa Image by Aleia Kim Figure 6.40 - Allosteric regulation of GPb Image by
Aleia Kim lase kinase is dephosphorylated by phosphoprotein phosphatase, the same enzyme that removes phosphate from GPa.
PKA and cAMPcAMP

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PKA is activated by cAMP, which is, in turn, produced by adenylate cyclase after activation by a G-protein (See HERE for
overview). G-proteins are activated ultimately by binding of ligands to specific membrane receptors called 7-TM receptors, also
known as Gprotein coupled receptors. These are discussed in greater detail HERE. Common ligands for 7-TM receptors include
epinephrine (binds β- adrenergic receptor) and glucagon (binds glucagon receptor). Epinephrine exerts its greatest effects on
muscle and glucagon works preferentially on the liver. Thus, epinephrine and glucagon can activate glycogen breakdown by
stimulating synthesis of cAMP followed by the cascade of events described above.
Turning off glycogen breakdown
Turning off signals is as important, if not more so, than turning them on. Glycogen is a precious resource. If its breakdown is not
controlled, a lot of energy used in its synthesis is wasted. The steps in the glycogen breakdown regulatory pathway can be reversed
at every level. First, the ligand (epinephrine or glucagon) can leave the receptor, turning off the stimulus. Second, the G-proteins
have an inherent GTPase activity. GTP, of course, is what activates Gproteins, so a GTPase activity converts the GTP it is carrying
to GDP and the G-protein becomes inactive. Thus, G-proteins turn off Figure 6.41 - Activation of phosphorylase kinase Image by
Aleia Kim their own activity. Interfering with their ability to convert GTP to GDP can have dire consequences, including cancer in
some cases.
Third, cells have phosphodiesterase enzymes (inhibited by caffeine) for breaking down cAMP. cAMP is needed to activate PKA, so
breaking it down stops PKA from activating phosphorylase kinase. Fourth, the enzyme known as phosphoprotein phosphatase (also
called PP1) plays a major role. It can remove phosphates from phosphorylase kinase (inactivating it) and form GPa, converting it to
the less likely to be active GPb. Regulation of phosphoprotein phosphatase activity occurs at several levels. Two of these are shown
in Figures 6.42 & 6.43.
In Figure 6.42, phosphoprotein phosphatase is shown being inactivated by phosphorylation of an inhibitor (called PI-1 - see below).
This happens as a result of cascading actions from binding of epinephrine (or glucagon) to a cell’s β-adrenergic receptor. Reversal
of these actions occurs when insulin binds to the cell’s insulin receptor, resulting in activation of phosphoprotein phosphatase.
PI-1
The inhibitor PI-1 can block activity of phosphpoprotein phosphatase only if it (PI-1) is phosphorylated. When PI-1 gets
dephosphorylated, it no longer functions as an inhibitor, so phosphoprotein phosphatase be- Figure 6.42 - Inactivation of
phosphoprotein phosphatase by protein kinase A via phosphorylation of PI-1 (Inhibitor) and the GM binding protein Image by Pehr
Jacobson Interactive Learning Module HERE comes active. Now, here is the clincher - PI-1 gets phosphorylated by PKA (thus,
when epinephrine or glucagon binds to a cell) and gets dephosphorylated when insulin binds to a cell.
Another regulatory mechanism
Another way to regulate phosphoprotein phosphatase in the liver involves GPa directly (Figure 6.43). In liver cells, phosphoprotein
phosphatase is bound to a protein called GL. GL can also bind to GPa. As shown in the figure, if the three proteins are complexed
together (top of figure), then PP1 (phosphoprotein phosphatase) is inactive. When glucose is present (such as when the liver has
made too much glucose), then the free glucose binds to the GPa and causes GPa to be released from the GL.
This has the effect of activating phosphoprotein phosphatase, which begins dephosphorylating enzymes. As shown in the figure,
two such enzymes are GPa (making GPb) and glycogen synthase b, making glycogen synthase a. These dephosphorylations have
opposite effects on the two enzymes, making GPb, which is less active and glycogen synthase a, which is much more active.
Glycogen synthesis
The anabolic pathway opposing glycogen breakdown is that of glycogen synthesis. Just Figure 6.43 - Regulation of phosphoprotein
phosphatase (PP-1) activity by GPa Image by Penelope Irving as cells reciprocally regulate glycolysis and gluconeogenesis to
prevent a futile cycle between these pathways, so too do cells use reciprocal schemes to regulate glycogen breakdown and
synthesis.
Synthesis of glycogen starts with G1P, which is converted to an 'activated' intermediate, UDPglucose. This activated intermediate is
what 'adds' the glucose to the growing glycogen chain in a reaction catalyzed by the enzyme known as glycogen synthase (Figure
6.44). Once the glucose is added to glycogen, the glycogen molecule may need to have branches inserted in it by the enzyme
known as branching enzyme (Figure 6.45).
Steps

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Let us first consider the steps in glycogen synthesis. 1) Glycogen synthesis from glucose involves phosphorylation to form G6P,
and isomerization to form G1P (using phospho- igure 6.45 - Branch formation in glycogen by branching enzyme Image by
Penelope Irving Figure 6.44 - Catalytic activity of glycogen synthase Image by Penelope Irving glucomutase, common to glycogen
breakdown). G1P is reacted with UTP to form UDP-glucose in a reaction catalyzed by UDP-glucose pyrophosphorylase. Glycogen
synthase catalyzes synthesis of glycogen by joining carbon #1 of the UDP-derived glucose onto the carbon #4 of the non-reducing
end of a glycogen chain, to form the familiar α(1,4) glycogen links. Another product of the reaction is UDP.
“Primer” requirements
It is also worth noting, in passing, that glycogen synthase will only add glucose units from UDP-Glucose onto a preexisting
glycogen chain that has at least four glucose residues. Linkage of the first few glucose units to form the minimal "primer" needed
for glycogen synthase recognition is catalyzed by a protein called glycogenin, which attaches to the first glucose and catalyzes
linkage of the first eight glucoses by α(1,4) bonds. 3) The characteristic α(1,6) branches of glycogen are the products of the enzyme
known as branching enzyme. Branching enzyme breaks α(1,4) chains and carries the broken chain to the carbon #6 and forms an
α(1,6) linkage (Figure 6.45).
Regulation of glycogen synthesis
The regulation of glycogen biosynthesis is reciprocal to that of glycogen breakdown. It also has a cascading covalent modification
system similar to the glycogen breakdown system described above. In fact, part of the system is identical to glycogen breakdown.
Epinephrine or glucagon signaling stimulates adenylate cyclase to make cAMP, which activates PKA. Figure 6.46 - Reciprocal
regulation by the phosphorylation cascade - glycogen breakdown activated / glycogen synthesis inhibited Image by Penelope Irving
Effect of phosphorylation
In glycogen synthesis, protein kinase A phosphorylates the active form of glycogen synthase (GSa), and converts it into the usually
inactive b form (called GSb).
Note the conventions for glycogen synthase and glycogen phosphorylase. For both enzymes, the more active forms are called the 'a'
forms (GPa and GSa) and the less active forms are called the 'b' forms (GPb and GSb). The major difference, however, is that GPa
has a phosphate, but GSa does not and GPb has no phosphate, but GSb does.
Thus phosphorylation and dephosphorylation have opposite effects on the enzymes of glycogen metabolism (Figure 6.46). This is
the hallmark of reciprocal regulation. It is of note that the less active glycogen synthase form, GSb, can be activated by G6P. Recall
that G6P had the exactly opposite effect on GPb.
Glycogen synthase, glycogen phosphorylase (and phosphorylase kinase) can all be dephosphorylated by the same enzyme -
phosphoprotein phosphatase - and it is activated when insulin binds to its receptor in the cell membrane.
Big picture
In the big picture, binding of epinephrine or glucagon to appropriate cell receptors stimulates a phosphorylation cascade which
simultaneously activates breakdown of glycogen by glycogen phosphorylase and inhibits synthesis of glycogen by glycogen
synthase. Epinephrine, is also known as adrenalin, and the properties that adrenalin gives arise from a large temporary increase of
blood glucose, which powers muscles.
On the other hand, insulin stimulates dephosphorylation by activating phosphoprotein phosphatase. Dephosphorylation reduces
action of glycogen phosphorylase (less glycogen breakdown) and activates glycogen synthase (starts glycogen synthesis). Our
bodies make glycogen when blood glucose levels rise. Since high blood glucose levels are harmful, insulin stimulates cells to take
up glucose. In the liver and in muscle cells, the uptaken glucose is made into glycogen. Figure 6.47 - Cotton - the purest natural
form of cellulose Wikipedia Interactive Learning Module HERE
Cellulose synthesis
Cellulose is synthesized as a result of catalysis by cellulose synthase. Like glycogen synthesis it requires an activated intermediate
to add glucose residues and there are two possible ones - GDP-glucose and UDPglucose, depending on which cellulose synthase is
involved. In plants, cellulose provides support to cell walls.
The reaction catalyzed is shown next where Cellulosen = a polymer of [(1→4)-β-Dglucosyl] n units long.
The GDP-glucose reaction is the same except with substitution of GDP-glucose for UDP-Figure 6.48 - The Pentose Phosphate
Pathway - Enzymes - 1 = G6P dehydrogenase / 2 = 6-Phosphogluconolactonase / 3 = 6-PG dehydrogenase / 4a = Ribose 5-

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phosphate isomerase / 4b = Ribulose 5-phosphate 3-epimerase / 5,7 = Transketolase / 6 = Transaldolase UDP-glucose + Cellulosen
UDP + Cellulosen+1 glucose. UDP-glucose for the reaction is obtained by catalysis of sucrose synthase. The enzyme is named for
the reverse reaction.
Pentose phosphate pathway
The pentose phosphate pathway (PPP - also called the hexose monophosphate shunt) is an oxidative pathway involving sugars that
is sometimes described as a parallel pathway to glycolysis. It is, in fact, a pathway with multiple inputs and outputs (Figure 6.48).
PPP is also a major source of NADPH for biosynthetic reactions and can provide ribose-5-phosphate for nucleotide synthesis.
Though when drawn out, the pathway’s “starting point” is often shown as glucose-6-phosphate (G6P), in fact there are multiple
entry points including other glycolysis intermediates, such as fructose-6-phosphate (F6P) and glyceraldehyde-3-phosphate
(GLYAL-3-P), as well as less common sugar compounds with 4,5, and 7 carbons.
The multiple entry points and multiple outputs gives the cell tremendous flexibility to meet its needs by allowing it to use a variety
of materials to make any of these products.
Oxidation #1
Beginning with G6P, PPP proceeds through its oxidative phase as follows:
The enzyme catalyzing the reaction is G6P dehydrogenase. It is the rate limiting step of the pathway and the enzyme is inhibited
both by NADPH and acetyl-CoA. NADPH is important for anabolic pathways, such as fatty acid synthesis and also for maintaining
glutathione in a reduced state. The latter is important in protection against damage from reactive oxygen species.
Deficiency of the G6P dehydrogenase enzyme is not rare, leading to acute hemolytic anemia, due to reduced NADPH
concentration, and a reduced ability of the cell to disarm reactive oxygen species with glutathione. Reduced activity of the enzyme
appears to have a protective effect against malarial infection, likely due to the increased fragility of the red blood cell membrane,
which is then unable to sustain an infection by the parasite. Hydrolysis Reaction #2 is a hydrolysis and it is catalyzed by
Hydrolysis
Reaction #2 is a hydrolysis and it is catalyzed by 6-phosphogluconolactonase. The reac- Sucrose + UDP UDP-glucose + Fructose
G6P + NADP+ 6-Phosphoglucono-δ-lactone + NADPH tion converts the circular 6-phosphoglucono- δ-lactone into the linear 6-
phosphogluconate (6-PG) in preparation for oxidation in the next step.
Decarboxylation
Reaction #3 is the only decarboxylation in the PPP and the last oxidative step. It is catalyzed by 6-phosphogluconate
dehydrogenase.
Mutations disabling the protein made from this gene negatively impact red blood cells. At this point, the oxidative phase of PPP is
complete and the remaining reactions involve molecular rearrangements. Ru5P has two possible fates and these are each described
below.
Isomerization
Reaction 4a: The enzyme catalyzing this reversible reaction is Ru5P isomerase (top of next column). It is important because this is
the way cells make R-5-P for nucleotide synthesis. The R-5-P can also be used in other PPP reactions shown elsewhere.
Epimerization
Reaction 4b (catalyzed by Ru-5-P epimerase) is another source of a pentose sugars and provides an important substrate for
subsequent reactions.
Transketolase reactions
The other reactions don’t really have an order to them and whether they occur or not depends on cellular needs. The first enzyme,
transketolase, is flexible in terms of its substrate/product combinations and is used not only in PPP, but also in the Calvin cycle of
plants. It catalyzes the next two reactions
In the first reaction (above), two phosphorylated sugars of 5 carbons each are converted into one phosphorylated sugar of 3 carbons
and one of 7 carbons. In the second (next page), a five carbon sugar phosphate and aRu-5-P Xylulose-5-phosphate (Xu-5-P) Xu-5-
P + R-5-P GLYAL-3-P + Sedoheptulose-7-phosphate (S-7-P) 6-PG + NADP+ Ribulose-5-phosphate (Ru-5-P) + NADPH + CO2 6-

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Phosphoglucono-δ-lactone + H2O 6-phosphogluconate (6-PG) + H+ Ru-5-P Ribose-5-phosphate (R-5-P) four carbon sugar
phosphate are rearranged into sugar phosphates with 3 and 6 carbons.
Glycolysis intermediates
In the reversible reactions of the pentose phosphate pathway, one can see how glycolysis intermediates can easily be rearranged and
made into other sugars. Thus, GLYAL-3-P and F6P can be readily made into Ribose-5- phosphate for nucleotide synthesis.
Involvement of F6P in the pathway permits cells to continue making nucleotides (by making R-5-P) or tryptophan (by making E-
4-P) even if the oxidative reactions of PPP are inhibited.
The last reaction is catalyzed by the enzyme known as transaldolase.
TPP co-factor
Transketolase uses thiamine pyrophosphate (TPP) to catalyze reactions. TPP’s thiaFigure 6.49 - Intermediates of the pentose
phosphate pathway Xu-5-P + Erythrose-4-phosphate (E-4-P) GLYAL-3-P + F6P GLYAL-3-P + S-7-P E-4-P + F6P zole ring’s
nitrogen and sulfur atoms on either side of a carbon, allow it to donate a proton and act as an acid, thus forming a carbanion, which
gets stabilized by the adjacent tetravalent nitrogen (Figures 6.50 & 6.51)).
The stabilized carbanion plays important roles in the reaction mechanism of enzymes, such as transketolase that use TPP as a
cofactor. Commonly, the carbanion acts as a nucleophile that attacks the carbonyl carbon of the substrate. Such is the case with
transketolase. Attack by the carbanion breaks the carbonyl bond on the substrate and covalently links it to the ionized carbon of
TPP, thus allowing it to “carry” the carbonyl group to the other substrate for attachment. In this way, two carbons are moved from
Xu- 5-P to E-4-P to make F6P (from E-4-P) and GLYAL-3-P (from Xu-5-P). Similarly, S-7-P and GLYAL-3-P are made from R-5-
P and Xu-5-P, respectively.
Thiamines
Thiamines are a class of compounds involved in catalysis of important respiration-related The Pentose Phosphate Pathway by
Kevin Ahern I need erythrose phosphate And don’t know what to do My cells are full of G-6-P And NADP too But I just hit upon a
plan As simple as can be I’ll run reactions through the path That’s known as PPP In just two oxidations There’s ribulose-5P Which
morphs to other pentoses Each one attached to P The next step it is simple Deserving of some praise The pentose carbons mix and
match Thanks to transketolase Glyceraldehyde’s a product Sedoheptulose is too Each with a trailing phosphate But we are not quite
through Now three plus seven is the same As adding six and four By swapping carbons back and forth There’s erythrose-P and
more At last I’ve got the thing I need From carbons trading places I’m happy that my cells are full Of some transaldolases Figure
6.50 - Thiamine pyrophosphate reactions in the citric acid cycle, pyruvate metabolism, the pentose phosphate pathway, and the
Calvin cycle. Thiamine was the first water-soluble vitamin (B1) to be discovered via association with the peripheral nervous
system disease known as Beriberi. Thiamine pyrophosphate (TPP) is an enzyme cofactor found in all living systems derived from
thiamine by action of the enzyme thiamine diphosphokinase. TPP facilitates catalysis of several biochemical reactions essential for
tissue respiration.
Deficiency of the vitamin is rare today, though people suffering from Crohn’s disease, anorexia, alcoholism or undergoing kidney
dialysis may develop deficiencies. TPP is required for the oxidative decarboxylation of pyruvate to form acetyl-CoA and similar
reactions. Transketolase, an important enzyme in the pentose phosphate pathway, also uses it as a coenzyme. Besides these
reactions, TPP is also required for oxidative decarboxylation of α-keto acids like α-ketoglutarate and branched-chain α-keto acids
arising from metabolism of valine, isoleucine, and leucine. Figure 6.51 - Mechanism of action of thiamine pyrophosphate (TPP) -
1) Carbanion formation; 2) Nucleophilic attack; 3) Covalent attachment of carbonyl; 4) Transfer to second group; 5) Release of
product and regeneration of TPP
TPP acts in the pyruvate dehydrogenase complex to assist in decarboxylation of pyruvate and “carrying” the activated acetaldehyde
molecule to its attachment (and subsequent oxidation) to lipoamide. Central to TPP’s function is the thiazolium ring, which
stabilizes carbanion intermediates (through resonance) by acting as an electron sink (Figure 6.51). Such action facilitates breaking
of carbon-carbon bonds such as occurs during decarboxylation of pyruvate to produce the activated acetaldehyde.
Thiamine deficiency
Thiamine is integral to respiration and is needed in every cell. Acute deficiency of thiamine leads to numerous problems - the best
known condition is beriberi, whose symptoms include weight loss, weakness, swelling, neurological issues, and irregular heart
rhythms. Figure 6.52 - The Calvin cycle - The resynthesis phase has multiple steps and is described below. Image by Aleia Kim

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Causes of deficiency include poor nutrition, significant intake of foods containing the enzyme known as thiaminase, foods with
compounds that counter thiamine action (tea, coffee), and chronic diseases, including diabetes, gastrointestinal diseases, persistent
vomiting. People with severe alcoholism often are deficient in thiamine.
Calvin cycle
The Calvin cycle (Figure 6.52) is a metabolic pathway occurring exclusively in photosynthetic organisms. Commonly referred to as
the “Dark Cycle” or the Light-Independent Cycle, the Calvin cycle does not actually occur in the dark. The cell/chloroplast simply
is not directly using light energy to drive it.
Assimilation
It is in the Calvin cycle of photosynthesis that carbon dioxide is taken from the atmosphere and ultimately built into glucose (or
other sugars). Reactions of the Calvin cycle take place in regions of the chloroplast known as the stroma, the fluid areas outside of
the thylakoid membranes. The cycle can be broken into three phases
1) assimilation of CO2
2) reduction reactions
3) regeneration of the starting material, ribulose 1,5 bisphosphate (Ru1,5BP).
Though reduction of carbon dioxide to glucose ultimately requires electrons from twelve molecules of NADPH (and 18 ATPs), it is
confusing because one reduction occurs 12 times (1,3 BPG to GLYAL-3P) to input the overall reduction necessary to make one
glucose.
Carbon dioxide
Another reason students find the pathway confusing is because the carbon dioxide molecules are absorbed one at a time into six
different molecules of Ru1,5BP. At no point are the six carbons ever together in the same molecule to make a single glucose.
Instead, six molecules of Ru1,5BP (30 carbons) gain six more carbons via carbon dioxide and then split into 12 molecules of 3-
phosphoglycerate (36 carbons). The gain of six carbons allows two three carbon molecules to be produced in excess for each turn
of the cycle. These two molecules molecules are then converted into glucose using the enzymes of gluconeogenesis. The other ten
molecules of 3-PG are used to regenerate the six molecules of Ru1,5BP. Figure 6.53 - Rubisco, the most abundant enzyme on Earth
Cyclic pathway
Like the citric acid cycle, the Calvin cycle doesn’t really have a starting or ending point, but can we think of the first reaction as the
fixation of carbon dioxide to Ru1,5BP. This reaction is catalyzed by the enzyme known as ribulose-1,5 bisphosphate carboxylase
(RUBISCO - Figure 6.53). The resulting six carbon intermediate is unstable and is rapidly converted to two molecules of 3-
phosphoglycerate.
As noted, if one starts with 6 molecules of Ru1,5BP and makes 12 molecules of 3-PG, the extra 6 carbons that are a part of the
cycle can be shunted off as two three-carbon molecules of glyceraldehyde-3-phosphate (GLYAL3P) to gluconeogenesis, leaving
behind 10 molecules to be reconverted into 6 moleFigure 6.54 - Resynthesis phase of the Calvin cycle - All paths lead to
regenerating Ru1,5BP, which is the aim of the resynthesis phase. Glycolysis/gluconeogenesis intermediates shown in blue. Enzyme
numbers explained in text. cules of Ru1,5BP. This occurs in what is called the resynthesis phase.
Resynthesis phase
The resynthesis phase (Figure 6.54) requires multiple steps, but only utilizes two enzymes unique to plants - sedoheptulose-1,7
bisphosphatase and phosphoribulokinase. RUBISCO is the third (and only other) enzyme of the pathway that is unique to plants.
All of the other enzymes of the pathway are common to plants and animals and include some found in the pentose phosphate
pathway and gluconeogenesis. Enzymes shown as numbers in Figure 6.54 are as follows (enzymes unique to plants in green):
1 - Phosphoglycerate kinase
2 - G3PDH
3 - Triosephosphate Isomerase
4 - Aldolase
5 - Fructose 1,6 bisphosphatase

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6 - Transketolase
7 - Phosphopentose Epimerase
8 - Phosphoribulokinase
9 - Sedoheptulose 1,7 bisphosphatase
10 - Phosphopentose Isomerase
Reactions
The resynthesis phase begins with conversion of the 3-PG molecules into GLYAL3P (there are actually 10 GLYAL3P molecules
involved in resynthesis, as noted above, but we are omitting numbers to try to help students to see the bigger picture. Suffice it to
say that there are sufficient quantities of all of the molecules to complete the reactions described). Some GLYAL3P is converted to
DHAP by triose phosphate isomerase. Some DHAPs are converted (via gluconeogenesis) to F6P (one phosphate is lost for each
F6P).
Two carbons from F6P are given to GLYAL3P to create E-4P and Xu-5P (reversal of PPP reaction). E- 4P combines with DHAP to
form sedoheptulose-1,7 bisphosphate (S1,7BP). The phosphate at position #1 is Figure 6.55 - Use of CO2 (Calvin cycle) vs. O2
(photorespiration) by RUBISCO. Image by Pehr Jacobson cleaved by sedoheptulose-1,7 bisphosphatase to yield S-7-P.
Transketolase (another PPP enzyme) catalyzes transfer to two carbons from S-7-P to GLYAL3P to yield Xu-5P and R5P.
Phosphopentose isomerase catalyzes conversion of R5P to Ru5P and phosphopentose epimerase similarly converts Xu-5P to Ru5P.
Finally, phosphoribulokinase transfers a phosphate to Ru5P (from ATP) to yield Ru1,5BP.
Photorespiration
In the Calvin cycle of photosynthesis, the enzyme ribulose-1,5-bisphosphate carboxylase (RUBISCO) catalyzes the addition of
carbon dioxide to ribulose-1,5- bisphosphate (Ru1,5BP) to create two molecules of 3-phosphoglycerate. Molecular oxygen (O2),
however, competes with CO2 for this enzyme, so about 25% of the time, the molecule that gets added is not CO2, but rather O2
(Figure 6.55). When this happens, the following reaction occurs
This is the first step in the process known as photorespiration. The process of photorespiration is inefficient relative to the
carboxylation of Ru1,5BP. Phosphoglycolate is converted to glyoxylate in the glyoxysome and then transamination of that yields
glycine. Two glycines can combine in a complicated coupled set of reactions in the mitochondrion shown next. Figure 6.56 - Maize
- a C4 plant Ru1,5BP + O2 Phosphoglycolate + 3-phosphoglycerate + 2H+ 2 Glycine + NAD+ + H2O Serine + CO2 + NH3 +
NADH + H+
Deamination and reduction of serine yields pyruvate, which can be then be converted back to 3-phosphoglycerate. The end point of
oxygenation of Ru1,5BP is the same as the carboxylation of Ru1,5BP reactions, but there are significant energy costs associated
with it, making the process less efficient.
C4 plants
The Calvin cycle is the means by which plants assimilate carbon dioxide from the atmosphere, ultimately into glucose. Plants use
two general strategies for doing so. The first is employed by plants called C3 plants (most plants) and it simply involves the
pathway described above. They are called C3 plants because the first stable intermediate after absorbing carbon dioxide contains
three carbons - 3-phosphoglycerate. Another class of plants, called C4 plants (Figure 6.56) employ a novel strategy for
concentrating the Figure 6.57 - Assimilation of CO2 by C4 plants Image by Aleia Kim CO2 prior to assimilation. C4 plants are
generally found in hot, dry environments where conditions would otherwise favor the wasteful photorespiration reactions of
RUBISCO and loss of water.
Capture by PEP
In C4 plants, carbon dioxide is captured in special mesophyll cells first by phosphoenolpyruvate (PEP) to make oxaloacetate
(contains four carbons and gives the C4 plants their name - Figure 6.57). The oxaloacetate is converted to malate and transported
into bundle sheath cells where the carbon dioxide is released and captured by Ru1,5BP, as in C3 plants. The Calvin cycle proceeds
from there. The advantage of the C4 plant scheme is that it allows concentration of carbon dioxide while minimizing loss of water
and photorespiration.
Peptidoglycan synthesis
Bacterial cell walls contain a layer of protection known as the peptidoglycan layer. Assembly of the layer begins in the cytoplasm.

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Steps in the process follow
1. Donation of an amine from glutamine to fructose-6- phosphate and isomerization to make glucosamine-6- phosphate.
2. Donation of an acetyl group from acetyl-CoA to make N-acetylglucosamine-6- phosphate
3. Isomerization of N-acetylglucosamine-6- phosphate makes N-acetylglucosamine-1- phosphate Figure 6.58 - Peptidoglycan layer
in a bacterial outer cell wall Wikipedia
4. UTP combines with N-acetylglucosamine-1-phosphate to make UDP-N-acetyl-glucosamine-1- phosphate
5. Addition of PEP and electrons from NADPH yields UDP-Nacetylmuramic acid
6. A pentapeptide or tetrapeptide chain is attached to the UDP-Nacetylmuramic acid. The sequence varies a bit between species,
but commonly is L-Ala - D-Glu - L-Lys - DAla - D-Ala
7. Dolichol phosphate replaces UMP on the UDP-N-acetylmuramic acid-pentapeptide.
8. UDP-N-acetyl-glucosamine donates a glucose to the Nacetylmuramic acid part of the Dolichol-PP-N-acetylmuramic
acidpentapeptide
9. A pentapeptide chain of glycines (pentaglycine) is linked to lysine of the pentapeptide chain to create a Dolichol-PP-
Nacetylmuramic acid-N-acetylglucosaminedecapeptide. The pentaglycine serves as cross links in the overall structure.
10. Dolichol-PP is removed to yield Nacetylmuramic acid-N-acetylglucosaminedecapeptide Figure 6.60 - Catalytic activity of
DDtranspeptidase Wikipedia Figure 6.59 - Penicillin
11. This last group is added to the growing peptidoglycan network by joining the pentaglycine of one chain to the tetrapeptide/
pentapeptide of another.
The enzyme catalyzing the addition of the N-acetylmuramic acid-N-acetylglucosaminedecapeptide to the network in the last step is
DD-transpeptidase. This is the cellular enzyme targeted by penicillin and its derivatives. One reason penicillin is so effective is
because synthesis of a peptidoglycan cell wall for a single bacterium requires millions of cycles of reactions above. Even slowing
down the process can have a major effect on bacterial growth. On the flip side, resistance to penicillin and derivatives arises as a
result of mutations in one enzyme - the transpeptidase.
Metabolons
At this point, it is appropriate to bring up the concept of metabolons. Metabolons are cellular complexes containing multiple
enzymes of a metabolic pathway that appear to be arranged so that the product of one enzymatic reaction is passed directly as
substrate to the enzyme that catalyzes the next reaction in the metabolic pathway. The structural complexes are temporary and are
held together by non-covalent forces.
Metabolons appear to offer advantages of reducing the amount of water needed to hydrate enzymes. Activity of enzymes in the
complex is increased. Most of the basic metabolic pathways are thought to use metabolons. They include glycolysis, the citric acid
cycle, nucleotide metabolism, glycogen synthesis, steroid synthesis, DNA synthesis, RNA synthesis, the urea cycle, and the process
of electron transport.
Hypoxia
Hypoxia occurs when the body or a region of it has an insufficient oxygen supply. Varia- Figure 6.61 - Hypoxia inducible factor
tions in arterial oxygen concentrations in normal physiology may lead to hypoxia, for example, during hypoventilation training or
strenuous physical exercise. Generalized hypoxia may appear in healthy people when at high altitudes. Cancer cells, which may be
undergoing faster respiration than surrounding tissues may also tend to be hypoxic. Hypoxia is an important consideration for sugar
metabolism due to the ability of cells to change their sugar metabolism (fermentation) when these conditions exist.
The body’s response to hypoxia is to produce Hypoxia-Inducible Factors (HIFs), which are transcription factors that induce
expression of genes to help cells adapt to the hypoxic conditions. Many of the genes activated by HIFs are enzymes of glycolysis
and GLUTs (glucose transport proteins). The combination of these gene products allows cells to 1) import more glucose and 2)
metabolize it more rapidly when it arrives. This is to be expected because anaerobic sugar metabolism is only about 1/15th as
efficient as aerobic metabolism. Consequently, it requires much more sugar metabolism to keep the cancer cells alive. A recently
discovered protein called cytoglobin is believed to help assist in hypoxia by facilitating transfer of oxygen from arteries to the
brain.

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Covalent modification
HIFs are regulated partly by an interesting covalent modification. When oxygen concentration is high, the enzyme prolyl
hydroxylase will hydroxylate proline residues in HIFs. This stimulates the protein degradation system (proteasome) to degrade
them. When oxygen concentration is low, the hydroxylation occurs to a much lower extent (or does not occur at all),
reducing/stopping degradation of HIFs and allowing them to activate genes. In this way, the concentration of HIFs is kept high
under low oxygen concentration (to activate HIF genes) and low under high oxygen concentrations (to stop synthesis of HIF
genes).

This page titled 6.1: Metabolism - Sugars is shared under a CC BY-NC-SA license and was authored, remixed, and/or curated by Kevin Ahern,
Indira Rajagopal, & Taralyn Tan.

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6.2: Citric Acid Cycle & Related Pathways
Source: BiochemFFA_6_2.pdf. The entire textbook is available for free from the authors at
http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy

Citric acid cycle

The primary catabolic pathway in the body is the citric acid cycle because it is here that oxidation to carbon dioxide occurs for
breakdown products of the cell’s major building blocks - sugars, fatty acids, and amino acids. The pathway is cyclic (Figure 6.63)
and thus, doesn’t really have a starting or ending point. All of the reactions occur in mitochondria, though one enzyme is embedded
in the organelle’s inner membrane. As needs change, cells may use a subset of the reactions of the cycle to produce a desired
molecule rather than to run the entire cycle (see HERE).

Figure 6.63 - Amino acid metabolism and the citric acid cycle. Amino acids boxed in yellow are made from the indicated
intermediate. Amino acids in blue are made into the intermediate in catabolism. Image by Aleia Kim

Acetyl-CoA
The molecule “feeding” the citric acid cycle is acetyl-CoA and it can be obtained from pyruvate (from glycolysis), from fatty acid
β-oxidation, from ketone bodies, and from amino acid metabolism. Molecules from other pathways feeding into the citric acid
cycle for catabolism make the citric acid cycle ‘cataplerotic’. It is worth noting that acetyl-CoA has very different fates, depending
on the cell’s energy status/needs (see HERE). The description below describes oxidation (catabolism) in citric acid cycle.
Anabolically, acetyl-CoA is also very important for providing building blocks for synthesis of fatty acids, ketone bodies, amino
acids and cholesterol. Other citric acid cycle intermediates are also important in amino acid metabolism (Figure 6.63), heme
synthesis, electron shuttling, and shuttling of acetyl-CoA across the mitochondrial inner membrane. The ability of the citric acid
cycle to supply intermediates to pathways gives rise to the term ‘anaplerotic.’ It means ‘to fill up.’ Before discussing the citric acid
cycle, it is important to first describe one important enzyme complex that is a major source of acetyl-CoA for the cycle.

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 Figure 6.64 - E1 Subunit of Pyruvate Dehydrogenase. Wikipedia

Pyruvate decarboxylation
The pyruvate dehydrogenase enzyme is a complex of multiple copies of three subunits that catalyze the decarboxylation of
pyruvate to form acetyl-CoA. The reaction mechanism requires use of five coenzymes. Pyruvate dehydrogenase is an enormous
complex in mammals with a size five times greater than ribosomes.

Subunits
The three subunits are designated by E1, E2, and E3. E2 is also referred to as dihydrolipoamide acetyltransferase and E3 is more
precisely called dihydrolipoyl dehydrogenase. Confusion arises with the name for E1. Some call it pyruvate dehydrogenase and
others give it the name pyruvate decarboxylase. We will use pyruvate decarboxylase solely to refer to E1 and pyruvate
dehydrogenase only to refer to the complex of E1, E2, and E3.
The catalytic actions of pyruvate dehydrogenase can be broken down into three steps, each taking place on one of the subunits. The
steps, sequentially occurring on E1, E2, and E3, are 1) decarboxylation of pyruvate; 2) oxidation of the decarboxylated product;
and 3) transfer of electrons to ultimately form NADH (Figure 6.65).

Figure 6.65 - Mechanism of action of pyruvate decarboxylation and oxidation by pyruvate dehydrogenase.

Catalysis
The catalytic process begins after binding of the pyruvate substrate with activation of the thiamine pyrophosphate coenzyme
through formation of an ylide intermediate. The nucleophilic carbanion of the ylide attacks the electrophilic ketone carbon on the
pyruvate, releasing carbon dioxide and creating an enol that loses a proton on the carbon to become a 1,3 dipole that includes the

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positively charged nitrogen of the thiamine. The reaction (step A in Figure 6.65) is a non-oxidative decarboxylation. Oxidation of
the two carbon hydroxyethyl unit occurs in the transfer to the lipoamide.

Reductive acetylation
Reductive acetylation occurs next (Step B) as the 2-carbon hydroxyethyl unit is transferred to lipoamide on E2. (Lipoamide is the
name for a molecule of lipoic acid covalently attached to a lysine side chain in the E2 subunit). In prokaryotes in the absence of
oxygen, the hydroxyethyl group is not passed to lipoamide, but instead is released as free acetaldehyde , which can accept electrons
from NADH (catalyzed by alcohol dehydrogenase) and become ethanol in the process of fermentation. In the presence of oxygen in
almost all aerobic organisms, the process continues with transfer of the hydroxyethyl unit to E2 and continuation of the cycle
below.

Figure 6.66 - Oxidized and reduced structures of lipoamide (lipoic acid linked to lysine)

Oxidation step
Transfer of the hydroxyethyl group from E1 to the lipoamide coenzyme in E2 is an oxidation, with transfer of electrons from the
hydroxyethyl group to lipoamide’s disulfide (reducing it) and formation on the lipoamide of an acetyl-thioester (oxidizing it).
The acetyl group is then transferred from lipoamide to coenzyme A in E2 (Step C in Figure 6.65), forming acetyl-CoA, which is
released and leaving reduced sulfhydryls on the lipoamide. In order for the enzyme to return to its original state, the disulfide bond
on lipoamide must be re-formed. This occurs with transfer of electrons from reduced lipoamide to an FAD covalently bound to E3
(Step D). This reduces FAD to FADH2.

Formation of NADH
In the last step in the process, electrons from FADH2 are transferred to external NAD+, forming NADH (Step E) and completing
the overall cycle. Then enzyme can then begin another catalytic round by binding to a pyruvate.

Pyruvate dehydrogenase regulation

Pyruvate deyhdrogenase is regulated both allosterically and by covalent modification - phosphorylation / dephosphorylation.
Regulation of pyruvate dehydrogenase, whether by allosteric or covalent mechanisms has the same strategy. Indicators of high
energy shut down the enzyme, whereas indicators of low energy stimulate it. For allosteric regulation, the high energy indicators
affecting the enzyme are ATP, acetyl-CoA, NADH, and fatty acids, which inhibit it. AMP, Coenzyme A, NAD+, and calcium, on
the other hand, stimulate it (Figure 6.67).

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 Figure 6.67 - Regulation scheme for pyruvate dehydrogenase (PD). Image by Aleia Kim

Covalent modification
Covalent modification regulation of pyruvate dehydrogenase is a bit more complicated. It occurs as a result of phosphorylation by
pyruvate dehydrogenase kinase (PDK - Figure 6.67) or dephosphorylation by pyruvate dehydrogenase phosphatase (PDP).
PDK puts phosphate on any one of three serine residues on the E1 subunit, which causes pyruvate kinase to not be able to perform
its first step of catalysis - the decarboxylation of pyruvate. PDP can remove those phosphates. PDK is allosterically activated in the
mitochondrial matrix when NADH and acetyl-CoA concentrations rise.

Product inhibition
Thus, the products of the pyruvate dehydrogenase reaction inhibit the production of more products by favoring its phosphorylation
by PDK. Pyruvate, a substrate of pyruvate dehydrogenase, inhibits PDK, so increasing concentrations of substrate activate pyruvate
dehydrogenase by reducing its phosphorylation by PDK. As concentrations of NADH and acetyl-CoA fall, PDP associates with
pyruvate kinase and removes the phosphate on the serine on the E1 subunit.

Figure 6.68 - Pyruvate dehydrogenase complex with three phosphorylation sites in red marked by arrows.Wikipedia
Low concentrations of NADH and acetyl-CoA are necessary for PDP to remain on the enzyme. When those concentrations rise,
PDP dissociates and PDK gains access to the serine for phosphorylation. Insulin and calcium can also activate the PDP. This is very
important in muscle tissue, since calcium is a signal for muscular contraction, which requires energy. Insulin also also activates
pyruvate kinase and the glycolysis pathway to use internalized glucose. It should be noted that the cAMP phosphorylation cascade
from the β-adrenergic receptor has no effect on pyruvate kinase, though the insulin cascade does, in fact, affect PDP and pyruvate
kinase.

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 Figure 6.69 - The citric acid cycle. Image by Aleia Kim

Citric acid cycle reactions

Focusing on the pathway itself (Figure 6.69), the usual point to start discussion is addition of acetyl-CoA to oxaloacetate (OAA) to
form citrate.
Acetyl-CoA for the pathway can come from a variety of sources. The reaction joining it to OAA is catalyzed by citrate synthase
and the ∆G°’ is fairly negative. This, in turn, helps to “pull” the malate dehydrogenase reaction preceding it in the cycle.
In the next reaction, citrate is isomerized to isocitrate by action of the enzyme called aconitase.
Isocitrate is a branch point in plants and bacteria for the glyoxylate cycle (see HERE). Oxidative decarboxylation of isocitrate by
isocitrate dehydrogenase produces the first NADH and yields α-ketoglutarate.
This five carbon intermediate is a branch point for synthesis of the amino acid glutamate. In addition, glutamate can also be made
easily into this intermediate in the reverse reaction. Decarboxylation of α-ketoglutarate produces succinyl-CoA and is catalyzed by
α-ketoglutarate dehydrogenase.
The enzyme α-ketoglutarate dehydrogenase is structurally very similar to pyruvate dehydrogenase and employs the same five
coenzymes – NAD+, FAD, CoA-SH, thiamine pyrophosphate, and lipoamide.

Regeneration of oxaloacetate
The remainder of the citric acid cycle involves conversion of the four carbon succinyl-CoA into oxaloacetate. Succinyl-CoA is a
branch point for the synthesis of heme (see HERE). Succinyl-CoA is converted to succinate in a reaction catalyzed by succinyl-
CoA synthetase (named for the reverse reaction) and a GTP is produced, as well – the only substrate level phosphorylation in the
cycle.

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The energy for the synthesis of the GTP comes from hydrolysis of the high energy thioester bond between succinate and the CoA-
SH. Evidence for the high energy of a thioester bond is also evident in the citrate synthase reaction, which is also very energetically
favorable. Succinate is also produced by metabolism of odd-chain fatty acids (see HERE).

Succinate Oxidation
Oxidation of succinate occurs in the next step, catalyzed by succinate dehydrogenase. This interesting enzyme both catalyzes this
reaction and participates in the electron transport system, funneling electrons from the FADH2 it gains in the reaction to coenzyme
Q. The product of the reaction, fumarate, gains a water across its trans double bond in the next reaction, catalyzed by fumarase to
form malate.
Fumarate is also a byproduct of nucleotide metabolism and of the urea cycle. Malate is important also for transporting electrons
across membranes in the malate-aspartate shuttle (see HERE) and in ferrying carbon dioxide from mesophyll cells to bundle sheath
cells in C4 plants (see HERE).

Figure 6.70 - Succinyl-CoA synthetase mechanism

Figure 6.71 - Succinate dehydrogenase embedded in the mitochondrial inner membrane (top). Wikipedia

Figure 6.72 - Succinate dehydrogenase reaction. Image by Aleia Kim

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Rare oxidation
Conversion of malate to oxaloacetate by malate dehydrogenase is a rare biological oxidation that has a ∆G°’ with a positive value
(29.7 kJ/mol).
The reaction is ‘pulled’ by the energetically favorable conversion of oxaloacetate to citrate in the citrate synthase reaction described
above. Oxaloacetate intersects other important pathways, including amino acid metabolism (readily converted to aspartic acid),
transamination (nitrogen movement) and gluconeogenesis.
It is worth noting that reversal of the citric acid cycle theoretically provides a mechanism for assimilating CO2. In fact, this reversal
has been noted in both anaerobic and microaerobic bacteria, where it is called the Arnon-Buchanan cycle (Figure 6.73).

Figure 6.73 - Arnon-Buchanon cycle. Alternative enzymes shown on right in lavender. Fd = ferredoxin. Wikipedia

Regulation of the citric acid cycle

Allosteric regulation of the citric acid cycle is pretty straightforward. The molecules involved are all substrates/products of the
pathway or molecules involved in energy transfer. Substrates/products that regulate or affect the pathway include acetyl-CoA and
succinyl-CoA .

Inhibitors and activators

High energy molecular indicators, such as ATP and NADH will tend to inhibit the cycle and low energy indicators (NAD+, AMP,
and ADP) will tend to activate the cycle. Pyruvate dehydrogenase, which catalyzes formation of acetyl-CoA for entry into the cycle
is allosterically inhibited by its product (acetyl-CoA), as well as by NADH and ATP.

Regulated enzymes
Regulated enzymes in the cycle include citrate synthase (inhibited by NADH, ATP, and succinyl-CoA), isocitrate dehydrogenase
(inhibited by ATP, activated by ADP and NAD+), and α-ketoglutarate dehydrogenase (inhibited by NADH and succinyl-CoA and
activated by AMP).

Anaplerotic/cataplerotic pathway
The citric acid cycle is an important catabolic pathway oxidizing acetyl-CoA into CO2 and generating ATP, but it is also an
important source of molecules needed by cells and a mechanism for extracting energy from amino acids in protein breakdown and
other breakdown products. This ability of the citric acid cycle to supply molecules as needed and to absorb metabolic byproducts
gives great flexibility to cells. When citric acid cycle intermediates are taken from the pathway to make other molecules, the term
used to describe this is cataplerotic, whereas when molecules are added to the pathway, the process is described as anaplerotic.

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Cataplerotic molecules
The citric acid cycle’s primary cataplerotic molecules include α-ketoglutarate, succinyl-CoA, and oxaloacetate. Transamination of
α-ketoglutarate and oxaloacetate produces the amino acids glutamate and aspartic acid, respectively. Oxaloacetate is important for
the production of glucose in gluconeogenesis.
Glutamate plays a very important role in the movement of nitrogen through cells via glutamine and other molecules and is also
needed for purine synthesis. Aspartate is a precursor of other amino acids and for production of pyrimidine nucleotides. Succinyl-
CoA is necessary for the synthesis of porphyrins, such as the heme groups in hemoglobin, myoglobin and cytochromes.
Citrate is an important source of acetyl-CoA for making fatty acids. When the citrate concentration is high (as when the citric acid
cycle is moving slowly or is stopped), it gets shuttled across the mitochondrial membrane into the cytoplasm and broken down by
the enzyme citrate lyase to oxaloacetate and acetyl-CoA. The latter is a precursor for fatty acid synthesis in the cytoplasm.

Anaplerotic molecules
Anaplerotic molecules replenishing citric acid cycle intermediates include acetyl-CoA (made in many pathways, including fatty
acid oxidation, pyruvate decarboxylation, amino acid catabolism, and breakdown of ketone bodies), α-ketoglutarate (from amino
acid metabolism), succinyl-CoA (from propionic acid metabolism), fumarate (from the urea cycle and purine metabolism), malate
(carboxylation of PEP in plants), and oxaloacetate (many sources, including amino acid catabolism and pyruvate carboxylase
action on pyruvate in gluconeogenesis)

Glyoxylate cycle

Figure 6.74 - Overview of the glyoxylate cycle. Image by Aleia Kim

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 Figure 6.75 - Reactions of the glyoxylate cycle. Wikipedia
A pathway related to the citric acid cycle found only in plants and bacteria is the glyoxylate cycle (Figures 6.74 & 6.75). The
glyoxylate cycle, which bypasses the decarboxylation reactions while using most of the non-decarboxylation reactions of the citric
acid cycle, does not operate in animals, because they lack two enzymes necessary for it – isocitrate lyase and malate synthase. The
cycle occurs in specialized plant peroxisomes called glyoxysomes. Isocitrate lyase catalyzes the conversion of isocitrate into
succinate and glyoxylate. Because of this, all six carbons of the citric acid cycle survive each turn of the cycle and do not end up as
carbon dioxide.
Succinate continues through the remaining reactions to produce oxaloacetate. Glyoxylate combines with another acetyl-CoA (one
acetyl-CoA was used to start the cycle) to create malate (catalyzed by malate synthase). Malate can, in turn, be oxidized to
oxaloacetate.
It is at this point that the glyoxylate pathway’s contrast with the citric acid cycle is apparent. After one turn of the citric acid cycle,
a single oxaloacetate is produced and it balances the single one used in the first reaction of the cycle. Thus, in the citric acid cycle,
there is no net production of oxaloacetate in each turn of the cycle.

Net oxaloacetate production

On the other hand, thanks to assimilation of carbons from two acetyl-CoA molecules, each turn of the glyoxylate cycle results in
two oxaloacetates being produced, after starting with one. The extra oxaloacetate of the glyoxylate cycle can be used to make other
molecules, including glucose in gluconeogenesis. This is particularly important for plant seed germination (Figure 6.76), since the
seedling is not exposed to sunlight. With the glyoxylate cycle, seeds can make glucose from stored lipids.
Because animals do not run the glyoxylate cycle, they cannot produce glucose from acetyl-CoA in net amounts, but plants and
bacteria can. As a result, plants and bacteria can turn acetyl-CoA from fat into glucose, while animals can’t. Bypassing the
oxidative decarboxylations (and substrate level phosphorylation) has energy costs, but, there are also benefits. Each turn of the

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glyoxylate cycle produces one FADH2 and one NADH instead of the three NADHs, one FADH2, and one GTP made in each turn
of the citric acid cycle.

Figure 6.76 - A gingko seed embryo. Wikipedia

Carbohydrate needs
Organisms that make cell walls, such as plants, fungi, and bacteria, need large quantities of carbohydrates as they grow to support
the biosynthesis of the complex structural polysaccharides of the walls. These include cellulose, glucans, and chitin. Notably, each
of the organisms can operate the glyoxylate cycle using acetyl-CoA from β-oxidation.

Coordination of the glyoxylate cycle and the citric acid cycle

The citric acid cycle is a major catabolic pathway producing a considerable amount of energy for cells, whereas the glyoxylate
cycle’s main function is anabolic - to allow production of glucose from fatty acids in plants and bacteria. The two pathways are
physically separated from each other (glyoxylate cycle in glyoxysomes / citric acid cycle in mitochondria), but nonetheless a
coordinated regulation of them is important.
The enzyme that appears to provide controls for the cycle is isocitrate dehydrogenase. In plants and bacteria, the enzyme can be
inactivated by phosphorylation by a kinase found only in those cells. Inactivation causes isocitrate to accumulate in the
mitochondrion and when this happens, it gets shunted to the glyoxysomes, favoring the glyoxylate cycle. Removal of the phosphate
from isocitrate dehydrogenase is catalyzed by an isocitrate dehydrognease-specific phosphoprotein phosphatase and restores
activity to the enzyme.
When this happens, isocitrate oxidation resumes in the mitochondrion along with the rest of the citric acid cycle reactions. In
bacteria, where the enzymes for both cycles are present together in the cytoplasm, accumulation of citric acid cycle intermediates
and glycolysis intermediates will tend to favor the citric acid cycle by activating the phosphatase, whereas high energy conditions
will tend to favor the glyoxylate cycle by inhibiting it.

Figure 6.77 - Acetyl-CoA metabolism. Image by Aleia Kim

Acetyl-CoA metabolism
Acetyl-CoA is one of the most “connected” metabolites in biochemistry, appearing in fatty acid oxidation/synthesis, pyruvate
oxidation, the citric acid cycle, amino acid anabolism/catabolism, ketone body metabolism, steroid/bile acid synthesis, and (by
extension from fatty acid metabolism) prostaglandin synthesis . Most of these pathways will be dealt with separately. Here we will
cover ketone body metabolism.

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Ketone body metabolism
Ketone bodies are molecules made when the blood levels of glucose fall very low. Ketone bodies can be converted to acetyl-CoA
by reversing the reaction of the pathway that makes them (Figure 6.78). Acetyl CoA, of course, can be used for ATP synthesis via
the citric acid cycle. People who are very hypoglycemic (including some diabetics) will produce ketone bodies (Figure 6.79) and
these are often first detected by the smell of acetone on their breath.

Figure 6.78 Ketone body metabolism. Image by Pehr Jacobson

Figure 6.79 - Three ketone bodies - acetone (top), acetoacetic acid (middle), and β-hydroxybutyrate (bottom)

Overlapping pathways
The pathways for ketone body synthesis and cholesterol biosynthesis (Figure 6.80 and see HERE) overlap at the beginning. Each of
these starts by combining two acetyl-CoAs together to make acetoacetyl-CoA. Not coincidentally, that is the next to last product of
β-oxidation of fatty acids with even numbers of carbons (see HERE for fatty acid oxidation). In fact, the enzyme that catalyzes the

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joining is the same as the one that catalyzes its breakage in fatty acid oxidation – thiolase. Thus, these pathways start by reversing
the last step of the last round of fatty acid oxidation.

Figure 6.80 - Diverging biosynthetic pathways for ketone bodies (left) and cholesterol biosynthesis (right). Image by Penelope
Irving

HMG-CoA formation
Both pathways also include addition of two more carbons to acetoacetyl-CoA from a third acetyl-CoA to form hydroxy-methyl-
glutaryl-CoA, or HMG-CoA, as it is more commonly known. It is at this point that the two pathways diverge. HMG-CoA is a
branch point between the two pathway and can either go on to become cholesterol or ketone bodies. In the latter pathway, HMG-
CoA is broken down into acetyl-CoA and acetoacetate.
Acetoacetate is itself a ketone body and can be reduced to form another one, D-β-hydroxybutyrate (not actually a ketone, though).
Alternatively, acetoacetate can be converted into acetone. This latter reaction can occur either spontaneously or via catalysis by
acetoacetate decarboxylase. Acetone can be converted into pyruvate and pyruvate can be made into glucose.
D-β-hydroxybutyrate travels readily in the blood and crosses the blood-brain barrier. It can be oxidized back to acetoacetate,
converted to acetoacetyl-CoA, and then broken down to two molecules of acetyl-CoA for oxidation in the citric acid cycle.

Ketosis
When a body is producing ketone bodies for its energy, this state in the body is known as ketosis. Formation of ketone bodies in the
liver is critical. Normally glucose is the body’s primary energy source. It comes from the diet, from the breakdown of storage
carbohydrates, such as glycogen, or from glucose synthesis (gluconeogenesis). Since the primary stores of glycogen are in muscles
and liver and since gluconeogenesis occurs only in liver, kidney, and gametes, when the supply of glucose is interrupted for any
reason, the liver must supply an alternate energy source.

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From fatty acid breakdown
In contrast to glucose, ketone bodies can be made in animals from the breakdown of fat/fatty acids. Most cells of the body can use
ketone bodies as energy sources. Ketosis may arise from fasting, a very low carbohydrate diet or, in some cases, diabetes.

Acidosis
The term acidosis refers to conditions in the body where the pH of arterial blood drops below 7.35. It is the opposite of the
condition of alkalosis, where the pH of the arterial blood rises above 7.45. Normally, the pH of the blood stays in this narrow pH
range. pH values of the blood lower than 6.8 or higher than 7.8 can cause irreversible damage and may be fatal. Acidosis may have
roots in metabolism (metabolic acidosis) or in respiration (respiratory acidosis).
There are several causes of acidosis. In metabolic acidosis, production of excess lactic acid or failure of the kidneys to excrete acid
can cause blood pH to drop. Lactic acid is produced in the body when oxygen is limiting, so anything that interferes with oxygen
delivery may create conditions favoring production of excess lactic acid. These may include restrictions in the movement of blood
to target tissues, resulting in hypoxia (low oxygen conditions) or decreases in blood volume. Issues with blood movement can
result from heart problems, low blood pressure, or hemorrhaging.
Strenuous exercise can also result in production of lactic acid due to the inability of the blood supply to deliver oxygen as fast as
tissues require it (hypovolemic shock). At the end of the exercise, though, the oxygen supply via the blood system quickly catches
up.
Respiratory acidosis arises from accumulation of carbon dioxide in the blood. Causes include hypoventilation, pulmonary
problems, emphysema, asthma, and severe pneumonia.

Figure 6.81 - Symptoms of acidosis

This page titled 6.2: Citric Acid Cycle & Related Pathways is shared under a CC BY-NC-SA license and was authored, remixed, and/or curated
by Kevin Ahern, Indira Rajagopal, & Taralyn Tan.

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6.3: Fats and Fatty Acids
Source: BiochemFFA_6_3.pdf. The entire textbook is available for free from the authors at
http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy
In the modern Western world, which is fat and getting fatter, there is a tremendous amount of interest in the metabolism of fat and
fatty acids. Fat is the most important energy storage form of animals, storing considerably more energy per carbon than
carbohydrates, but its insolubility in water requires the body to package it specially for transport. Surprisingly, fat/fatty acid
metabolism is not nearly as tightly regulated as that of carbohydrates. Neither are the metabolic pathways of breakdown and
synthesis particularly complicated, either.
Movement of dietary fat
Before we discuss the breakdown and synthesis of fat, let us first discuss the movement of dietary fat and oil (triglycerides - Figure
6.82) in the body. Upon consumption of triglycerides in the diet, they first are solubilized in the digestive system by the churning
action of the stomach and the emulsifying properties of the bile acids.
Upon passing into the lumen of the intestine, the triglycerides are acted on first by enzymes known as lipases that use water twice
on each triglyceride to release two fatty acids, leaving behind a monoacylglyceride. As shown in Figure 6.83, the fatty acids and
the monoacylglyceride are moved across the intestinal wall into the lymph system where they are reassembled back into a
triglyceride. In the lymph system triglycerides and other insoluble lipids are packaged into lipoprotein complexes called
chylomicrons that enter the blood stream and travel to target cells. The journey of lipids in the body after leaving the digestive
system is long and is discussed in more depth HERE.
In the body, fat is stored in specialized cells known as adipocytes. When these cells receive appropriate signals, they begin the
breakdown of fat into glycerol and fatty acids.
Breakdown of fat
Breakdown of fat in adipocytes requires catalytic action of three enzymes. The first of these is controlled by binding of hormones
to the cell membrane (Figure 6.84). It is the only regulated enzyme of fat breakdown and is known as hormone sensitive
triacylglycerol lipase. It removes the first fatty acid from the fat. Diacylglyceride lipase removes the second one and
monoacylglyceride lipase removes the third. As noted, only the first one is regulated and it appears to be the rate limiting reaction
when active.
Epinephrine activation
As shown in Figure 6.84, activation of hormone sensitive triacylglycerol lipase (HSTL) is accomplished by epinephrine stimulation
process and that it overlaps with the same activation that stimulates glycogen breakdown and gluconeogenesis.
This coordination is very important. Each of the pathways stimulated by the epinephrine signaling system aims to provide the body
with more materials to catabolize for energy - sugars and fatty acids. The HSTL is inhibited by dephospohrylation and this is
stimulated by binding of insulin to its cell membrane receptor.

Perilipin
A protein playing an important roles in regulation of fat breakdown is perilipin. Perilipin associates with fat droplets and helps
regulate action of HSTL, the enzyme catalyzing the first reaction in fat catabolism. When perilipin is not phosphorylated, it coats
the fat droplet and prevents HSTL from getting access to it. Activation of protein kinase A in the epinephrine cascade, however,
results in phosphorylation of both perilipin and HSTL. When this occurs, perilipin loosens its tight binding to the fat droplet,
allowing digestion of the fat to begin by HSTL.
Perilipin expression is high in obese organisms and some mutational variants have been associated with obesity in women. Another
mutation reduces perilipin expression and is associated with greater lipolysis (fat breakdown) in women. Mice lacking perilipin eat
more food than wild-type mice, but gain 1/3 less weight when on the same diet.

Fat synthesis
Synthesis of fat requires action of acyl transferase enzymes, such as glycerol-3 O-phosphate acyl transferase, which catalyzes
addition of fatty acids to the glycerol backbone (reaction #1 above). The process requires glycerol-3-phosphate (or DHAP) and
three fatty acids. In the first reaction, glycerol-3-phosphate is esterified at position 1 with a fatty acid, followed by a duplicate

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reaction at position 2 to make phosphatidic acid (diacylglycerol phosphate). This molecule, which is an intermediate in the
synthesis of both fats and phosphoglycerides, gets dephosphorylated to form diacylglycerol before the esterification of the third
fatty acid to the molecule to make a fat.
Fatty acids released from adipocytes travel in the bloodstream bound to serum albumin. Arriving at target cells, fatty acids are
taken up by membrane-associated fatty acid binding proteins, which help control cellular fatty acid uptake by transport proteins.
Players in this process include CD36, plasma membrane-associated fatty acid-binding protein, and a family of fatty acid transport
proteins (called FATP1-6).
Fatty acid oxidation
Upon arrival inside of target cells, fatty acids are oxidized in a process that chops off two carbons at a time to make acetyl-CoA,
which is subsequently oxidized in the citric acid cycle. Depending on the size of the fatty acid, this process (called β-oxidation) will
begin in either the mitochondrion (Figure 6.86) or the peroxisomes (see HERE).

Transport
To be oxidized in the mitochondrion, fatty acids must first be attached to coenzyme A (CoA-SH or CoA) and transported through
the cytoplasm and the outer mitochondrial membrane. In the mitochondrion’s intermembrane space, the CoA on the fatty acid is
replaced by a carnitine (Figure 6.87) in order to be moved into the matrix. After this is done, the fatty acid linked to carnitine is
transported into the mitochondrial matrix and in the matrix the carnitine is replaced again by coenzyme A. It is in the mitochondrial
matrix where the oxidation occurs. The fatty acid linked to CoA (called an acyl-CoA) is the substrate for fatty acid oxidation.
Steps
The process of fatty acid oxidation (Figure 6.88) is fairly simple. The reactions all occur between carbons 2 and 3 (with #1 being
the one linked to the CoA) and sequentially include the following steps 1) dehydrogenation to create FADH2 and a fatty acyl group
with a double bond between carbons 2 and 3 in the trans configuration; 2) hydration across the double bond to put a hydroxyl group
on carbon 3 in the L configuration; 3) oxidation of the hydroxyl group to make a ketone; and 4) thiolytic cleavage to release acetyl-
CoA and a fatty acid two carbons shorter than the starting one.
Enzymes of β-oxidation
Two of the enzymes of β-oxidation are notable. The first is acyl-CoA dehydrogenase, which catalyzes the dehydrogenation in the
first reaction and yields FADH2. The enzyme comes in three different forms – ones specific for long, medium, or short chain length
fatty acids. The first of these is sequestered in the peroxisomes of animals (see below) whereas the ones that work on medium and
shorter chain fatty acids are found in the mitochondria. Αction of all three enzymes is typically needed to oxidize a fatty acid.
Plants and yeast perform β-oxidation exclusively in peroxisomes.
The most interesting of the acyl-CoA dehydrogenases is the one that works on medium length fatty acids. This one, which is the
one most commonly deficient in animals, has been associated with sudden infant death syndrome. Reactions two and three in β-
oxidation are catalyzed by enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase, respectively. The latter reaction yields an
NADH.

Thiolase
The second notable enzyme of β-oxidation is thiolase because this enzyme not only catalyzes the formation of acetyl-CoAs in β-
oxidation, but also the joining of two acetyl-CoAs (essentially the reversal of the last step of β-oxidation) to form acetoacetyl-CoA
– essential for the pathways of ketone body synthesis and cholesterol biosynthesis.
Similarity to citric acid cycle oxidation
It is worth noting that oxidation of fatty acids is chemically very similar to oxidation of the four carbon compounds of the citric
acid cycle (Figure 6.89). In fatty acid oxidation, dehydrogenation between carbons 2 and 3 generates electrons which are donated to
FAD to make FADH2 and a trans-bonded intermediate is formed.
The same thing happens in the citric acid cycle reaction catalyzed by succinate dehydrogenase - the trans-bonded molecule is
fumarate. Addition of water in the second step of fatty acid oxidation occurs also in the next step of the citric acid cycle catalyzed
by fumarase to create malate. Oxidation of the hydroxyl on carbon 3 in β-oxidation is repeated in the citric acid cycle reaction
catalyzed by malate dehydrogenase yielding oxaloacetate.

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Oxidation of odd chain fatty acids
Though most fatty acids of biological origin have even numbers of carbons, not all of them do. Oxidation of fatty acids with odd
numbers of carbons ultimately produces an intermediate with three carbons called propionyl-CoA, which cannot be oxidized
further in the β-oxidation pathway.
Metabolism of this intermediate is odd. Sequentially, the following steps occur (Figure 6.90) – 1) carboxylation to make D-
methylmalonyl-CoA; 2) isomerization to L-methylmalonyl-CoA; 3) rearrangement to form succinyl-CoA. The last step of the
process utilizes the enzyme methylmalonyl-CoA mutase, which uses the B12 coenzyme in its catalytic cycle. Succinyl-CoA can be
metabolized in the citric acid cycle.

Peroxisomal oxidation
Long chain fatty acids (typically 22 carbons or more - Figure 6.91) have their oxidation initiated in the peroxisomes, due to the
localization of the long acyl-CoA dehydrogenase in that organelle. Peroxisomal fatty acid oxidation is chemically similar to β-
oxidation of mitochondria, but there are some differences in the overall process.
Differences
First, since there is no electron transport system in peroxisomes, the reduced electron carriers produced in oxidation there must
have their own recycling process. Peroxisomes accomplish this by transferring electrons and protons from FADH2 to O2 to form
hydrogen peroxide (H2O2). As a result of this, the lack of electron transport means no proton pump and, consequently, no ATP
produced from FADH2 for peroxisomal fatty acid oxidation, making it less efficient that mitochondrial β-oxidation.
Electrons from NADH produced in the third step of the fatty acid oxidation must be shuttled to the cytoplasm and ultimately to the
mitochondrion for ATP generation. Peroxisomal oxidation is increased for individuals on a high fat diet. In addition to long chain
fatty acids, peroxisomes are also involved in oxidation of branched chain fatty acids, leukotrienes, and some prostaglandins.
Unsaturated fatty acid oxidation
Unsaturated fatty acids complicate the oxidation process a bit (see below), primarily because they have cis bonds, for the most part,
if they are of biological origin, and these must be converted to the relevant trans intermediates for β-oxidation. Sometimes the bond
must be moved down the chain, as well, in order to be positioned properly. Two enzymes (described below) handle all the
necessary isomerizations and moves necessary to oxidize all of the unsaturated fatty acids (Figure 6.92).

Extra enzymes
As noted above, oxidation of unsaturated fatty acids requires two additional enzymes to the complement of enzymes for β-
oxidation. If the β-oxidation of the fatty acid produces an intermediate with a cis bond between carbons three and four, cis-∆3-
enoyl-CoA isomerase will convert the bond to a trans bond between carbons two and three and β-oxidation can proceed as normal.
On the other hand, if β-oxidation produces an intermediate with a cis double bond between carbons four and five, the first step of β-
oxidation (dehydrogenation between carbons two and three) occurs to produce an intermediate with a trans double bond between
carbons two and three and a cis double bond between carbons four and five.
2,4 dienoyl-CoA reductase
The enzyme 2,4 dienoyl CoA reductase reduces this intermediate (using NADPH) to one with a single cis bond between carbons
three and four. The newly created cis-bonded molecule is then identical to the one acted on by cis-∆3-enoyl-CoA isomerase above,
which converts it into a regular β-oxidation intermediate, as noted above.
α-oxidation
Yet another consideration for oxidation of fatty acids is α-oxidation. This pathway, which occurs in peroxisomes, is necessary for
catabolism of fatty acids that have branches in their chains. For example, breakdown of chlorophyll’s phytol group yields phytanic
acid (Figure 6.93), which undergoes hydroxylation and oxidation on carbon number two (in contrast to carbon three of β-
oxidation), followed by decarboxylation and production of an unbranched intermediate that can be further oxidized by the β-
oxidation pathway. Though α-oxidation is a relatively minor metabolic pathway, the inability to perform the reactions of the
pathway leads to Refsum’s disease where accumulation of phytanic acid leads to neurological damage.
ω-oxidation of fatty acids

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In addition to β-oxidation and α-oxidation of fatty acids, which occur in the mitochondria and peroxisomes of eukaryotic cells
respectively, another fatty acid oxidation pathway known as ω-oxidation also occurs in the smooth endoplasmic reticulum of liver
and kidney cells. It is normally a minor oxidation pathway operating on medium chain fatty acids (10-12 carbons), but gains
importance 1) when β-oxidation is not functional or 2) for production of long chain intermediates, such as 20-HETE (20-
hydroxyeicosatetraenoic acid), that can function in signaling.
Steps in the process involve 1) oxidation of the terminal methyl group of the fatty acid to an alcohol; 2) oxidation of the alcohol to
an aldehyde, and 3) oxidation of the aldehyde group to a carboxylic acid (Figure 6.94). The first oxidation is catalyzed by a mixed
function oxidase, and yields 20-HETE if the starting material is arachidonic acid. The last two reactions are catalyzed by alcohol
dehydrogenase and each requires NAD+. After the last oxidation, the fatty acid has carboxyl groups at each end and can be
attached to coenzyme A at either end and subsequently oxidized, ultimately yielding succinate.
Regulation of fatty acid oxidation
Breakdown of fatty acids is controlled at different levels. The first is by control of the availability of fatty acids from the
breakdown of fat. As noted above, this process is by regulating the activity of hormone-sensitive triacylglycerol lipase (HSTL)
activity by epinephrine (stimulates) and insulin (inhibits).
A second level of control of fatty acid availability is by regulation of carnitine acyl transferase (Figure 6.87 - see HERE). This
enzyme controls the swapping of CoA on an acyl-CoA molecule for carnitine, a necessary step for the fatty acid to be imported into
the mitochondrion for oxidation.
The enzyme is inhibited by malonyl-CoA, an intermediate in fatty acid synthesis. Thus, when fatty acids are being synthesized,
import of them into the mitochondrion for oxidation is inhibited. Last, the last enzyme in the β-oxidation cycle, thiolase, is
inhibited by acetyl-CoA.
Fatty acid synthesis
Synthesis of fatty acids occurs in the cytoplasm and endoplasmic reticulum of the cell and is chemically similar to the reverse of
the β-oxidation process, but with a couple of key differences (Figure 6.95). The first of these occur in preparing substrates for the
reactions that grow the fatty acid. Fatty acid synthesis occurs in the cytoplasm of eukaryotic cells. Transport of acetyl-CoA from
the mitochondrial matrix occurs when it begins to build up. This happens when the citric acid cycle slows or stops from lack of
exercise.
Two molecules can play roles in moving acetyl-CoA to the cytoplasm – citrate and acetylcarnitine. Joining of oxaloacetate with
acetyl-CoA in the mitochondrion creates citrate which gets transported across the membrane, followed by action of citrate lyase in
the cytoplasm of the cell to release acetyl-CoA and oxaloacetate. Additionally, when free acetyl-CoA accumulates in the
mitochondrion, it may combine with carnitine and be transported out to the cytoplasm.
Fatty acid synthase
In animals, six different catalytic activities necessary to fully make palmitoyl-CoA are contained in a single complex called Fatty
Acid Synthase. As shown in Figures 6.96 and 6.97, these include 1) transacylases (MAT) for swapping CoA-SH with ACP-SH on
acetyl-CoA and malonyl-CoA; 2) a synthase (KS) to catalyze addition of the two carbon unit from the three carbon malonyl-ACP
in the first step of the elongation process; 3) a reductase (KR) to reduce the ketone; 4) a dehydrase (DH) to catalyze removal of
water; 5) a reductase (ER) to reduce the trans double bond and 6) a thioesterase (TE) to cleave the finished palmitoyl -CoA into
palmitic acid and CoA-SH.
In the middle of the complex is a site for binding the ACP portion of the growing fatty acid chain to hold it as the other part of the
fatty acid is rotated into positions around the enzyme complex for each catalysis. In bacteria, these six activities are found on
separate enzymes and are not part of a complex.
Cytoplasmic reactions
The process of making a fatty acid in the cytoplasm starts with two acetyl-CoA molecules. One is converted to malonyl-CoA by
adding a carboxyl group. This reaction is catalyzed by the enzyme acetyl-CoA carboxylase (ACC), the only regulated enzyme of
fatty acid synthesis (see below) and the only one separate from the fatty acid synthase. Next, both acetyl-CoA and malonyl-CoA
have their CoA portions replaced by a carrier protein known as ACP (acyl-carrier protein) to form acetyl-ACP (catalyzed by acetyl-
CoA : ACP transacylase - MAT in Figure 6.97) and malonyl-ACP (catalyzed by malonyl-CoA : ACP transacylase - MAT in Figure

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6.97). Joining of a fatty acyl-ACP (in this case, acetyl-ACP) with malonyl-ACP splits out the carboxyl group from malonyl-ACP
that was added to it and creates the acetoacyl-ACP intermediate (catalyzed by β-ketoacyl-ACP synthase - KS on Figure 6.97) .
From this point forward, the chemical reactions resemble those of β-oxidation reversed. First, the ketone is reduced to a hydroxyl
using NADPH (catalyzed by β-ketoacyl-ACP reductase - KR on Figure 6.97). In contrast to the hydroxylated intermediate of β-
oxidation, the intermediate here (D-β- hydroxybutyryl-ACP) is in the D-configuration.
Dehydration
Next, water is removed from carbons 2 and 3 of the hydroxyl intermediate in a reaction catalyzed by 2,3-trans-enoyl-ACP
dehydrase - DH on Figure 6.97. This yields a trans doubled-bonded molecule. Last, the double bond is hydrogenated to yield a
saturated intermediate by 2,3-trans-enoyl-ACP reductase - ER on Figure 6.97. This completes the first cycle of synthesis.
Additional cycles involve addition of more two-carbon units from malonyl-ACP to the growing chain until ultimately an
intermediate with 16 carbons is produced (palmitoyl-ACP). At this point, a thioesterase cleaves the ACP from the palmitoyl-ACP
to yield palmitic acid and the cytoplasmic synthesis ceases.
Regulation of fatty acid synthesis
Acetyl-CoA carboxylase, which catalyzes synthesis of malonyl-CoA, is the only regulated enzyme in fatty acid synthesis. Its
regulation involves both allosteric control and covalent modification. The enzyme is known to be phosphorylated by both AMP
Kinase and Protein Kinase A.
Dephosphorylation is stimulated by phosphatases activated by insulin binding. Dephosphorylation activates the enzyme and favors
its assembly into a long polymer, while phosphorylation reverses the process. Citrate acts as an allosteric activator and may also
favor polymerization. Palmitoyl-CoA allosterically inactivates it.
Elongation past 16 carbons
Elongation to make fatty acids longer than 16 carbons occurs in the endoplasmic reticulum and is catalyzed by enzymes described
as elongases. Mitochondria also can elongate fatty acids, but their starting materials are generally longer than 16 carbons long.
The mechanisms in both environments are similar to those in the cytoplasm (a malonyl group is used to add two carbons, for
example), but CoA is attached to the intermediates, not ACP. Further, whereas cytoplasmic synthesis employs the fatty acid
synthase complex, the enzymes in these organelles are separable and not part of a complex.
Desaturation of fatty acids
Fatty acids are synthesized in the saturated form and desaturation occurs later - in the endoplasmic reticulum. Reactions to elongate
the fatty acid (with elongases) may also occur to make unsaturated fatty acids of varying lengths. Desaturases are named according
to the location of the double bonds they introduce in fatty acids. The delta (Δ) system numbers the carbon at the carboxyl end as
number 1 and the omega (ω) number system numbers the carbon at the methyl end as number 1 (Figure 6.98). Humans have
desaturases named as Δ5, Δ6, and Δ9. A Δ9 desaturase, for example, could convert stearic acid into oleic acid, because stearic acid
(see HERE) is a saturated 18 carbon fatty acid and oleic acid is an 18 carbon fatty acid with only one double bond - at position Δ9.
Polyunsaturated fatty acids
Polyunsaturated fatty acids require the action of multiple enzymes and (in some cases) the action of elongases. Arachidonic acid,
for example, is a 20 carbon fatty acid with four double bonds and its synthesis requires both an elongase (to increase the length of
the fatty acid from 16 to 20) and multiple desaturases - one for each desaturated double bond.
Animals are limited in the fatty acids they can make, due to an inability of their desaturases to catalyze reactions beyond carbons
Δ9. Thus, humans can make oleic acid, but cannot synthesize linoleic acid (Δ9,12) or linolenic acid (Δ9,12,15). Consequently,
these two must be provided in the diet and are referred to as essential fatty acids.
Almost all desaturases make cis, not trans double bonds. There are a few minor exceptions to this, in cattle, for example (Figure
6.99). The trans fatty acids found in trans fat of prepared food are produced not by biological processes, but rather by the process of
partial hydrogenation of unsaturated fats.
Unusual oxidation reaction
Removal of electrons and protons from a fatty acid to create a double bond is an oxidation reaction and these electrons, must have a
destination. The path they take is a bit complex. It involves NAD(P)H, O2, two membrane-bound cytochromes, the membrane
bound desaturase, and the fatty acid.

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In the electron transfer, the O2 is reduced to two molecules of H2O. This reduction requires four electrons and four protons. Two
electrons and two protons come from the fatty acid to form the double bond on it. Two electrons come from the NAD(P)H via the
cytochromes and two protons come from the aqueous solution.
Prostaglandin synthesis
The pathway for making prostaglandins and related molecules, such as the leukotrienes, prostacyclin, and thromboxanes is an
extension of the synthesis of fatty acids (Figure 6.100).
Prostaglandins, known as eicosanoids because they contain 20 carbons, are synthesized in cells from arachidonic acid whenever it
has been cleaved from membrane lipids. Prostaglandins are important for many physiological phenomena in the body, including
swelling and pain and reduction of their levels is a strategy of some painkillers, such as aspirin (see below). Inflammation arising
from bee stings, for example, occurs because bee (and snake) venom contains mellitin, an activator of PLA2 activity (Figure
6.100). There are two strategies for reducing prostaglandin production and the pain associated with it.
Phospholipase A2
Action of phospholipase enzymes on glycerophospholipids produces fatty acids and either glycerol-3-phosphate or other
substances. Figure 6.101 shows cleavage sites on phospholipids that are targeted by different phospholipases. Phospholipase A1
(PLA1), for example, cleaves the fatty acid from position one of the glycerophospholipid and phospholipiase D (PLD) cleaves the
R group from the phosphate part of the molecule.
Since the fatty acid on position #2 (where PLA2 cuts) is most commonly unsaturated, PLA2 is an important phosopholipase for
hydrolyzing the unsaturated fatty acid known as arachidonic acid from glycerophospholipids. Release of arachidonic acid from
membranes is necessary for synthesis of prostaglandins.
Inhibition of the release of arachidonic acid from membranes is the mechanism of action of steroidal anti-inflammatory drugs.
They block action of phospholipase A2 (PLA2 - Figure 6.101) which cleaves arachidonic acid from membrane lipids.
Lipocortin
Lipocortin (also called annexin) is a protein that inhibits action of PLA2. Synthesis of lipocortin is stimulated by glucocorticoid
hormones, such as cortisol, and is used in some treatments to reduce swelling/inflammation when it is severe and untreatable by
non-steroidal drugs.
Second strategy
Synthesis of the prostanoid compounds (prostaglandins, prostacyclin, and thromboxanes) depends on conversion of arachidonic
acid to prostaglandins G2 and H2 by COX enzymes. A non-steroidal strategy for decreasing production of prostaglandins then is to
inhibit the enzyme that catalyzes their synthesis from arachidonic acid (Figure 6.102). This enzyme is known as prostaglandin
synthase, but is more commonly referred to as a cyclooxygenase (or COX) enzyme.
COX enzymes come in at least two forms in humans - COX-1, COX-2. A third form known as COX-3 has been reported as a splice
variant of COX-1, but information about it is unclear. COX-1 and COX-2 are very similar in structure (70 kD and 72 kD,
respectively, and 65% amino acid sequence homology), but coded by different genes.
COX-1 is synthesized constitutively whereas COX-2 displays inducible expression behavior and has a more specific pattern of
tissue expression. COX-2 enzymes are expressed in increasing amounts in areas of growth and inflammation.
Non-steroidal drugs
Molecules inhibiting cyclooxygenases are known as non-steroidal anti-inflammatory drugs (NSAIDs). Molecules in this class
include aspirin, ibuprofen, vioxx, and celebrex.
Targeting inhibitors
Some NSAID inhibitors, such as aspirin, bind to all types of COX enzymes. Newer COX inhibitors target the COX-2 enzyme
specifically because it was believed to be a better target for relief of joint pain than COX-1 enzymes which are synthesized by most
cells. COX-2 enzymes are found more specifically in joints so the thinking was that specific inhibition of them would not affect the
COX-1 enzymes which are important for producing prostaglandins that help maintain gastric tissue.
Numerous COX-2 - specific inhibitors were developed - celecoxib, etoricoxib, and rofecoxib (Vioxx), for example. Unfortunately,
the COX-2 specific inhibitors are associated with some serious side effects, including a 37% increase in incidence of major
cardiovascular events in addition to some of the gastrointestinal problems of NSAIDs.

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Imbalance
The increased risk of heart attack, thrombosis, and stroke are apparently due to an imbalance between prostacyclin (reduced by
inhibitors) and thromboxanes (not reduced by the inhibitors). Prostacyclin (made from prostaglandin H2 by prostacyclin synthase)
is a special prostaglandin that inhibits activation of blood platelets in the blood clotting process and acts as a vasodilator.
Thromboxanes counter prostacyclin, causing vasoconstriction and activating blood platelets for clotting. Due to imbalances in these
opposite acting molecules resulting from COX-2-specific inhibition, Vioxx, was withdrawn from the market in September, 2004,
due to health concerns.
Other compounds known to inhibit COX enzymes include some flavonoids, some components of fish oil, hyperforin, and vitamin
D.
Connections to other pathways
There are several connections between fats and fatty acid metabolism and other metabolic pathways. Diacylglycerol (DAG - Figure
6.105), which is produced by removal of a phosphate from phosphatidic acid, is an intermediate in fat synthesis and also a
messenger in some signaling systems. Phosphatidic acid, of course, is a branch intermediate in the synthesis of triacylglycerols and
other lipids, including phosphoglycerides.
Fatty acids twenty carbons long based on arachidonic acid (also called eicosanoids) are precursors of the leukotrienes,
prostaglandins, thromboxanes, and endocannabinoids.
Acetyl-CoA from β-oxidation can be assembled by the enzyme thiolase to make acetoacetyl-CoA, which is a precursor of both
ketone bodies and the isoprenoids, a broad category of compounds that include steroid hormones, cholesterol, bile acids, and the fat
soluble vitamins. In plants, acetyl-CoA can be made into carbohydrates in net amounts via the glyoxylate cycle.
Fat, obesity, and hunger
Obesity is an increasing problem in the western world. It is, in fact, the leading preventable cause of death worldwide. In 2014,
over 600 million adults and 42 million children in the world were classified as obese, a condition when their body mass index is
over 30 kg/m2 (Figure 6.106). The body mass index of a person is obtained by dividing a person’s weight by the square of their
height. At a simple level, obesity arises from consumption of calories in excess of metabolic need, but there are many molecular
factors to consider.
Adipokines
Adipokines are adipose tissue-synthesized cytokines. The class of molecules includes leptin (first discovered adipokine) and
hundreds of other such compounds. These include adiponectin (regulates glucose levels and fatty acid oxidation), apelin (control of
blood pressure, angiogenesis promotion, vasodilator release, increased water intake), chemerin (stimulation of lipolysis, adipocyte
differentiation, link to insulin resistance), and resistin (links to obesity, type II diabetes, LDL production in liver), among others.
Resistin
Resistin is an adipokine peptide hormone with numerous associated negative health effects. Injection of the hormone into mice
results in increased resistance to insulin, a phenomenon of type 2 diabetes.
Resistin is linked to increased inflammation and serum levels of it correlate with increased obesity, though direct linkage of it to
obesity is controversial. Resistin stimulates production of LDLs in the liver, supporting increased levels in the arteries. Resistin also
adversely impacts the effects of statin drugs used to control levels of cholesterol in the body.
Leptin
Leptin is a peptide hormone (adipokine) made in adipose cells that negatively impacts hunger and regulates energy balance. It is
countered by ghrelin, also known as the hunger hormone. Both hormones act in the hypothalamus where hunger is controlled.
When leptin levels are higher due to higher levels of body fat, hunger is suppressed, but when levels of leptin are lower (less body
fat), then appetite increases.
Notably, leptin is also made in places besides adipose tissue and leptin receptors are found in places besides the hypothalamus, so
the hormone has other effects in the body. When sensitivity to leptin changes, increased obesity can result. In mice, deletion of
leptin function by mutation results in mice with voracious appetites and extreme obesity. Deletion of the leptin receptor gene in
mice results in the same phenotype. Eight humans with leptin mutations all suffer from extreme obesity in infancy.
Physiology

6.3.7 https://bio.libretexts.org/@go/page/7838
Leptin is produced primarily by cells in white adipose tissue, but is also made in brown adipose tissue, ovaries, skeletal muscle,
stomach, mammary epithelial cells and bone marrow.
Leptin levels
Leptin levels in the body are highest between midnight and early morning, presumably to suppress appetite. Though it is produced
by fat cells, levels of leptin in humans do not strictly reflect levels of fat. For example, early in fasting, leptin levels fall before fat
levels fall. Sleep deprivation can reduce leptin levels, as can increasing levels of testosterone and physical exercise.
Increasing estrogen, however, increases leptin levels. Emotional stress and insulin can increase leptin levels. Obesity increases
leptin levels, but doesn’t fully suppress appetite. Leptin resistance in these individuals is an important consideration, lessening the
effects of the hormone on appetite.
Blocking leptin action
In the medial hypothalamus, leptin stimulates satiety and in the lateral hypothalamus, leptin inhibits hunger. Lesions in the lateral
hypothalamus that block the ability to sense hunger result in anorexia (there are other causes of anorexia, though) and lesions in the
medial hypothalamus cause excess hunger (no satiety). Neuropeptide Y is a potent hunger promoter whose receptors in the arcuate
nucleus can be bound and blocked by leptin. Leptin levels are more sensitive to decreasing food intake than increasing food intake
meaning that in humans the hormone plays a bigger role with respect to appetite than to levels of fat in the body.
At the molecular level, binding of leptin to the Ob-Rb receptor causes down-regulation of synthesis of endocannabinoids, whose
normal function is to increase hunger. High fructose diets have been associated with reduced levels of leptin and of leptin receptor.
Ghrelin
Ghrelin is a peptide hormone made by cells in the gastrointestinal tract when the stomach is empty. Stretching of the stomach
reduces the expression of the hormone. Ghrelin exerts its effects on the central nervous system to increase appetite and it is an
unusual peptide in being able to cross the blood-brain barrier. The ghrelin receptor in the brain is found on the same cells as the
leptin receptor (arcuate nucleus). Leptin can counter the ghrelin effect by decreasing hunger.
Behavioral effects
Activation of ghrelin occurs after processing the zymogen form of the hormone (pre-proghrelin) followed by linkage of an octanoic
acid to a serine at position 3. Circulating levels of ghrelin increase before eating and decrease afterwards. There appears to be a
dose dependence for ghrelin on the amount of food consumed. Ghrelin increases food seeking behavior and there is a negative
correlation between levels of ghrelin and weight.
Neuropeptide Y
Neuropeptide Y is a neuropeptide neurotransmitter produced by neurons of the sympathetic nervous system. It acts as a
vasoconstrictor and favors growth of fat tissue. It appears to stimulate food intake, fat storage, relieve anxiety/stress, reduce pain
perception, and lower blood pressure. Blockage of neuropeptide Y receptors in the brain of rats decreases food intake.
Stress effects
In mice and monkeys, repeated stress and high fat, high sugar diets stimulate neuropeptide Y levels and cause abdominal fat to
increase.
High levels of neuropeptide Y may also help individuals to recover from post-traumatic stress disorder and to reduce the fear
response. It may also protect against alcoholism. Mice lacking the ability to make neuropeptide Y have a higher voluntary
consumption of alcohol and are less sensitive to its effects. The neuropeptide Y receptor is a G-protein-coupled receptor in the 7-
transmembrane domain family.
Metabolism: Fats and Fatty Acids
564
YouTube Lectures
by Kevin
HERE & HERE
565

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Figure 6.83 - Movement of dietary triglycerides
Image by Aleia Kim
Figure 6.82 - Trimyristin - A triacylglyceride
566
Figure 6.84 - Breakdown of fat in adipocytes
Image by Pehr Jacobson
567
Figure 6.85 - Synthesis of fat from phosphatidic acid (phosphatidate)
Image by Penelope Irving
Synthesis of Phosphatidic Acid from Glycerol-3-phosphate
1. Glycerol-3-phosphate + Acyl-CoA <=> Monoacylglycerol phosphate + CoA-SH
2. Monoacylglycerol phosphate + Acyl-CoA <=> Phosphatidic acid + CoA-SH
568
Figure 6.86 - Mitochondria - site of β-oxidation
Figure 6.87 - Transport of fatty acid (acyl group) across mitochondrial inner membrane
Image by Aleia Kim
569
Figure 6.88 - Four reactions in β-oxidation
Image by Aleia Kim
YouTube Lectures
by Kevin
HERE & HERE
570
Figure 6.89 - Similar reactions for fatty acid oxidation and oxidation of 4-carbon compounds in the citric acid cycle
Image by Aleia Kim
571
Figure 6.90 - Metabolism of propionyl-CoA
Image by Pehr Jacobson
572
In beta oxidation, it just occurred to me
The process all takes place ‘tween carbons two and three
Some hydrogens are first removed to FADH2
Then water adds across the bond, the H to carbon two
Hydroxyl oxidation’s next, a ketone carbon three
Then thiolase catalysis dissects the last two C’s
The products of the path, of course, are acetyl-CoAs
Unless there were odd carbons, hence propionyl-CoA
Figure 6.91 - Cerotic acid - A long chain fatty acid with 26 carbons

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573
Figure 6.92 - Unsaturated fatty acid oxidation
YouTube Lectures
by Kevin
HERE & HERE
574
Figure 6.93 - Phytanic acid
575
Figure 6.94 - ω Oxidation
Wikipedia
576
Figure 6.95 - Fatty acid synthesis is the reverse of fatty acid oxidation chemically
577
Figure 6.96 - One round of fatty acid synthesis
image by Aleia Kim
Figure 6.97 - Fatty acid synthase complex
578
579
For fatty acid synthesis, I must reverse the path
Of breaking fatty acids, though you’ll wonder ‘bout the math
Each cycle of addition starts with carbons one two three
Yet products of reactions number carbons evenly
The reason is that CO2 plays peek-a-boo like games
By linking to an Ac-CoA then popping off again
Reactions are like oxidations ‘cept they’re backwards here
Reduction, dehydration, then two hydrogens appear
The product of the process is a 16 carbon chain
The bonds are saturated. No double ones remain
For them desaturases toil to put in links of cis
In animals to delta nine, but no more go past this
And last there’s making longer ones eicosanoidic fun
They’re made by elongases in the e. reticulum
Kevin Ahern˙
YouTube Lectures
by Kevin
HERE & HERE
Figure 6.98 - Carbon numbering schemes for fatty acids
Image by Pehr Jacobson

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580
Stearoyl-CoA + 2 Cytochrome b5 (red) + O2 + 3 H+ + NADPH
Oleoyl-CoA + 2 Ferricytochrome b5 (ox) + 2 H2O + NADP+
Desaturase Reaction to Oxidize Stearic Acid
Figure 6.99 - Elaidic acid - A rare trans fatty acid in biology
581
Figure 6.100 - Eicosanoid synthesis pathways
Image by Pehr Jacobson
582
583
Figure 6.101 - Cleavage sites for four phospholipiases on a glycerophospholipid - phospholipases A1 (PLA1), A2 (PLA2), C
(PLC), and D (PLD)
YouTube Lectures
by Kevin
HERE & HERE
584
Figure 6.102 - Catalytic activity of cyclooxygenase and peroxidase in making prostaglandins
Image by Pehr Jacobson
585
Figure 6.103 - Synthesis of prostaglandins from prostaglandin H2 (red)
Image by Pehr Jacobson
Figure 6.104 - Two NSAIDs
586
Figure 6.105 - Diacylglycerol
Figure 6.106 - Obesity worldwide - females (top) and males (bottom)
Wikipedia
587
Figure 6.107 Leptin
Wikipedia
588
YouTube Lectures
by Kevin
HERE & HERE
Figure 6.108 Neuropeptide Y
589
590
Figure 6.109 - Pre-proghrelin
591

6.3.11 https://bio.libretexts.org/@go/page/7838
Graphic images in this book were products of the work of several talented students. Links to their Web pages are below
Click HERE for
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Web Page
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Pehr Jacobson’s
Web Page
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Aleia Kim’s
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Web Page
Problem set related to this section HERE
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Email Kevin Ahern / Indira Rajagopal / Taralyn Tan
592
When Acids Get Oxidized
To the tune of "When Johnny Comes Marching Home"
Metabolic Melodies Website HERE
The fatty acids carried by
CoA, CoA
Are oxidized inside the
mi-to-chon-dri-ay

They get to there as you have seen

By hitching rides on carnitine

6.3.12 https://bio.libretexts.org/@go/page/7838
Then it goes away
When acids get oxidized
Electrons move through membranes, yes
It’s true, it’s true
They jump from complex I onto
Co-Q, Co-Q

The action can be quite intense

When building proton gradients
And its good for you
When acids get oxidized
The protons pass through complex V
You see, you see
They do this to make lots of
A-TP, TP

The mechanism you should know

Goes through the stages L-T-O
So there's energy
When acids get oxidized
Recording by Tim Karplus
Lyrics by Kevin Ahern
Recording by Tim Karplus Lyrics by Kevin Ahern
593
When Acids Are Synthesized
To the tune of “When Johnny Comes Marching Home”
Metabolic Melodies Website HERE
The 16 carbon fatty acid, palmitate
Gets all the carbons that it needs from acetate
Which citric acid helps release
From mitochondri - matrices
Oh a shuttle's great
When acids are synthesized
Carboxylase takes substrate and it puts within
Dioxy carbon carried on a biotin
CoA's all gain a quick release
Replaced by larger ACPs
And it all begins
When acids are synthesized
A malonate contributes to the growing chain
Two carbons seven times around again, again
For saturated acyl-ates
There's lots of N-A-DPH
That you must obtain
When acids are synthesized
Palmitic acid made this way all gets released
Desaturases act to make omega-threes

6.3.13 https://bio.libretexts.org/@go/page/7838
The finished products big and small
Form esters with a glycerol
So you get obese
When acids are synthesized
Recording by Tim Karplus
Lyrics by Kevin Ahern
Recording by Tim Karplus Lyrics by Kevin Ahern

This page titled 6.3: Fats and Fatty Acids is shared under a CC BY-NC-SA license and was authored, remixed, and/or curated by Kevin Ahern,
Indira Rajagopal, & Taralyn Tan.

6.3.14 https://bio.libretexts.org/@go/page/7838
6.4: Other Lipids
Source: BiochemFFA_6_4.pdf. The entire textbook is available for free from the authors at
http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy
Sugars are the building blocks of carbohydrates, amino acids are the building blocks of proteins and nucleotides are the building
blocks of the nucleic acids - DNA and RNA. Another crucial building block is acetyl-CoA, which is used to build many lipid
substances, including fatty acids, cholesterol, fat soluble vitamins, steroid hormones, prostaglandins, endocannabinoids, and the
bile acids. Indeed, acetyl-CoA goes into more different classes of molecule than any other building block.

Isoprenoids
We focus our attention here on a group of molecules made from acetyl-CoA that are known as as the isoprenoids. Isoprenoids are a
large, diverse and ancient group of molecules that are found in all three domains of life. As noted earlier, they are components of
membrane lipids in the cell membranes of archaebacteria, but beyond this, they serve an astonishing variety of functions. From
photosynthetic pigments to plant defense compounds, from flavor compounds in cinnamon, mint, ginger and cloves to plant and
animal hormones, from the cannabinoids in marijuana to the lycopene that gives tomatoes their color, and from heme to the
quinones in the electron transport chain, isoprenoids are ubiquitous in cells. Isoprenoids derive their name from the fact that they
are, in fact, made from five carbon building blocks called isoprenes that are derived from acetyl-CoA. The synthesis of the two
isoprene units - isopentenyl pyrophosphate and dimethylallyl pyrophosphate is shown in Figure 6.111 and Figure 6.112.
The pathway leading up to isoprene synthesis overlaps with that of ketone body synthesis, for the two reactions (Figure 6.112), as
has been discussed earlier in this book (see HERE). Thiolase catalyzes the initial reaction, joining together two acetyl-CoA
molecules to make acetoacetyl-CoA. In the second reaction catalyzed by HMG-CoA synthase, a third acetyl-CoA is joined to form
the six carbon compound known as hydroxymethyl glutaryl-CoA (HMG-CoA). Reaction three is an important one biologically and
medically because of the enzyme catalyzing it - HMG-CoA reductase.

Statins
Medically, HMG-CoA reductase is the target of a class of drugs known as statins (Figure 6.113 & Movie 6.1), which are used to
reduce cholesterol levels in people. These competitive inhibitors, which compete with HMG-CoA for binding have two effects.
First, they reduce the production of mevalonate, which restricts the amount of substrate available to make cholesterol. Second, and
perhaps more importantly, they increase production of LDL receptors in the liver, which favors uptake and destruction of LDLs,
thus lowering serum cholesterol levels.

Regulation
Biologically, the HMG-CoA reductase enzyme is also of importance because it is the primary regulatory point in cholesterol
synthesis. Control of it is complex. First, it is feedback inhibited by cholesterol itself. High levels of glucose in the blood activate
the enzyme. Phosphorylation by AMP-activated protein kinase inhibits its activity. Interestingly, the same enzyme phosphorylates
and inactivates acetyl-CoA carboxylase - the only regulatory enzyme controlling fatty acid synthesis. Transcription of the gene
encoding HMG-CoA reductase
is enhanced by binding of the sterol regulatory element binding protein (SREBP) to the sterol recognition element (SRE ) located
near the gene coding sequence. As cholesterol levels rise, SREBP is proteolytically cleaved and transcription stops.
From HMG-CoA, the enzyme HMG-CoA reductase catalyzes the formation of mevalonate. This reaction requires NADPH and
results in release of coenzyme A. Mevalonate gets phosphorylated twice and then decarboxylated to yield the five carbon
intermediate known as isopentenyl-pyrophosphate (IPP). IPP is readily converted to the other important isoprenoid unit,
dimethylallylpyrophosphate (DMAPP).

Isoprenes
These two five carbon compounds, IPP and DMAPP, are also called isoprenes (Figure 6.115) and are the building blocks for the
synthesis of cholesterol and related compounds. This pathway proceeds in the direction of cholesterol starting with the joining of
IPP and DMAPP to form geranyl-pyrophosphate. Geranyl-pyrophosphate combines with another IPP to make farnesyl-
pyrophosphate, a 15-carbon compound.

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Squalene
Two farnesyl-pyrophosphates join to create the 30-carbon compound known as squalene. Squalene, in a complicated rearrangement
involving reduction and molecular oxygen forms a cyclic intermediate known as lanosterol (Figure 6.116) that resembles
cholesterol. Conversion of lanosterol to cholesterol is a lengthy process involving 19 steps that occur in the endoplasmic reticulum.
The cholesterol biosynthesis pathway from lanosterol is a long one and requires significant amounts of reductive and ATP energy.
As noted earlier (see HERE), cholesterol has an important role in membranes. It is also a precursor of steroid hormones and bile
acids and its immediate metabolic precursor, 7-dehydrocholesterol (Figure 6.117), branches to form vitamin D (Figure 6.118).
All steroid hormones in animals are made from cholesterol and include the progestagens, androgens, estrogens, mineralocorticoids,
and the glucocorticoids. The branch molecule for all of the steroid hormones is the cholesterol metabolite (and progestagen) known
as pregnenalone (Figure 6.119). The progestagens are thus precursors of all of the other classes of steroid hormones.
The estrogens are derived from the androgens in an interesting reaction that required formation of an aromatic ring (Figure 6.120).
The enzyme catalyzing this reaction is known as an aromatase and it is of medical significance. The growth of some tumors is
stimulated by estrogens, so aromatase inhibitors are prescribed to prevent the formation of estrogens and slow tumor growth. Two
commonly used inhibitors include exemestane (a suicide inhibitor - Figure 6.121) and anastrozole (a competitive inhibitor).

Other fat-soluble vitamins

Synthesis of other fat soluble vitamins and chlorophyll also branches from the isoprenoid synthesis pathway at geranyl
pyrophosphate. Joining of two geranylgeranyl pyrophosphates occurs in plants and bacteria and leads to synthesis of lycopene,
which, in turn is a precursor of β-carotene, the final precursor of Vitamin A (see below also). Vitamins E and K, as well as
chlorophyll are all also synthesized from geranylgeranyl pyrophosphate.

Bile acid metabolism

Another metabolic pathway from cholesterol leads to the polar bile acids, which are important for the solubilization of dietary fat
during digestion. Converting the very non-polar cholesterol to a bile acid involves oxidation of the terminal carbon on the side
chain off the rings. Other alterations to increase the polarity of these compounds include hydroxylation of the rings and linkage to
other polar compounds.
Common bile acids include cholic acid, chenodeoxycholic acid, glycocholic acid, taurocholic acid, and deoxycholic acid (Figure
6.123). Another important consideration about bile acids is that their synthesis reduces the amount of cholesterol available and
promotes uptake of LDLs by the liver. Normally bile acids are recycled efficiently resulting in limited reduction of cholesterol
levels. However, inhibitors of the recycling promote reduction of cholesterol levels.

Vitamin A Synthesis
Vitamin A is important for many cellular functions related to growth, differentiation and organogenesis during embryonic
development, tissue maintenance, and vision, to name a few.
There are three main active forms of the vitamin, retinal, retinol and retinoic acid, each with its own set of functions. Retinal,
complexed with the protein, opsin, is found in the rod cells of the retina and is necessary for vision. Retinol and retinoic acid both
function as signaling molecules that can modulate gene expression during development.
Synthesis of vitamin A occurs as a branch in synthesis of isoprenoids. Addition of isopentenyl pyrophosphate to farnesyl
pyrophosphate creates a 20-carbon intermediate, geranylgeranyl pyrophosphate (GGPP - Figure 6.124).
Joining of two GGPPs creates a 40 carbon intermediate that is unstable and decomposes to phytoene. Desaturases oxidize two
single bonds in phytoene, creating lycopene.
Lycopene is a linear 40 carbon unsaturated molecule found in tomatoes and other red vegetables and it gives them their color.
Cyclization of end portions of lycopene give rise to β-carotene, the precursor of vitamin A (retinal/retinol - Figure 6.124).
β-carotene is found in carrots and other orange vegetables, and is converted in the body to vitamin A. Catalytic action by β-
Carotene 15,15’ monooxygenase cleaves β-carotene to form retinal (the aldehyde form used in vision).
The enzyme retinol dehydrogenase catalyzes reduction of retinal to retinol (storage form). Oxidation of retinal creates another
important retinoid known as retinoic acid. This form of vitamin A cannot be reduced back to retinal and thus cannot be used for
vision or storage.

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Instead, retinoic acid has roles in embryonic development. Retinoic acid acts through binding to the Retinoic Acid Receptor
(RAR). RAR binds to DNA and affects transcription of several important sets of genes important for differentiation. These include
the Hox genes, which control anterior/posterior patterning in early embryonic development.

Sphingolipid synthesis
Synthesis of sphingolipids, which are found primarily in brain and nerve tissue, begins with palmitoyl-CoA and serine that
combine to make an 18-carbon amine called 3-keto-sphinganine (Figure 6.125). Reduction of that by NADPH yields
dihydrosphingosine and addition of a fatty acid from an acyl-CoA yields N-acylsphinganine, which is a ceramide (Figure 6.126). A
ceramide can be converted into a cerebroside by addition of a glucose from UDP-glucose (Figure 6.127).
If a few other simple sugars are added to the cerebroside, a globoside is created. If, instead of adding sugar, a phosphocholine is
added from phosphatidylcholine, then sphingomyelin is created (Figure 6.127). If a complex set of sugars are added to to a
cerebroside, then a ganglioside results (Figure 6.127).

Sphingolipid breakdown
In the overall metabolism of sphingolipids, the greatest problems arise with their catabolism. Figure 6.128 illustrates the numerous
genetic diseases arising from mutations in DNA coding for some of these enzymes. All are lysosomal storage diseases and many of
these are quite severe. GM1 glandiosidoses (arising from inability to breakdown GM1 gangliosides) cause severe
neurodegeneration and seizures. Individuals suffering from them typically die by age 3. Tay-Sachs disease usually causes death by
age 4, though late-onset forms of the disease in adults are known.
With Gaucher’s disease, three different types have been described with widely varying effects. In some, the disease is fatal by age
four and in others, it does not manifest until teens or even adulthood. Fabry’s disease patients can live into their 50s, on average.

Glycerophospholipid metabolism
Glycerophospholipids are the major components of membranes. Synthesis of glycerophospholipids begins with glycerol-3-
phosphate. In the first reaction, glycerol-3-phosphate gains a fatty acid at position one from an acyl-CoA, followed by a duplicate
reaction at position two to make phosphatidic acid (Figure 6.129). This molecule, which can branch to other reactions to form fats,
is an important intermediate in the synthesis of many glycerophospholipids. Glycerophospholipid compounds can often be made by
more than one pathway. The nucleotide CDP plays an important role in glycerophospholipid synthesis, serving as part of an
activated intermediate for synthesis of phosphatidyl compounds. This is necessary, because formation of the phosphodiester bonds
of these compounds requires higher energy input.
Cells use two strategies to accomplish this. Both involve CDP. In the first, CTP combines with phosphatidic acid to make CDP-
diacylglcyerol with release of a pyrophosphate. The reaction is catalyzed by phosphatidate cytidylyltransferase.
CDP-diacylglycerol then serves as an activated intermediate to donate the phosphotidate part of itself to another molecule. The
reaction below illustrates one example
The second strategy is to make a CDP derivative of the group being added to phosphatidic acid. An example is shown next
Then the CDP donates the phosphocholine to a diacylglycerol to made phosphatidylcholine and CMP
Synthesis of other important glycerophospholipids follows from these basic strategies. Phosphatidylethanolamine can be easily
made from phosphatidylserine by decarboxylation.
Phosphatidylethanolamine can serve as a precursor in an alternative pathway for making phosphatidylcholine (SAM = S-Adenosyl
Methioinine / SAH = S-Adenosyl Homocysteine)
Phosphatidylserine and phosphatidylethanolamine can swap groups reversibly in the reaction below
Similarly, phosphatidylserine and phosphatidylcholine can be interchanged as follows:
Phosphatidylglycerol can be made from glycerol-3-phosphate and CDP-diacylglycerol
Cardiolipin, which is essentially a diphosphatidyl compound can be made by joining CDP-diacylglyerol with phosphatidylglycerol
Phosphtidylinositol can be made from CDP-diacylglycerol and inositol .

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Heme synthesis
The porphyrin ring found in the hemes of animals, fungi, and protozoa (Figure 6.130) is synthesized starting from very simple
compounds (Figure 6.131). The process is a bit complicated, occurring between the cytoplasm and the mitochondrion. The first
step is the creation of δ-aminolevulinic acid (also called aminolevulinic acid or dALA) from glycine and Succinyl-CoA.
Joining of two δ-aminolevulinic acid molecules together with splitting out of two molecules of water yields porphobilinogen.
Joining of four molecules of porphobilinogen together yields hydroxylmethylbilane (Figure 6.132).
Next, a series of reactions involving 1) loss of water; 2) loss of four molecules of carbon dioxide; 3) loss of two more carbon
dioxides, loss of six protons and electrons and (finally) 4) addition of Fe++ with loss of two protons yields heme. Individual heme
molecules may be further processed.
Two enzymes in heme synthesis are sensitive to the presence of lead, and this is one of the primary causes of lead toxicity in
humans. Inhibition of the enzymes leads to 1) anemia and 2) accumulation of δ-aminolevulinic acid, which can be harmful to
neurons in development, resulting in learning deficiencies in children.

Porphyria
Defects in enzymes of the pathway can also lead to porphyrias, diseases in which one or more of the intermediates in the heme
synthesis pathway accumulate due to deficiency of the enzyme necessary to convert the accumulating material into the next
molecule in the pathway. The accumulation of purplish intermediates gave the diseases the name porphyria from the Greek word
for purple.
Severe porphyrias can lead to brain damage, nerve damage, and mental disturbances. The “madness” of King George III may have
been due to a form of porphyria. In other manifestations of the disease, cutaneous porphyrias cause skin problems on exposure to
light. This need, for patients with certain forms of porphyria, to avoid light, coupled with the fact that porphyrias can be treated by
blood tranfusions, may have led to the legend of vampires.

Breakdown of heme
Catabolism of heme (Figure 6.133) begins in macrophages within the spleen . Targets for degradation are hemes within damaged
red blood cells, which get removed from the blood supply due to their appearance. It is because of this system, for example, that
sickle cell anemia is classified as an anemia (decrease in red blood cells or hemoglobin in the blood). After cells have sickled, they
lose their shape and are more likely to be removed from the blood by this process, leaving the patient weakened from low blood
cell counts.
The first biochemical step in catabolism is conversion of heme to biliverdin. This reaction is catalyzed by heme oxygenase and
requires electrons from NADPH. In the process, Fe++ is released. Interestingly, carbon monoxide is also produced and it acts as a
vasodilator.
Next, biliverdin is converted to bilirubin by biliverdin reductase and is secreted from the liver into bile. Bacteria in the intestine
convert bilirubin to urobilinogens, some of which is absorbed intestinal cells and transported into kidneys and excreted. The yellow
color of urine arises from the compound known as urobilin, which is an oxidation product of urobilinogen. The remainder of the
urobilinogens are converted in the intestinal tract to stercobilinogen whose oxidation product is stercobilin and it gives the color
associated with feces.

This page titled 6.4: Other Lipids is shared under a CC BY-NC-SA license and was authored, remixed, and/or curated by Kevin Ahern, Indira
Rajagopal, & Taralyn Tan.

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6.5: Amino Acids and the Urea Cycle
Source: BiochemFFA_6_5.pdf. The entire textbook is available for free from the authors at
http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy
In contrast to some of the metabolic pathways described to this point, amino acid metabolism is not a single pathway. The 20 amino
acids have some parts of their metabolism that overlap with each other, but others are very different from the rest. In discussing
amino acid metabolism, we will group metabolic pathways according to common metabolic features they possess (where possible).
First, we shall consider the anabolic pathways.

Transamination
Before beginning discussion of the pathways, it is worthwhile to discuss a reaction common to the metabolism of most of the
amino acids and other nitrogen-containing compounds and that is transamination. In cells, nitrogen is a nutrient that moves from
one molecule to another in a sort of hand-off process. A common transamination reaction is shown on the next page.
A specific reaction of this type is shown in Figure 6.134.
Glutamate and glutamine play central roles in transamination, each containing one more amine group than α-ketoglutarate and
glutamate, respectively. Transamination reactions, as noted earlier, occur by a ping-pong mechanism and involve swaps of amines
and oxygens in Schiff base reactions. Two amino acids, glutamine and asparagine are the products of gaining an amine in their
respective R-groups in reactions involving ammonium ion.

Synthesis varies
It is also important to recognize that organisms differ considerably in the amino acids that they can synthesize. Humans, for
example, cannot make 9 of the 20 amino acids needed to make proteins, and the number of these that can be synthesized in needed
amounts varies between adults and children.
Amino acids that cannot be made by an organism must be in the diet and are called essential amino acids. Non-essential amino
acids are those an organism can make in sufficient quantities (Figure 6.135). Though amino acids do not have a common pathway
of metabolism, they are often organized in “families” of amino acids with overlapping metabolic reactions common to members of
each group. To designate amino acid families in the text we will use a blue font for headings to distinguish them.

α-ketoglutarate family
This family of amino acids arises from α-ketoglutarate of the citric acid cycle. It includes the amino acids glutamic acid, glutamine,
proline, and arginine. It is also called the glutamate family, since all the amino acids in it derive from glutamate.
Glutamate
α-ketoglutarate is readily converted to glutamate in transamination reactions, as noted above. It can also be produced by the
enzyme glutamate dehydrogenase, which catalyzes the reaction below (in reverse) to make glutamate.
In the forward direction, the reaction is a source of ammonium ion, which is important both for the urea cycle and for glutamine
metabolism. Because it is a byproduct of a citric acid cycle intermediate, glutamate can therefore trace its roots to any of the
intermediates of the cycle. Citrate and isocitrate, for example, can be thought of as precursors of glutamate. In addition, glutamate
can be made by transamination from α-ketoglutarate in numerous transamination reactions involving other amino acids.

Glutamine
Synthesis of glutamine proceeds from glutamate via catalysis of the enzyme glutamine synthetase, one of the most important
regulatory enzymes in all of amino acid metabolism (Figure 6.136).
Regulation of the enzyme is complex, with many allosteric effectors. It can also be controlled by covalent modification by
adenylylation of a tyrosine residue in the enzyme (Figure 6.137). In the figure, PA and PD are regulatory proteins facilitating
conversion of the enzyme.
Ammonia used in the reaction catalyzed by glutamate synthetase commonly arises from nitrite reduction, amino acid breakdown,
or photorespiration. Because it builds ammonia into an amino acid, glutamine synthetase helps reduce the concentration of toxic
ammonia - an important consideration in brain tissue. Some inhibitors of glutamine synthetase are, in fact, the products of
glutamine metabolism. They include histidine, tryptophan, carbamoyl phosphate, glucosamine-6-phosphate, CTP, and AMP. The

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glutamate substrate site is a target for the inhibitors alanine, glycine, and serine. The ATP substrate site is a target for the inhibitors
GDP, AMP, and ADP. Complete inhibition of the enzyme is observed when all of the substrate sites of the multi-subunit enzyme
are bound by inhibitors. Lower levels of inhibitors results in partial or full activity, depending on the actual amounts.

Proline
Synthesis of proline starts with several reactions acting on glutamate. They are shown below in the green text box.
The L-glutamate-5-semialdehyde, so produced, is a branch point for synthesis of proline or ornithine. In the path to make proline,
spontaneous cyclization results in formation of 1-pyrroline-5-carboxylic acid (Figure 6.138).
This, in turn, is reduced to form proline by pyrroline-5-carboxylate reductase.

Arginine
Arginine is a molecule synthesized in the urea cycle and, thus, all urea cycle molecules can be considered as precursors. Starting
with citrulline, synthesis of arginine can proceed as shown on the next page. The urea cycle can be seen HERE.
An alternate biosynthetic pathway for making arginine from citrulline involves reversing the reaction catalyzed by nitric oxide
synthase. It catalyzes an unusual five electron reduction reaction that proceeds in the following manner
Yet another way to synthesize arginine biologically is by reversal of the arginase reaction of the urea cycle
Arginine can also be made starting with glutamate. This 5 step pathway leading to ornithing is illustrated at the top of the next page
(enzymes in blue). Ornithine, as noted above can readily be converted to arginine.
The last means of making arginine is by reversing the methylation of asymmetric dimethylarginine (ADMA - Figure 6.140).
ADMA is a metabolic byproduct of protein modification. It interferes with production of nitric oxide and may play a role in
cardiovascular disease, diabetes mellitus, erectile dysfunction, and kidney disease.

Serine family
Serine is a non-essential amino acid synthesized from several sources. One starting point is the glycolysis intermediate, 3-
phosphoglycerate, (3-PG) in a reaction catalyzed by 3-PG dehydrogenase.
Transamination by phosphoserine aminotransferase produces O-phosphoserine. The phosphate is then removed by phosphoserine
phosphatase, to make serine. These reactions are shown below. Phosphoserine phosphatase is missing in the genetic disease known
as Williams-Beuren syndrome.
Serine can also be derived from glycine and vice versa. Their metabolic paths are intertwined as will be seen below. Serine is
important for metabolism of purines and pyrimidines, and is the precursor for glycine, cysteine, and tryptophan in bacteria, as well
as for sphingolipids and folate. Serine in the active site of serine proteases is essential for catalysis. A serine in the active site of
acetylcholinesterases is the target of nerve gases and insecticides.

Covalent modification target

Serine in proteins can be the target of glycosylation or phosphorylation. D-serine is the second D-amino acid known to function in
humans. It serves as a neuromodulator for NMDA receptors, by serving as a co-agonist, together with glutamate. D-serine is being
studied as a schizophrenia treatment in rodents and as a possible biomarker for Alzheimers.

Glycine
As noted, glycine’s metabolism is intertwined with that of serine. This is apparent in the reaction catalyzed by serine
hydroxymethyltransferase.
Notably, the previous reaction is also needed for recycling of folate molecules, which are important for single carbon reactions in
nucleotide synthesis.
Vertebrates can also synthesize glycine in their livers using the enzyme glycine synthase.
Glycine is a very abundant component of collagen. It is used in the synthesis of purine nucleotides and porphyrins. It is an
inhibitory neurotransmitter and is a co-agonist of NMDA receptors with glutamate. Glycine was detected in material from Comet
Wild 2.
Cysteine

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Cysteine can be synthesized from several sources. One source is the metabolism of the other sulfur-containing amino acid,
methionine. This begins with formation of S-Adenosyl-Methionine (SAM), catalyzed by methionine adenosyltransferase.
SAM is a methyl donor for methyl transfer reactions and that is the next step in the pathway - donation of a methyl group
(catalyzed by transmethylase)
SAH (S-Adenosylhomocysteine) is cleaved by S-adenosylhomocysteine hydrolase,
Homocysteine can be recycled back to methionine by action of methionine synthase
On the path to making cysteine, homocysteine reacts as follows (catalyzed by cystathionine β-synthase).
Last, cystathionase catalyzes release of cysteine
β-ketobutyrate can be metabolized to propionyl-CoA and then to succinyl-CoA to be used ultimately in the citric acid cycle.
Another route to making cysteine is a two-step process that begins with serine, catalyzed first by serine-O-acetyltransferase
and then by cysteine synthase
Cysteine can be also released from cystine by cystine reductase
Finally, cysteine can be made from cysteic acid by action of cysteine lyase

Aspartate family
Metabolism of aspartic acid is similar to that of glutamate. Aspartic acid can arise from transamination of a citric acid cycle
intermediate (oxaloacetate).
Aspartate can also be generated from asparagine by the enzyme asparaginase.
Further, aspartate can be produced by reversal of a reaction in the urea cycle (see HERE)
Aspartate is also a precursor to four amino acids that are essential in humans. They are methionine, isoleucine, threonine, and
lysine. Because oxaloacetate can be produced from aspartate, aspartate is an important intermediate for gluconeogenesis when
proteins are the energy source.

Asparagine
Asparagine, too, is an amino acid produced in a simple transamination reaction. In this case, the precursor is aspartate and the
amine donor is glutamine (catalyzed by asparagine synthetase)

Methionine
Metabolism of methionine overlaps with metabolism of the other sulfur-containing amino acid, cysteine. Methionine is not made in
humans (essential) so the pathway shown in Figure 6.141 is from bacteria.
The process begins with phosphorylation of aspartate. Numbers for each catalytic step in the figure are for the enzymes that follow:
1 - Aspartokinase
2 - Aspartate-semialdehyde dehydrogenase
3 - Homoserine dehydrogenase
4 - Homoserine O-transsuccinylase
5 - Cystathionine-γ-synthase
6 - Cystathionine-β-lyase
7 - Methionine synthase
Though humans cannot make methionine by the pathway shown in the figure, they can recycle methionine from homocysteine (a
product of S-adenosylmethionine metabolism). This reaction requires the enzyme methionine synthase and Vitamin B12 as a co-
factor.
An alternative pathway of converting homocysteine to methionine involves a prominent liver enzyme, betaine-homocysteine
methyltransferase. This enzyme catalyzes the reaction below.
In this reaction, a methyl group is transferred to homocysteine from glycine betaine to make the methionine. Glycine betaine is a
trimethylated amine of glycine found in plants. It is a byproduct of choline metabolism.

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Bacteria, mitochondria, and chloroplasts use a modified form of methionine, N-formyl-methionine (Figure 6.142), as the first
amino acid incorporated into their proteins. Formylation of methionine occurs only after methionine has been attached to its tRNA
for translation. Addition of the formyl group is catalyzed by the enzyme methionyl-tRNA formyltransferase

Threonine
Though threonine is chemically similar to serine, the metabolic pathway leading to threonine does not overlap with that of serine.
As seen in the figure, aspartate is a starting point for synthesis. Two phosphorylations/dephosphorylations and two reductions with
electrons from NADPH result in production of threonine.
Enzymes in Figure 6.143 are as follows:
1 Aspartokinase
2 β-aspartate semialdehyde dehydrogenase
3 Homoserine dehydrogenase
4 Homoserine kinase
5 Threonine synthase
Breakdown of threonine produces acetyl-CoA and glycine. It can also produce α-ketobutyrate, which can be converted to succinyl-
CoA for oxidation in the citric acid cycle.

Lysine
To get from aspartate to lysine, nine reactions and two non-enzymatic steps are involved, as seen in Figure 6.144. Enzymes
involved in lysine biosynthesis include (numbers correspond to numbered reactions in Figure 6.144):
1 - Aspartokinase
2 - Aspartate-semialdehyde dehydrogenase
3 - 4-hydroxy-tetrahydrodipicolinate synthase
4 - 4-hydroxy-tetrahydrodipicolinate reductase
5 - 2,3,4,5-tetrahydropyridine-2,6-dicarboxylate N-succinyltransferase
6 - Succinyl-diaminopimelate transaminase
7 - Succinyl-diaminopimelate desuccinylase
8 - Diaminopimelate epimerase
9 - Diaminopimelate decarboxylase
Low in cereal grains
Lysine is the essential amino acid found in the smallest quantity in cereal grains, but is found abundantly in legumes. Besides its
synthesis and breakdown, lysine can be methylated, acetylated, hydroxylated, ubiquitinated, sumoylated, neddylated, biotinylated,
pupylated, and carboxylated within proteins containing it. Hydroxylation of lysine is important for strengthening collagen and
acetylation/methylation of lysine in histone proteins play roles in control of gene expression and epigenetics. Besides being used to
make proteins, lysine is important for calcium absorption, recovery from injuries, and for production of hormones.
Oral lysine has been used as a treatment for herpes infections (cold sores) but its efficacy is not established and it is not clear by
what mechanism is would reduce the duration of the infection or reduce the number of outbreaks of viral infection..

Aromatic amino acids

The aromatic amino acids, tryptophan, phenylalanine, and tyrosine can all be made starting with two simple molecules - PEP and
erythrose-4-phosphate (Figure 6.145). All three aromatic amino acids are also important sources of hormones, neurotransmitters,
and even the skin pigment melanin.

Tryptophan synthesis
The proteogenic amino acid with the largest R-group, tryptophan is an essential amino acid distinguished structurally by its indole
group. The amino acid is made in bacteria and plants from shikimic acid or anthranilate and serine is used in its synthesis.
Erythrose-4-phosphate and phosphenolpyruvate (PEP) also serve as building blocks of tryptophan. The pathway of its synthesis is
shown in Figures 6.146 to 6.148.

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Erythrose-4-phosphate and phosphoenolpyruvate (PEP) are joined and then, after one hydrolysis, one dehydration, one oxidation
and one reduction, the product is shikimic acid (Figure 6.147).
Shikimic acid is converted to chorismic acid in three steps, as shown in Figure 6.147. Finally, synthesis of tryptophan from
chorismic acid is shown in Figure 6.148.
Regulation
Regulation of tryptophan synthesis in bacteria occurs partly via a process called attenuation that operates through the trp operon. In
this mechanism, low levels of tryptophan slow ribosomal movement (and translation) through the operon. This is particularly
important because bacteria can have transcription and translation occurring simultaneously. Slowing translation due to low
tryptophan levels allows a transcription termination mechanism to be inhibited. Since translation only slows when tryptophan is in
short supply, premature termination of transcription occurs when tryptophan is abundant (see also HERE).
Besides its importance for making proteins, tryptophan is an important precursor of serotonin (neurotransmitter), melatonin
(hormone), niacin (vitamin), and auxin (plant hormone). The two pathways leading from tryptophan to three of these molecules is
shown in Figure 6.149.
Melatonin
Melatonin is a compound made from tryptophan that is found in a wide spectrum of biological systems, including plants, animals,
fungi, and bacteria. In animals, it acts as a hormone for circadian rhythm synchronization, signaling the onset of darkness each day.
It has effects on the timing of sleep, seasonal effects, and can affect blood pressure, among other physiological phenomena. It can
cross cell membranes, as well as the blood-brain barrier. Melatonin is a potent anti-oxidant and provides protective functions for
nucleic acids. It is used sometimes to help in treatment of sleep disorders. Some reports have indicated that children with autism
have abnormal melatonin pathways with low levels of the hormone.
Blue light
Melatonin production is affected by blue light and may be linked to sleep abnormalities for people using computer monitors after
dark. To protect against this, some computer programs are available that reduce the screen’s blue light output in the evenings.
Special eyeglasses that block blue light are also available. Though melatonin is linked to sleep in some animals (including
humans), nocturnal animals are activated by increasing melatonin levels. Varying day/night lengths during the year alter melatonin
production and provide biological signals of the seasons. These are especially important in the seasonal coloring and breeding
habits of some animals. Melatonin is present in cherries, bananas, grapes, rice, cereals, olive oil, wine, and beer.
Serotonin
Serotonin, or 5-hydroxytryptamine, is a monoamine neurotransmitter derived from tryptophan. Blood platelets store serotonin and
release it when they bind to a clot, causing vasoconstriction. Serotonin plays a role in cognitive functions and enhances memory
and learning. Serotonin is widely thought to be a contributor to feelings of happiness and well-being. Some common anti-
depressant drugs, including Prozac, Paxil and Zoloft, act to modulate action of serotonin at synapses.
Niacin
Niacin is also known as Vitamin B3 and nicotinic acid. Niacin can be made from tryptophan and people who have the inability to
absorb tryptophan in the digestive system exhibit symptoms similar to niacin deficiency.
Extreme deficiency of niacin in the diet leads to the disease known as pellagra, while insufficient amounts of niacin in the diet are
linked with nausea, anemia, headaches, and tiredness. A diet that is primarily composed of grains like corn can lead to niacin
deficiency, because the niacin in these sources is not readily bioavailable. Treatment of the grain with alkali, as in the traditional
Mexican practice of soaking corn in lime, can make the niacin more easily absorbed from food.
Niacin is related to pyridine and the amide form of it is nicotinamide, an important component of NAD+/NADH and
NADP+/NADPH. The last pairs of molecules are essential as electron acceptors/carriers for most cellular oxidation-reduction
reactions.
Auxins
Auxins are plant growth hormones derived from tryptophan. The most important of these is indole-3-acetic acid (Figure 6.151).
Auxins are involved in almost every aspect of plant growth and development. They activate proteins, such as expansins and various
enzymes that modify the structure of cell wall components, to loosen the cell walls of a plant and stimulate elongation of cells. In

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the presence of cytokinins, auxins stimulate cell division. Auxins are also involved in the maintenance of meristems and in cell
patterning and organogenesis. Auxins are crucial for establishing root primordia as well as for elongation of root hairs. Auxins play
important roles in organizing the xylem and phloem of plants, and it has long been known that plant callus tissue can be made to
differentiate into shoots or roots, depending on the relative concentrations of auxins and cytokinins supplied in the medium.
Agrobacterium tumefaciens, a bacterium which infects a wide variety of plants, inserts its own DNA, including genes necessary for
the synthesis of plant hormones, into its host’s cells. The subsequent overproduction of auxins stimulates the growth of tumors
(called crown galls) on the plant (Figure 6.153).
Phenylalanine
Phenylalanine is an essential, hydrophobic amino acid in humans that is a precursor of tyrosine and since tyrosine is a precursor of
several important catecholamines, phenylalanine is, thus, a precursor of them as well.
PKU
Phenylalanine is linked to the genetic disease phenylketonuria (PKU) which arises from an inability to metabolize the amino acid
in people lacking (or deficient in) the enzyme phenylalanine hydroxylase. If left untreated, the disease can cause brain damage and
even death, but if detected early, it can be easily managed by carefully monitoring dietary intake of the amino acid. Because of this,
newborns are routinely tested for PKU. Phenylalanine is a component of the artificial sweetener known as aspartame (Nutrasweet -
Figure 6.154) and is consequently dangerous for people suffering from this disorder.
Biosynthesis of phenylalanine in bacteria overlaps with synthesis of tryptophan. The branch occurs at chorismic acid where the
enzyme chorismate mutase catalyzes a molecular rearrangement to produce prephenate.
Proton attack on prephenate results in loss of water and carbon dioxide to yield phenylpyruvate.
Transamination of phenylpyruvate yields phenylalanine.
Alternatively, phenylalanine can obtain its amine group in a transamination reaction from alanine.
Hydroxylation of phenylalanine by aromatic amino acid hydroxylase (phenylalanine hydroxylase) yields tyrosine.

Tyrosine
Because tyrosine is made from phenylalanine and the latter is an essential amino acid in humans, it is not clear whether to classify
tyrosine as essential or non-essential. Some define it as a conditionally essential amino acid. Others simply categorize it as non-
essential.
As noted above, tyrosine can arise as a result of hydroxylation of phenylalanine. In addition, plants can synthesize tyrosine by
oxidation of prephenate followed by transamination of the resulting 4-hydroxyphenylpyruvate (Figure 6.155).
The hydroxyl group on tyrosine is a target for phosphorylation by protein kinase enzymes involved in signal transduction pathways
(Figure 6.156). When located in membranes, these enzymes are referred to as receptor tyrosine kinases and they play important
roles in controlling cellular behavior/response.
In photosystem II of chloroplasts, tyrosine, at the heart of the system, acts as an electron donor to reduce oxidized chlorophyll. The
hydrogen from the hydroxyl group of tyrosine is lost in the process, requiring re-reduction by four core manganese clusters.
Tyrosine is also important in the small subunit of class I ribonucleotide reductases where it forms a stable radical in the catalytic
action of the enzyme (see HERE).
Tyrosine metabolites
Tyrosine is a precursor of catecholamines, such as L-dopa, dopamine, norepinephrine, and epinephrine (Figure 6.157). The thyroid
hormones triiodothyronine (T3) and thyroxine (T4) are also synthesized from tyrosine. As shown in Figure 6.158, this involves a
series of iodinations of tyrosines side-chains of a protein known as thyroglobulin. Combinations of iodinated tyrosines give rise to
thyroxine and triiodothyronine. These are subsequently cleaved from the protein and released into the bloodstream.
Oxidation and polymerization of tyrosine is involved in synthesis of the family of melanin pigments. Tyrosine is involved in the
synthesis of at least two types - eumelanin and pheomelanin (Figure 6.159).
Another molecule derived from tyrosine is the benzoquinone portion of Coenzyme Q (CoQ). This pathway requires the enzyme
HMG-CoA Reductase and since this enzyme is inhibited by cholesterol-lowering statin drugs, CoQ can be limited in people being
treated for high cholesterol levels.

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Dopamine
Dopamine plays several important roles in the brain and body. A member of the catecholamine and phenethylamine families, its
name comes from the fact that it is an amine made by removing a carboxyl group from L-DOPA. Dopamine is synthesized in the
brain and kidneys. It is also made in plants, though its function in plants is not clear. Conversion of dopamine to norepinephrine
(Figure 6.157) requires vitamin C.
Dopamine is a neurotransmitter, being released by one nerve cell and then traveling across a synapse to signal an adjacent nerve
cell. Dopamine plays a major role in the brain’s reward-mediated behavior. Rewards, such as food or social interaction, increase
dopamine levels in the brain, as do addictive drugs. Other brain dopamine pathways are involved in motor control and in managing
the release of various hormones.
Chemical messenger
Outside the nervous system, dopamine is a local chemical messenger. In blood vessels, it inhibits norepinephrine release and causes
vasodilation. In the kidneys, it increases sodium excretion and urine output. It reduces gastrointestinal motility and protects
intestinal mucosa in the digestive system and in the immune system, it reduces lymphocyte activity. The effect dopamine has on the
pancreas is to reduce insulin production. With the exception of the blood vessels, dopamine is synthesized locally and exerts its
effects near the cells that release it.
Epinephrine
Epinephrine (also called adrenalin) is a catecholamine chemically related to norepinephrine that is a hormone with medical
applications. It is used to treat anaphylaxis, cardiac arrest, croup, and, in some cases, asthma, when other treatments are not
working, due to its ability to favor bronchodilation.
Epinephrine is the drug of choice for treating anaphylaxis. The compound may be given through inhalation, by intravenous
injection, or subcutaneous injection and exerts effects through the α- and β-adrenergic receptors. In the body, it is produced and
released by adrenal glands and some neurons.
Effects
Physiological effects of epinephrine may include rapid heart beat, increased blood pressure, heart output, pupil dilation, blood
sugar concentration and increased sweating. Other physical effects may include shakiness, increased anxiety, and an abnormal heart
rhythm.
Norepinephrine
Norepinephrine (also called noradrenalin) is a catecholamine molecule that acts as a hormone and neurotransmitter. It is chemically
similar to epinephrine, differing only in the absence of a methyl group on its amine. Norepinephrine is made and released by the
central nervous system (locus coeruleus of the brain) and the sympathetic nervous system. The compound is released into the blood
stream from adrenal glands and affects α- and β-adrenergic receptors.
Norepinephrine is at its lowest levels during sleep and at its highest levels during stress (fight or flight response). The primary
function of norepinephrine is to prepare the body for action. It increases alertness, enhances memory functions, and helps to focus
attention. Norepinephrine increases heart rate and blood pressure, increases blood glucose and blood flow to skeletal muscle and
decreases flow of blood to the gastrointestinal system.
Medical considerations
Norepinephrine may be injected to overcome critically low blood pressure and drugs countering its effects are used to treat heart
conditions. α-blockers, for example, are used to battle cardiovascular and psychiatric disorders. β-blockers counter a different set of
norepinephrine’s effects than α-blockers and are used to treat glaucoma, migraine headaches and other cardiovascular problems.
Pyruvate family
The family of amino acids derived from pyruvate has four members, each with a simple aliphatic side chain no longer than four
carbons. The simplest of these is alanine.
Alanine
Alanine is the amino acid that is most easily produced from pyruvate. The simple transamination catalyzed by alanine transaminase
produces alanine from pyruvate.

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Alternative pathways for synthesis of alanine include catabolism of valine, leucine, and isoleucine.
Glucose-alanine cycle
The glucose-alanine cycle is an important nitrogen cycle related to the Cori cycle that occurs between muscle and liver cells in the
body (see HERE). In it, breakdown of glucose in muscles leads to pyruvate. When nitrogen levels are high, pyruvate is
transaminated to alanine, which is exported to hepatocytes.
In the liver cells, the last transamination of the glucose-alanine cycle occurs. The amine group of alanine is transferred to α-
ketoglutarate to produce pyruvate and glutamate. Glucose can then be made by gluconeogenesis from pyruvate. Importantly,
breakdown of glutamate yields ammonium ion, which can be made into urea for excretion, thus reducing the body’s load of
potentially toxic amines. This pathway may be particularly important in the brain.
Another way of removing excess ammonium from a tissue is by attaching it to glutamate to make glutamine. Glutamate is a
neurotransmitter, so having an alternative way of removing amines (glucose-alanine cycle) is important, especially in the brain.
Leucine
Like valine and isoleucine, leucine is an essential amino acid in humans. In adipose tissue and muscle, leucine is used in sterol
synthesis. It is the only amino acid to stimulate muscle protein synthesis, and as a dietary supplement in aged rats, it slows muscle
degradation. Leucine is an activator of mTOR, a protein which, when inhibited, has been shown to increase life span in
Saccharomyces cerevisiae, C. elegans, and Drosophila melanogaster.
Metabolism of leucine, valine, and isoleucine (also called Branched Chain Amino Acids - BCAAs) starts with decarboxylation of
pyruvate and attachment of the two-carbon hydroxyethyl fragment to thiamine pyrophosphate (Figure 6.161). Metabolism of
isoleucine proceeds with attachment of the hydroxylated two carbon piece (hydroxyethyl-TPP) to α-ketobutyrate and is covered in
the section describing that amino acid (see HERE).
Metabolism of valine and leucine proceeds with attachment of the hydroxyethyl piece from TPP to another pyruvate to create α-
acetolactate. Rearrangement of α-acetolactate by acetolactate mutase makes 3-hydroxy-3-methyl-2-oxobutanoate.
Reduction with NAD(P)H by acetohydroxy acid isomeroreductase yields α,β-dihydroxyisovalerate.
Loss of water, catalyzed by dihydroxyacid dehydratase produces α-ketoisovalerate.
This molecule is a branch point for synthesis of leucine and valine. Addition of an acetyl group from acetyl-CoA yields α-
isopropylmalate (catalyzed by α-isopropylmalate synthase).
Rearrangement, catalyzed by isopropylmalate dehydratase, gives rise to β-isopropylmalate.
Oxidation by isopropylmalate dehydrogenase and NAD+, gives α-ketoisocaproate.
Transamination of it (catalyzed by leucine aminotransferase and using glutamate) gives the final product of leucine (top of next
column).
Valine
An essential amino acid in humans, valine is derived in plants from pyruvate and shares part of its metabolic synthesis pathway
with leucine and a small slice of it with isoleucine. Metabolism of all three amino acids starts with decarboxylation of pyruvate and
attachment of the two-carbon hydroxyethyl fragment to thiamine pyrophosphate (Figure 6.161), as noted above.
As seen earlier, α-ketoisovalerate is the molecule at the point in the metabolic pathway where synthesis of valine branches from
that of leucine. In fact, α-ketoisovalerate is only one step away from valine. Transamination of α-ketoisovalerate catalyzed by
valine isoleucine aminotransferase gives valine.
Isoleucine
Synthesis of isoeleucine (an essential amino acid in humans) begins in plants and microorganisms with pyruvate and α-ketobutyrate
(a byproduct of threonine metabolism - threonine deaminase - Figure 6.162).
Metabolism of isoleucine proceeds with attachment to α-ketobutyrate of the hydroxyethyl-TPP product of pyruvate
decarboxylation to form α-aceto-α-hydroxybutyrate. The reaction is catalyzed by acetolactate synthase. Rearrangement and
reduction by acetohydroxy acid isomeroreductase and NAD(P)H yields α,β-dihydroxy-β-methylvalerate. Shown on next page.
Loss of water (catalyzed by dihydroxy acid dehydratase) gives α-keto-β-methylvalerate.

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Τransamination (using glutamate and valine isoleucine transaminase) yields isoleucine.
Interestingly, several of the enzymes of valine metabolism catalyze reactions in the isoleucine pathway. Though the substrates are
slightly different, they are enough like the valine intermediates that they are recognized as substrates.
Isoleucine has a second asymmetric center within it, but only one isomeric form of the four possible ones from the two centers is
found biologically.
Regulation of synthesis
Regulation of synthesis of the branched chain amino acids (BCAAs - valine, leucine, and isoleucine) is complex. The key molecule
in the regulation is α-ketobutyrate, which is synthesized in cells as a breakdown product of threonine. The enzyme catalyzing its
synthesis is threonine deaminase (Figure 6.162), which is allosterically regulated. The enzyme is inhibited by its own product
(isoleucine) and activated by valine, a product of a parallel pathway.
Thus, when valine concentration is high, the balances shifts in favor of production of isoleucine and since isoleucine competes with
valine and leucine for hydroxyethyl-TPP, synthesis of these two amino acids goes down. When isoleucine concentration increases,
threonine deaminase is inhibited, shifting the balance back to production of valine and leucine.

Attenuation
Another control mechanism for regulation of leucine synthesis occurs in bacteria and is known as attenuation. In this method,
accumulation of leucine speeds the process of translation of a portion of the mRNA copy of the leucine operon (coding sequences
for enzymes necessary to make leucine). This, in turn, causes transcription of the genes of the leucine operon to terminate
prematurely, thus stopping production of the enzymes necessary to make leucine.
When leucine levels fall, translation slows, preventing transcription from terminating prematurely and allowing leucine metabolic
enzymes to be made. Thus, leucine levels in the cell control the synthesis of enzymes necessary to make it.

Histidine family
Synthesis of histidine literally occurs in a class by itself - there are no other amino acids in its synthesis family. The amino acid is
made in plants (Arabidopsis, in this case) by a pathway that begins with ribose-5-phosphate. The overall pathway is show in the
green text boxes on the next two pages. Abbreviations used in the boxes are shown below.
Enzyme names
1 = Ribose-phosphate diphosphokinase
2= ATP-phosphoribosyltransferase
3 = Phosphoribosyl-ATP pyrophospohydrolase
4 = Phosphoribosyl-AMP cyclohydrolase
5 = ProFAR-I (N’-[(5’phosphoribosyl)formimino]-5-aminoimidazole-4-carboxamide ribonucleotide isomerase)
6 = Imidazole glycerol-phosphate synthase (IGPS)
7 = Ιmidazole glycerol-phosphate dehydratase
8 = Histidinol-phosphate aminotransferase
9 = Histidinol-phosphate phosphatase
10 = Histidinol dehydrogenase
Abbreviations used
1 - PRPP = Phosphoribosyl Pyrophosphate
2. PRATP = Phosphoribosyl ATP
3. PRAMP = Phosphoribosyl AMP
4. ProFAR = (N′-[(5′-phosphoribosyl)formimino]-5-aminoimidazole-4-carboxamide) ribonucleotide
5. PRFAR = (N′-[(5-phosphoribulosyl)formimino]-5-aminoimidazole-4-carboxamide) ribonucleotide
6. IGP = Imidazole glycerol-phosphate
7. AICAR = 5′-phosphoribosyl-4-carboximide-5-aminoimidazole
8. IAP = Imidazole acetol-phosphate
9. α-KG = α-ketoglutarate

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Histidine is a feedback inhibitor of ATP-phosphoribosyltransferase and thus helps to regulate its own synthesis. Histidine is the
only amino acid to contain an imidazole ring. It is ionizable and has a pKa of about 6. As a result, histidine’s R-group can gain/lose
a proton at pH values close to cellular conditions.

Selenocysteine
A cysteine analog commonly referred to as the 21st amino acid, selenocysteine (Figure 6.163) is an unusual amino acid
occasionally found in proteins. Although it is rare, selenocysteine has been found in proteins in bacteria, archaea and eukaryotes.
In contrast to amino acids such as phosphoserine, hydroxyproline, or acetyl-lysine, which arise as a result of post-translational
modifications, selenocysteine is actually built into growing peptide chains in ribosomes during the process of translation.
No codon specifies selenocysteine, so to incorporate it into a protein, a tRNA carrying it must bind to a codon that normally
specifies STOP (UGA). This alternative reading of the UGA is dependent on formation of a special hairpin loop structure in the
mRNA encoding selenoproteins.
Selenium is rather toxic, so cellular and dietary concentrations are typically exceedingly low. About 25 human proteins are known
to contain the amino acid. These include five glutathione peroxidases, and three thioredoxin reductases. Iodothyronine deiodinase,
a key enzyme that converts thyroxine to the active T3 form, also contains selenocysteine in its active site. All of these proteins
contain a single selenocysteine.
A eukaryotic protein known as selenoprotein P, found in the blood plasma of animals, contains ten selenocysteine residues and is
thought to function as an antioxidant and/or in heavy metal detoxification. Besides selenocysteine, at least two other biological
forms of a seleno-amino acid are known. These include 1) selenomethionine (Figure 6.164), a naturally occurring amino acid in
Brazil nuts, cereal grains, soybeans, and grassland legumes and 2) methylated forms of selenocysteine, such as Se-
methylselenocysteine, are found in Astragalus, Allium, and Brassica species.
Stop codon
The specifics of the process of translation will be described elsewhere in the book, but to get selenocysteine into a protein, the
tRNA carrying selenocysteine pairs with a stop codon (UGA) in the mRNA in the ribosome. Thus, instead of stopping translation,
selenocysteine can incorporated into a growing protein and translation continues instead of stopping.
Four genes are involved in preparation of selenocysteine for incorporation into proteins. They are known as sel A, sel B, sel C, and
sel D. Sel C codes for the special tRNA that carries selenocysteine. The amino acid initially put onto the selenocysteine tRNA is
not selenocysteine, but rather serine. Action of sel A and sel D are necessary to convert the serine to a selenocysteine.
An intermediate in the process is selenophosphate, which is the selenium donor. It is derived from H2Se, the form in which
selenium is found in the cell. The tRNA carrying selenocysteine has a slightly different structure than other tRNAs, so it requires
assistance in translation. The sel B gene encodes for an EF-Tu-like protein that helps incorporate the selenocysteine into the protein
during translation.
Recoding the UGA
Using UGA codons to incorporate selenocysteine into proteins could wreak havoc if done routinely, as UGA, in fact, almost always
functions as a stop codon and is only rarely used to code for selenocysteine. Fortunately, there is a mechanism to ensure that the
reading of a UGA codon as selenocysteine occurs only when the mRNA encodes a selenoprotein.
Unusual structures in mRNAs
The mRNAs for selenocysteine-containing proteins form unusual mRNA structures around the UGA codon that make the ribosome
“miss” it as a stop codon and permit the tRNA with selenocysteine to be incorporated instead.
Pyrrolysine
Like selenocysteine, pyrrolysine is a rare, unusual, genetically encoded amino acid found in some cells. Proteins containing it are
enzymes involved in methane metabolism and so far have been found only methanogenic archaeans and one species of bacterium.
The amino acid is found in the active site of the enzymes containing it. It is sometimes referred to as the 22nd amino acid.
Synthesis of the amino acid biologically begins with two lysines. One is converted to (3R)-3-Methyl-D-ornithine, which is attached
to the second lysine. After elimination of an amine group, cyclization, and dehydration, L-pyrrolysine is produced. Pyrrolysine is
attached to an unusual tRNA (pylT gene product) by action of the aminoacyl tRNA synthetase encoded by the pylS gene. This

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unusual tRNA can pair with the UAG stop codon during translation and allow for incorporation of pyrrolysine into the growing
polypeptide chain during translation in a manner similar to incorporation of selenocysteine.
Urea cycle
The urea cycle holds the distinction of being the first metabolic cycle discovered - in 1932, five years before the citric acid cycle. It
is an important metabolic pathway for balancing nitrogen in the bodies of animals and it takes place primarily in the liver and
kidney.
Organisms, like humans, that excrete urea are called ureotelic. Those that excrete uric acid (birds, for example) are called uricotelic
and those that excrete ammonia (fish) are ammonotelic. Ammonia, of course, is generated by metabolism of amines and is toxic, so
managing levels of it is critical for any organism. Excretion of ammonia by fish is one reason that an aquarium periodically
requires cleaning and replacement of water.
Liver failure can lead to accumulation of nitrogenous waste and exacerbates the problem. As shown in Figure 1.166, the cycle
contains five reactions, with each turn of the cycle producing a molecule of urea. Of the five reactions, three occur in the cytoplasm
and two take place in the mitochondrion. (The reaction making carbamoyl phosphate, catalyzed by carbamoyl phosphate synthetase
is not shown in the figure.)
Ornithine synthesis
Though the cycle doesn’t really have a starting point, a common place to begin discussion is with the molecule of ornithine. As
discussed elsewhere in this book, ornithine intersects the metabolic pathways of arginine and proline.
Ornithine is found in the cytoplasm and is transported into the mitochondrion by the ornithine-citrulline antiport of the inner
mitochonrial membrane. In the matrix of the mitochondrion, two reactions occur relevant to the cycle. The first is formation of
carbamoyl phosphate from bicarbonate, ammonia, and ATP catalyzed by carbamoyl phosphate synthetase I.
Carbamoyl phosphate then combines with ornithine in a reaction catalyzed by ornithine transcarbamoylase to make citrulline.
The citrulline is transported out to the cytoplasm by the ornithine-citrulline antiport mentioned above. In the cytoplasm, citrulline
combines with L-aspartate using energy of ATP to make citrullyl-AMP (an intermediate) followed by argininosuccinate. The
reaction is catalyzed by argininosuccinate synthase.
Next, fumarate is split from argininosuccinate by argininosuccinate lyase to form arginine.
Water is used by arginase to cleave arginine into urea and ornithine, completing the cycle.
Urea is less toxic than ammonia and is released in the urine. Some organisms make uric acid for the same reason.
It is worth noting that aspartic acid, ammonia, and bicarbonate enter the cycle and fumarate and urea are produced by it. Points to
take away include 1) ammonia is converted to urea using bicarbonate and the amine from aspartate; 2) aspartate is converted to
fumarate which releases more energy than if aspartate were converted to oxaloacetate, since conversion of fumarate to malate to
oxaloacetate in the citric acid cycle generates an NADH, but direct conversion of aspartate to oxaloacetate does not; and 3)
glutamate and aspartate are acting as shuttles to funnel ammonia into the cycle. Glutamate, as will be seen below, is a scavenger of
ammonia.
Urea cycle regulation
The urea cycle is controlled both allosterically and by substrate concentration. The cycle requires N-acetylglutamate (NAG) for
allosteric activation of carbamoyl phosphate synthetase I. The enzyme that catalyzes synthesis of NAG, NAG synthetase, is
activated by arginine and glutamate. Thus, an indicator of high amine levels, arginine, and an important shuttler of amine groups,
glutamate, stimulates the enzyme that activates the cycle.
The reaction catalyzed by NAG synthetase is
At the substrate level, all of the other enzymes of the urea cycle are controlled by the concentrations of substrates they act upon.
Only at high concentrations are the enzymes fully utilized.
Complete deficiency of any urea cycle enzyme is fatal at birth, but mutations resulting in reduced expression of enzymes can have
mixed effects. Since the enzymes are usually not limiting for these reactions, increasing substrate can often overcome reduced
enzyme amounts to a point by simply fully activating enzymes present in reduced quantities.
Ammonia accumulation

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However, if the deficiencies are sufficient, ammonium can accumulate and this can be quite problematic, especially in the brain,
where mental deficiencies or lethargy can result. Reduction of ammonium concentration relies on the glutamate dehydrogenase
reaction (named for the reverse reaction).
Additional ammonia can be taken up by glutamate in the glutamine synthetase reaction.
The result of these reactions is that α-ketoglutarate and glutamate concentrations will be reduced and the concentration of
glutamine will increase. For the brain, this is a yin/yang situation. Removal of ammonia is good, but reduction of α-ketoglutarate
concentration means less energy can be generated by the citric acid cycle. Further, glutamate is, itself, an important
neurotransmitter and a precursor of another neurotransmitter - γ-aminobutyric acid (GABA).
Energy generation
From an energy perspective, the urea cycle can be said to break even or generate a small amount of energy, if one includes the
energy produced in releasing ammonia from glutamate (one NADH). There are two NADHs produced (including the one for
converting fumarate to oxaloacetate), which give 4-6 ATPs, depending on how efficiently the cell performs electron transport and
oxidative phosphorylation.
The cycle takes in 3 ATPs and produces 2 ADPs and one AMP. Since AMP is equivalent to 2 ATP, the cycle uses 4 ATP. Thus, the
cycle either breaks even in the worst case or generates 2 ATPs in the best case.

Amino acid catabolism

Amino acids are divided according to the pathways involved in their degradation. There are three general categories. Ones that
yield intermediates in the glycolysis pathway are called glucogenic and those that yield intermediates of acetyl-CoA or acetoacetate
are called ketogenic. Those that involve both are called glucogenic and ketogenic. These are shown in Figures 6.167 and 6.168.
As seen in the two figures, amino acids largely produce breakdown products related to intermediates of the citric acid cycle or
glycolysis, but this isn’t the complete picture. Some amino acids, like tryptophan, phenylalanine, and tyrosine yield hormones or
neurotransmitters on further metabolism (as noted earlier). Others like cysteine and methionine must dispose of their sulfur and all
of the amino acids must rid themselves of nitrogen, which can happen via the urea cycle, transamination, or both.

Tyrosine catabolism
Breakdown of tyrosine (Figure 6.169) is a five step process that yields acetoacetate and fumarate. Enzymes involved include 1)
tyrosine transaminase; 2) p-hydroxylphenylpyruvate dioxygenase; 3) homogentisate dioxygenase; 4) maleylacetoacetate cis-trans-
isomerase; and 5) 4-fumaryl acetoacetate hydrolase.
Breakdown of leucine is a multi-step process ultimately yielding the ketone body acetoacetate and acetyl-CoA. Branched chain
amino acids (BCAAs - valine, leucine, and isoleucine) rely on Branched Chain AminoTransferase (BCAT) followed by Branched
Chain α-ketoacid dehydrogenase (BCKD) for catabolism.
Breakdown of isoleucine yields intermediates that are both ketogenic and glucogenic. These include acetyl-CoA and propionyl-
CoA.
Breakdown of valine is a multi-step process ultimately yielding propionyl-CoA.

This page titled 6.5: Amino Acids and the Urea Cycle is shared under a CC BY-NC-SA license and was authored, remixed, and/or curated by
Kevin Ahern, Indira Rajagopal, & Taralyn Tan.

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6.6: Nucleotides
Source: BiochemFFA_6_6.pdf. The entire textbook is available for free from the authors at
http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy

Diverse functions of nucleotides

Nucleotides are most often thought of as the building blocks of the nucleic acids, DNA and RNA. While this, is, of course, a vital
function, nucleotides also play other important roles in cells. Ribonucleoside triphosphates like ATP, CTP, GTP and UTP are
necessary, not just for the synthesis of RNA, but as part of activated intermediates like UDP-glucose in biosynthetic pathways. ATP
is also the universal “energy currency” of cells, and coupling of energetically unfavorable reactions with the hydrolysis of ATP
makes possible the many reactions in our cells that require an input of energy. Adenine nucleotides serve as components of
NAD(P)+ and FAD. Nucleotides can also serve as allosteric and metabolic regulators. The synthesis and breakdown pathways for
nucleotides and the molecules derived from them are thus, of vital importance to cells. Regulation of nucleotide synthesis,
especially for deoxyribonucleotides, is important to ensure that the four nucleotides are made in the right proportions, as
imbalances in nucleotide concentrations can lead to increases in mutation rates.
Pathways of nucleotide metabolism are organized in two major groups and one minor one. These include, respectively, metabolism
of 1) purines; 2) pyrimidines; and 3) deoxyribonucleotides. Each group can be further subdivided into pathways that make
nucleotides from simple precursors (de novo pathways) and others that use pieces of nucleotides to reassemble full ones (salvage
pathways). Notably, de novo synthesis pathways for all of the nucleotides begin with synthesis of ribonucleotides.
Deoxyribonucleotides are made from the ribonucleotides.

Purine nucleotide metabolism

Synthesis of purine nucleotides by the de novo pathway begins with addition of a pyrophosphate to carbon 1 of ribose-5-phosphate,
creating phosphoribosylpyrophosphate (PRPP). The reaction is catalyzed by PRPP synthetase. Some number the purine metabolic
pathway starting with the next reaction. We have therefore given this reaction the number of zero in Figure 6.172.
In the next step (reaction 1 in Figure 6.172), the pyrophosphate is replaced by an amine from glutamine in a reaction catalyzed by
PRPP amidotransferase (PPAT). The product is 5-phosphoribosylamine (5-PRA).
PPAT is an important regulatory enzyme for purine biosynthesis. The end products of the pathway, AMP and GMP both inhibit the
enzyme and PRPP activates it. Interestingly, full inhibition of the enzyme requires binding of both AMP and GMP.
Binding of only one of the two nucleotides allows the enzyme to remain partially active so that the missing nucleotide can be
synthesized. Through this enzyme, the relative amounts of ATP and GTP are controlled.
5-PRA is very unstable chemically (half-life of 38 seconds at 37°C), so it has been proposed that it is shuttled directly from PRPP
amidotransferase to GAR synthetase for the next reaction.
In this reaction (#2), glycine is added to the growing structure above the ribose-5-phosphate to create glycineamide ribonucleotide
(GAR). This reaction, which requires ATP, is catalyzed, as noted, by the enzyme GAR synthetase.
In reaction #3, a formyl group is transferred onto the GAR from N10-formyl-tetrahydrofolate (N10-formyl-THF or fTHF) by
phosphoribosylglycinamide formyltransferase (GART).
Next, the double bonded oxygen in the ring is replaced with an amine in a reaction catalyzed by
phosphoribosylformylglycinamidine synthase (PFAS) that uses glutamine and produces glutamate. The reaction requires energy
from ATP (top of next column).
In humans the GAR synthetase, phosphoribosylglycinamide formyltransferase, and the enzyme catalyzing the next reaction (#5),
AIR synthetase activities are all on the same protein known as trifunctional purine biosynthetic protein adenosine-3.
In reaction #6, carboxylation of AIR occurs, catalyzed by phosphoribosylaminoimidazole carboxylase (PAIC)
Aspartic acid is then added to donate its amine group and fumarate will be lost in the reaction that follows this one. The enzyme
involved here is phosphoribosyl-aminoimidazole-succinocarboxamide synthase (PAICS)
In the next reaction, the carbon shell of aspartate is released (as fumarate) and the amine is left behind. The reaction is catalyzed by
adenylosuccinate lyase (ADSL).

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Reaction #9 involves another formylation reaction, catalyzed by phosphoribosylaminoimidazolecarboxamide formyltransferase
(ATIC-E1).
Next, inosine monophosphate synthase (ATIC-E2) catalyzes release of water to form the first molecule classified as a purine -
inosine monophosphate or IMP).
Though it doesn’t appear in DNA, IMP does, in fact, occur in the anticodon of many tRNAs where its ability to pair with numerous
bases is valuable in reading the genetic code.
IMP is a branch point between pathways that lead to GMP or AMP. The pathway to GMP proceeds via catalysis by IMP
dehydrogenase as follows:
In the last step of GMP synthesis, GMP synthase catalyzes a transamination to form GMP using energy from ATP.
The energy source being ATP makes sense, since the cell is presumably making GMP because it needs guanine nucleotides. If the
cell is low on guanine nucleotides, GTP would be in short supply.

Adenine nucleotide synthesis

Synthesis of AMP from IMP follows. First, adenylosuccinate synthetase catalyzes the addition of aspartate to IMP, using energy
from GTP.
Then, adenylosuccinate lyase splits fumarate off to yield AMP.
In humans, the bifunctional purine biosynthesis protein known as PURH contains activities of the last two enzymes above.
Abbreviations used above
PRPP = Phosphoribosyl Pyrophosphate
5-PRA = 5-phosphoribosylamine
GAR = glycineamide ribonucleotide
fGAR = Phosphoribosyl-N-formylglycineamide
THF = Tetrahydrofolate
fTHF = N10-formyl-Tetrahydrofolate
fGAM = 5'-Phosphoribosylformylglycinamidine
AIR = 5-Aminoimidazole ribotide
CAIR = 5'-Phosphoribosyl-4-carboxy-5-aminoimidazole
SAICAR = Phosphoribosylamino-imidazolesuccinocarboxamide
AICAR = 5-Aminoimidazole-4-carboxamide ribonucleotide
FAICAR = 5-Formamidoimidazole-4-carboxamide ribotide
IMP = inosine monophosphate

Regulation
It is worth repeating that synthesis of GMP from IMP requires energy from ATP and that synthesis of AMP from IMP requires
energy from GTP. In addition, the enzymes converting IMP into intermediates in the AMP and GMP pathways are each feedback
inhibited by the respective monophosphate nucleotide. Thus, IMP dehydrogenase is inhibited by GMP (end product of pathway
branch) and adenylosuccinate synthetase is inhibited by AMP, the end product of that pathway branch.
Purine nucleotide levels are balanced by the combined regulation of PRPP amidotransferase , IMP dehydrogenase,
adenylosuccinate synthetase and the nucleotides AMP and GMP. The importance of the regulatory scheme of purines is illustrated
by two examples. First imagine both AMP and GMP are abundant. When this occurs, PRPP amidotransferase will be completely
inhibited and no purine synthesis will occur.

Partial activity
High levels of GMP and low levels of AMP would result in PRPP amidotransferase being slightly active, due to the fact GMP will
fill one allosteric site, but low AMP levels will mean second allosteric site will likely be unfilled. This lowered (but not completely
inhibited) activity of PRPP amidotransferase will allow for limited production of 5-PRA and the rest of the pathway intermediates,
so it will remain active.

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At the IMP branch, however, the high levels of GMP will inhibit IMP dehydrogenase, thus shutting off that branch and allowing all
of the intermediates to be funneled into making AMP. When the AMP level rises high enough, AMP binds to PRPP
amidotransferase and along with GMP, shuts off the enzyme. A reversal will occur if AMP levels are high, but GMP levels are low.

Proper balance
Regulated in this way, AMP and GMP levels can be maintained in a fairly narrow concentration range. Properly balancing
nucleotide levels in cells is critical. It is likely for this reason that cells have numerous controls on the amount of each nucleotide
made.
Other mechanisms
Cells have two other ways of balancing GMP and AMP nucleotides. First, the enzyme GMP reductase will convert GMP back to
IMP using electrons from NADPH.
The IMP, in turn, can then be made into AMP if its concentration is low. Second, AMP can be converted back to IMP by the
enzyme AMP deaminase. In this case, the IMP can then be made into GMP.
It is important to maintain appropriate proportions of the different nucleotides. Excess or scarcity of any nucleotide of any
nucleotide can result in an increased tendency to mutation.
To convert AMP to ATP and GMP to GTP requires action of kinase enzymes. Each monophosphate nucleotide form has its own
specific nucleoside monophosphate kinase. For adenine-containing nucleotides (ribose forms and deoxyribose forms), adenylate
kinase catalyzes the relevant reaction.
The adenylate kinase reaction is reversible and is used to generate ATP when the cell’s ATP concentration is low. When ATP is
made from 2 ADPs in this way, AMP levels increase and this is one way the cell senses that it is low on energy.
Guanosine monophosphates also have their own kinase and it catalyzes the reaction at the top of the next page.
Other monophosphate kinases for UMP and CMP use ATP in a similar fashion.
In going from the diphosphate form to the triphosphate form, the picture is simple - one enzyme catalyzes the reaction for all
diphosphates (ribose and deoxyribose forms). It is known as nucleoside diphosphate kinase or (more commonly) NDK or NDPK
and it catalyzes reactions of the form
where X and Y refer to any base.

Purine salvage reactions

Not all nucleotides in a cell are made from scratch. The alternatives to de novo syntheses are salvage pathways. Salvage reactions
to make purine nucleotides start with attachment of ribose to purine bases using phosphoribosylpyrophosphate (PRPP).
The enzyme catalyzing this reaction is known as hypoxanthine/guanine phosphoribosyltransferase (HGPRT - Figure 6.175) and is
interesting from an enzymological as well as a medical perspective. First, the enzyme is able to catalyze both of the next two
important salvage reactions - converting hypoxanthine to IMP or guanine to GMP.
HGPRT is able to bind a variety of substrates at its active site and even appears to bind non-natural substrates, such as acyclovir
preferentially over its natural ones.
Medical perspective
From a medical perspective, reduction in levels of HGPRT leads to hyperuricemia, a condition where uric acid concentration
increases in the body. Complete lack of HGPRT is linked to Lesch-Nyhan syndrome, a rare, inherited disease in high uric acid
concentration throughout the body is associated with severe accompanying neurological disorders.
Reduced production of HGPRT occurs frequently in males and has a smaller consequence (gout) than complete absence.
Interestingly, gout has been linked to a decreased likelihood of contracting multiple sclerosis, suggesting uric acid may help prevent
or ameliorate the disease.
Expression of HGPRT is stimulated by HIF-1, a transcription factor made in tissues when oxygen is limiting, suggesting a role for
HGPRT under these conditions.
Adenine salvage

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The enzyme known as adenine phosphoribosyltransferase (APRT) catalyzes the reaction corresponding to HGPRT for salvaging
adenine bases.

Pyrimidine nucleotide metabolism

The de novo pathway for synthesizing pyrimidine nucleotides has about the same number of reactions as the purine pathway, but
also has a different strategy. Whereas the purines were synthesized attached to the ribose sugar, pyrimidine bases are made apart
from the ribose and then attached later.
The first reaction is catalyzed by carbamoyl phosphate synthetase (Figure 6.176).
Two different forms are found in eukaryotic cells. Form I is found in mitochondria and form II is in the cytoplasm.
The reaction catalyzed by carbamoyl phosphate synthetase is the rate limiting step in pyrimidine biosynthesis and corresponds to
reaction 1 in Figure 6.178.

Balance
The enzyme is activated by ATP and PRPP and is inhibited by UMP. This helps to balance pyrimidine vs. purine concentrations.
High concentrations of a purine (ATP) activates the synthesis of pyrimidines. PRPP increases in concentration as purine
concentration increases, so it too helps to establish that balance. UMP is an end product of pyrimidine metabolism, so the process is
self-limiting. The next enzyme in the pathway, aspartate transcarbamoylase (ATCase) also plays a role in the same balance, as we
will see. The reaction it catalyzes is shown below and is reaction 2 in Figure 6.178.
ATCase is a classic enzyme exhibiting allosteric regulation and feedback inhibition, having both homotropic and heterotropic
effectors (Figure 6.179 and see HERE). With 12 subunits (6 regulatory and 6 catalytic units), the enzyme exists in two states - a
low activity T-state and a high activity R-state. Binding of the aspartate substrate to the active site shifts the equilibrium in favor of
the R-state.
Aspartate is a homotropic effector of the enzyme, because it acts allosterically on the enzyme and is a substrate for it as well.
Similarly, binding of ATP to the regulatory units favors the R-state, whereas binding of CTP to the regulatory units favors the T-
state. ATP and CTP are heterotropic effectors of the enzyme because they are not substrates for it, but act allosterically.
Regulation
As was seen with the first enzyme of the pathway, high concentration of purine nucleotides stimulates synthesis of pyrimidines and
high concentration of pyrimidines turns off the pathway that synthesizes them.
Dihydroorotase catalyzes reaction 3 and is found in the cytoplasm, as is ATCase.
Reaction 4 occurs in the mitochondrion, so the product of reaction 3, dihydroorotate, must be transported into the mitochondrion
from the cytoplasm. In reaction 4, dihydroorotate is oxidized to orotate. The enzyme catalyzing the reaction is dihydroorotate
dehydrogenase.
Reaction #5, catalyzed by orotate phosphoribosyl transferase, involves connection of orotate to ribose to yield a nucleotide -
orotidine-5’-monophosphate (OMP).
Last, OMP is converted to uridine-5’-monophosphate (UMP) by action of a fascinating enzyme known as OMP decarboxylase.
OMP decarboxylase is frequently cited as an example for the incredible ability of an enzyme to speed a reaction. The
decarboxylation of OMP, if allowed to proceed in the absence of an enzyme takes about 78 million years. In the presence of OMP
decarboxylase, the reaction takes place in 18 milliseconds, a speed increase of about 1017. Remarkably, the enzyme accomplishes
this without any cofactors or coenzymes of any kind.
The mechanism of action of the enzyme is shown in Figure 6.180. In mammals, the activities of OMP decarboxylase and orotate
phosphoribosyl transferase are contained on the same protein.
A monophosphate kinase (UMP/CMP kinase) catalyzes conversion of UMP to UDP.
The same enzyme will also phosphorylate CMP to CDP and dCMP to dCDP. Like the reaction of adenylate kinase, the reaction
above, when run in the reverse direction, can be a source of ATP when the cell is low on energy.
The next step, catalyzed by NDPK, uses energy of any triphosphate nucleotide (XTP) to produce UTP from UDP.
CTP Synthase

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UTP is the substrate for synthesis of CTP via catalysis by CTP synthase.
This enzyme is inhibited by its product, ensuring too much CTP is not made and activated by physiological concentrations of ATP,
GTP, and glutamine. One human isozyme, CTPS 1, has been shown to be inactivated by phosphorylation by glycogen synthase
kinase 3.
CTP synthase has two domains and is a heterodimer (Figure 6.183). It exists as an inactive monomer at low enzyme concentrations
or in the absence of UTP and ATP. One domain of the enzyme cleaves the amine group from glutamine and transfers it internally to
the UTP. The other domain (synthase domain) binds ATP and initiates the mechanism shown in Figure 6.184 for making CTP.
CTP is the only nucleotide synthesized de novo directly as a triphosphate, since it arises directly from UTP. Since
deoxyribonucleotides are made from ribonucleoside diphosphates, it means deoxycytidine nucleotides must either be made
preferentially from salvage nucleotides or CTP must be dephosphorylated first.
One enzyme that can do this is a membrane-bound enzyme known as apyrase, which sequentially converts CTP to CDP and then
CMP.
Pyrimidine salvage reactions
Pyrimidine salvage synthesis allows cells to remake pyrimidine triphosphate nucleotides starting from either the C or U pyrimidine
bases, nucleosides, or nucleotides. Figures 1.85 & 6.186 depict salvage pathway reactions. As is apparent in Figure 1.86, there are
multiple ways of making the same molecules. For example, uracil can be made into uridine by reaction 11 or by reaction 12.
The figure depicts not only the synthesis of CTP and UTP from basic components, but also shows how these nucleotides can be
broken down into smaller pieces.
In many cases, the same enzyme works on cytidine, uridine, and deoxycytidine molecules.
Enzymes of note
There are several enzymes of note in the salvage pathway. Seven enzymes, for example, work on both uracil and cytosine
containing nucleosides/nucleotides. These include NTP phosphatase (reaction 2), NDPK (reaction 3), apyrase (reaction 4), NDP
phosphatase (reaction 5), UMP/CMP kinase (reaction 6), pyrimidine-specific 5’ nucleotidase (reaction 7), and uridine/cytidine
kinase (reaction 8). The enzymes for reactions 6 and 8 can also use deoxyribonucleosides/deoxyribonucleotides as substrates.
Cytidine deaminase (reaction #9) converts cytidine to uridine by removing an amine group from the cytosine base and thus is a
counter for the UTP to CTP reaction catalyzed by CTP synthetase. Countered reactions allow cells to balance concentrations of
nucleosides/nucleotides in either direction if they should get out of balance.
Two other reactions in the figure are worth mentioning. Both UTP and CTP are converted in the breakdown process to UMP and
CMP, respectively. Both of these reactions are important for deoxyribonucleotide metabolism. In each case, the monophosphate
derivatives are phosphorylated, creating diphosphate derivatives (UDP and CDP) that are substrates for RNR that yield dUDP and
dCDP, respectively. dUDP is phosphorylated to dUTP and then pyrophosphate is removed by dUTPase to yield dUMP. dUMP is a
substrate for thymidine synthesis (see HERE). dCDP is converted to dCTP by NDPK
Deoxyribonucleotide metabolism
Deoxyribonucleotides, the building blocks of DNA, are made almost exclusively from ribonucleoside diphosphates. A single
enzyme called ribonucleotide reductase (RNR) is responsible for the conversion of each of these to a deoxy form (Figure 6.187).
The enzyme’s substrates are ribonucleoside diphosphates (ADP, GDP, CDP, or UDP) and the products are deoxyribonucleoside
diphosphates (dADP, dGDP, dCDP, or dUDP). Thymidine nucleotides are synthesized from dUDP.
RNR has two pairs of two identical subunits - R1 (large subunit) and R2 (small subunit). R1 has two allosteric binding sites and a
catalytic site. R2 forms a tyrosine radical necessary for the reaction mechanism of the enzyme.
There are three classes of RNR enzymes and they differ in the nature or means of generating a radical used in the enzyme’s
catalytic mechanism. Class I RNRs are found in eukaryotes, eubacteria, bacteriophages, and viruses. They all use a ferrous iron
center that loses an electron (converting to ferric iron) to generate a free radical on a tyrosine ring. These enzymes only work in
aerobic conditions.
Class II RNRs use 5’-deoxyadenosyl cobalamin (vitamin B12) to generate a radical and work under aerobic or anaerobic
conditions. They are found in eubacteria, archaebacteria, and bacteriophages. Class III RNRs generate a glycine radical using S-
adenosyl methionine (SAM) and an iron-sulfur center. They work under anaerobic conditions and are used by archaebacteria,

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eubacteria, and bacteriophages. Substrates for class I enzymes are ribonucleoside diphosphates. Class II enzymes work on
ribonucleoside diphosphates or ribonucleoside triphosphates. Class III enzymes work on ribonucleoside triphosphates.
In class I enzymes, RNR is an iron-dependent dimeric enzyme with each monomeric unit containing a large subunit (known as α or
R1) and a small subunit (known as β or R2). The R1 subunit contains regulatory binding sites for allosteric effectors (see below),
whereas the R2 subunit houses a tyrosine residue that forms a radical critical to the reaction mechanism of the enzyme. Electrons
needed in the reaction are transmitted from NADPH to the enzyme by one of two pathways, reducing a disulfide bond in the
enzyme to two sulfhydryls. In the first transfer mechanism, NADPH passes electrons to glutathione, which passes them to
glutaredoxin, which then donates them to the RNR enzyme used in the reaction. In the second mechanism, NADPH passes
electrons to FAD, which uses them to reduce thioredoxin, which then passes the electrons to RNR with the same end result as in the
first pathway - reduction of a suflhydryl in RNR.
In the reaction mechanism (Figure 6.188), a tyrosine side chain in the R2 unit must be radicalized to start. This electronic change is
transmitted through the small R2 subunit to the active site of the large R1 subunit. Several aromatic amino acid side chains are
thought to play a role in that process. Iron atoms in the R2 subunit assist in creation and stabilization of the radical. The tyrosine
radical contains an unpaired electron delocalized across its aromatic ring.
Transfer of the electronic instability to the R1 unit results in radicalization of a cysteine (to form a thiyl radical) at the active site.
The thiyl radical, thus formed, abstracts a hydrogen atom (proton plus electron) from carbon 3 of ribose on the bound
ribonucleoside diphosphate, creating a radical carbon atom. Radicalization of carbon #3 favors release of the hydroxyl group on
carbon #2 as water. The extra proton comes from the sulfhydryl of the enzyme’s cysteine. In the next step of the process, a proton
and two electrons from the same cysteine are transferred to carbon #2 and then carbon #3 takes back the proton originally removed
from it to yield a deoxyribonucleoside diphosphate. The enzyme’s thiyl group gains an electron from R2 and the disulfide bond
created in the reaction must be reduced by electrons from NADPH again in order to catalyze again.
Regulation
In addition to RNR’s unusual reaction mechanism, the enzyme also has a complex system of regulation, with two sets of allosteric
binding sites, both found in the R1 subunit. Because a single enzyme, RNR, is responsible for the synthesis of all four
deoxyribonucleotides, it is necessary to have mechanisms to ensure that the enzyme produces the correct amount of each dNDP.
This is a critical consideration, since imbalances in DNA precursors can lead to mutation.
Consequently, the enzyme must be responsive to the levels of the each deoxyribonucleotide, selectively making more of those that
are in short supply, and preventing additional synthesis of those that are abundant. These demands are met by having two separate
control mechanisms on the enzyme - one that determines which substrate will be acted on, and another that controls the enzyme’s
activity.
Two allosteric sites
RNR is allosterically regulated via two molecular binding sites - a specificity binding site (binds dNTPs and induces structural
changes in the enzyme that determines which substrates preferentially bind at the catalytic site and an activity control site (controls
whether or not enzyme is active). The activity control site functions like a simple on/off switch - ATP activates catalysis, dATP
inactivates it. (One subset of class I enzymes, however, is not affected by dATP.)
The inactivation of RNR by dATP is an important factor in the disease known as Severe Combined Immunodeficiency Disease
(SCID). In SCID, the salvage enzyme adenosine deaminase is deficient, leading to a rise in concentration of dATP in cells of the
immune system. dATP shuts down RNR in these cells, thus stopping their proliferation and leaving the affected individual with a
very weak or no immune system.
Allosteric effectors
When dTTP is abundant (Figure 6.189), it binds to RNR’s specificity site and inhibits binding and reduction of CDP and UDP but
stimulates binding and reduction of GDP at the active site of the enzyme. Conversely, binding of ATP or dATP at the specificity
site stimulates binding and reduction of CDP and UDP at the active site. Last, binding of dGTP to the specificity site (specificity
site B) induces binding and reduction of ADP at the active site.
Students sometimes confuse the active site of RNR with the activity control site (sometimes called the activity site). The active site
is where the reaction is catalyzed, and could better be called the catalytic site, whereas the activity site is an allosteric binding site
for ATP or dATP that controls whether the enzyme is active. High levels of dATP are an indicator that sufficient dNTPs are

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available, so the enzyme gets inhibited to stop production of more. Low levels of dATP allow binding of ATP and activation of the
enzyme.
In addition to regulation by deoxyribonucleotides and ATP, RNR can be directly inhibited by hydroxyurea.
dTTP synthesis
Synthesis of dTTP by the de novo pathway involves a multi-step process from UDP to dTTP. It begins with UDP, which is
converted to dUDP by RNR. dUDP is phosphorylated by NDPK to yield dUTP, which is quickly broken down by dUTPase to
produce dUMP. The remaining reactions are shown in Figure 6.190.
Important enzymes in the pathway include dUTPase and thymidylate synthetase. dUTPase is important for keeping the
concentration of dUTP low so it does not end up in DNA. DNA polymerase can use dUTP just as it does dTTP, and incorporate it
into a DNA strand, across from adenine nucleotides.
Thymidylate synthetase is important because it is a target (directly and indirectly) for anticancer therapies. As shown in Figure
6.191, a methyl group from N5,N10-methylene-tetrahydrofolate (often called tetrahydrofolate) is donated to dUMP, making dTMP
and dihydrofolate (DHF). Folate molecules are in limited quantities in cells and must be recycled, because if they are not, then the
reaction to make dTMP cannot occur. Recycling of dihydrofolate to tetrahydrofolate occurs by the reaction shown in Figure 6.192.
The enzyme involved in the conversion of dihydrofolate to tetrahydrofolate, dihydrofolate reductase (DHFR - Figure 6.192), is one
target of anticancer drugs because by stopping the regeneration of tetrahydrofolate from dihydrofolate (otherwise a dead end), one
can stop production of thymidine nucleotides and, as a result, halt DNA synthesis, thus preventing a cancer cell from dividing.
Competitive inhibitors of DHFR include methotrexate (Figure 6.194) or aminopterin. Cells contain numerous folates for
performing one carbon metabolism and the pathways by which they are all recycled is shown in Figure 6.193.
5-fluorouracil
Yet another important inhibitor of thymidine synthesis is used to treat cancer. This compound, 5-fluorouracil (Figure 6.195 and
Movie 6.3) is a suicide inhibitor of thymidylate synthase.
Salvage synthesis
Besides synthesis from simple precursors, nucleotides can also be made from pieces of existing ones. This is particularly relevant,
since consumption of food introduces to the body a large collection of proteins, lipids, and nucleic acids that are all more efficiently
recycled than degraded. For proteins, the process is simple. Digestion converts them into constituent building blocks (amino acids)
and these are re-assembled into proteins of the consuming organism using the genetic code.
Nucleotides
The multi-component structure of nucleotides, though (base, sugar, phosphate) means subsections of them may be re-utilized.
Phosphate is recycled simply by entering the phosphate pool of the cell. It is typically built back into triphosphate forms
(ultimately) by oxidative phosphorylation and kinase actions. Salvage of bases is different for purines and pyrimidines and is
discussed separately HERE and HERE.
Nucleotide catabolism
Besides salvage and being built into nucleic acids, nucleotides can also be broken down into simpler component molecules. Some
of these molecules, such as uric acid, can have significant impact on organisms (see HERE).
Purine catabolism
Breakdown of purine nucleotides starts with nucleoside monophosphates, which can be produced by breakdown of an RNA, for
example, by a nuclease (Figure 6.196).
Metabolism of AMP and GMP converge at xanthine. First, AMP is dephosphorylated by nucleotidase to create adenosine, which is
then deaminated by adenosine deaminase to yield inosine. Alternatively, AMP can be deaminated by AMP deaminase to yield IMP.
IMP is also an intermediate in the synthesis pathway for purine anabolism. Dephosphorylation of IMP (also by nucleotidase) yields
inosine. Inosine has ribose stripped from it by action of purine nucleotide phosphorylase to release hypoxanthine. Hypoxanthine is
oxidized to xanthine in a hydrogen peroxide-generating reaction catalyzed by xanthine oxidase.
Catabolism of GMP proceeds independently, though similarly. First, phosphate is removed by nucleotidase to yield guanosine.
Guanosine is stripped of ribose to yield free guanine base, which is deaminated by guanine deaminase (also called guanase) to

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produce xanthine.
Xanthine oxidase enters the picture a second time in the next reaction catalyzing a second reaction by a similar mechanism to the
hypoxanthine oxidation described previously. It is shown on the next page.
Uric acid
Uric acid is problematic in some higher organisms (including humans) because it is not very soluble in water. Consequently it
precipitates out of solution, forming crystals (Figure 6.198). Those crystals can accumulate in joints and (frequently) in the big toe.
Such a condition is known as gout.
Interestingly, there may be a negative correlation between gout and contracting multiple sclerosis. This protective effect may be
due to the antioxidant protection afforded by uric acid. Uric acid is the primary excretion form of nitrogen for birds. Dalmation
dogs also excrete uric acid instead of urea and may suffer from joint pain as a result of gout-like conditions.
Gout is treated with a hypoxanthine analog known as allopurinol (Figure 6.199). It inhibits action of xanthine oxidase, which favors
increase in the concentration of hypoxanthine. The latter is used in salvage synthesis to make additional purines.
Uric acid can be excreted into the urine (in humans) or broken down into allantoin by the uricase enzyme. Since humans lack the
enzyme to make allantoin (urea in humans is produced by the urea cycle), its presence in the body means it was produced by non-
enzymatic means. This is taken to be an indicator of oxidative stress, since it allantoin is produced non-enzymatically by oxidation
of uric acid.
Pyrimidine catabolism
Catabolism of uridine and thymidine nucleotides is shown above (Figure 6.200). Catabolism of cytidine nucleotides proceeds
through uridine by deamination of cytosine. The free bases, thymine and uracil, are released by the enzyme ribosylpyrimidine
nucleosidase In the reductive pathway, uracil and thymine reduction by NADPH gives dihydrothymine and dihydrouracil
respectively. Addition of water to these creates 3-ureidoisobutyrate and 3-ureidopropionate respectively. Hydrolysis of both these
intermediates yields ammonium ion and carbon dioxide (which are made into urea) plus 3-aminoisobutyrate for the thymine
pathway and β-alanine for the product of the uracil pathway. 3-aminoisobutyrate is produced during exercise and activates
expression of thermogenic genes in white fat cells.
β-alanine is a rate-limiting precursor of carnosine, a dipeptide of histidine and β-alanine (Figure 6.201). Carnosine functions as an
antioxidant that scavenges reactive oxygen species. It also acts as an anti-glycating agent to prevent against attachment of sugar
molecules to proteins. These are factors in degenerative diseases and may play a role in aging.
Sugars
Last, but not least, the sugars ribose and deoxyribose can be recycled (ribose) or catabolized (ribose and deoxyribose). In the case
of ribose, it can be reattached to bases by phosphorylase enzymes, such as uridine phosphorylase, or converted into PRPP for the
same purpose, to create nucleosides. Ribose-5-phosphate is an intermediate in the pentose phosphate pathway, allowing it to be
converted into other sugars or broken down in glycolysis.
Deoxyribose-5-phosphate can be broken into two pieces by deoxyribose-5-phosphate aldolase. The products of this reaction are
glyceraldehyde-3-phosphate and acetaldehyde. The former can be oxidized in glycolysis and the latter can be converted into acetyl-
CoA for further metabolism.

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Rajagopal, & Taralyn Tan.

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 CHAPTER OVERVIEW

7: Information Processing
The nature of biological information, how it is copied and passed on, how it is read and interpreted, and how it gives rise to the
cellular activities that we can observe, is the subject of this chapter. Another kind of information is also considered, towards the end
of the chapter- the molecular information that cells receive from, and send to, each other. Overlaid on the instructions in the genes,
this information provides cells with ongoing clues about both their own inner state and the environment around them. The interplay
of these two kinds of information is responsible for the form and behavior of all living organisms.
7.1: Prelude to Information Processing
7.2: Genes and Genomes
7.3: DNA Replication
7.4: DNA Repair
7.5: Transcription
7.6: RNA Processing
7.7: Translation
7.8: Gene Expression
7.9: Signaling

Thumbnail: DNA double helix. Image used with permission (public domain; NIH - Genome Research Institute).

This page titled 7: Information Processing is shared under a CC BY-NC-SA license and was authored, remixed, and/or curated by Kevin Ahern,
Indira Rajagopal, & Taralyn Tan.

1
7.1: Prelude to Information Processing
“The blueprints for the construction of one human being requires only a meter of DNA and one tiny cell. … even Mozart started
out this way.” — L.L. Larison Cudmore
As creatures used to regarding ourselves as exceptional, humans must surely be humbled to realize that the instructions, for making
one of our own, reside in a molecule so simple that scientists, for a very long time, did not believe could possibly contain enough
information to build even a simple cell. But a large body of evidence, built up over the past century, supports Larison Cudmore’s
assertion that the information for making you and me (and all the other kinds of living things in the world) is encoded in DNA.
Tying in with Mendel’s observations about how characteristics are passed on from one generation to the next, the discovery that
there was a molecule that carried this information, altered for ever how people thought about heredity.
The elucidation of the structure of DNA provided greater insights into how traits might be encoded in a molecule, and the ways in
which the information is used by cells. As we learn more about this topic, scientists have remarked on how the information in our
DNA resembles the programs that drive computers. While this analogy is a simplification, there is definitely a sense in which, as
Richard Dawkins put it, “the machine code of the genes is uncannily computer-like”, with information in our DNA directly
determining the properties of the proteins that run our cells. We know, as Ada Yonath described it, that, “DNA is a code of four
letters; proteins are made up of amino acids which come in 20 forms. So the ribosome is a very clever machine that reads one
language and operates in another. “
If this sounds strange, it is even more intriguing to realize DNA is copied and passed on from cell to cell, from one generation to
the next. There is an unbroken line of inheritance from the first cell to every organism alive today. In the words of Lewis Thomas,
“All of today’s DNA, strung through all the cells of the earth, is simply an extension and elaboration of [the] first molecule.”
The nature of this information, how it is copied and passed on, how it is read and interpreted, and how it gives rise to the cellular
activities that we can observe, is the subject of this chapter. Another kind of information is also considered, towards the end of the
chapter- the molecular information that cells receive from, and send to, each other. Overlaid on the instructions in the genes, this
information provides cells with ongoing clues about both their own inner state and the environment around them. The interplay of
these two kinds of information is responsible for the form and behavior of all living organisms.

This page titled 7.1: Prelude to Information Processing is shared under a CC BY-NC-SA license and was authored, remixed, and/or curated by
Kevin Ahern, Indira Rajagopal, & Taralyn Tan.

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7.2: Genes and Genomes
Source: BiochemFFA_7_1.pdf. The entire textbook is available for free from the authors at
http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy

Introduction
For many years, scientists wondered about the nature of the information that directed the activities of cells. What kind of molecules
carried the information, and how was the information passed on from one generation to the next? Key experiments, done between
the 1920s and the 1950s, established convincingly that this genetic information was carried by DNA. In 1953, with the elucidation
of the structure of DNA, it was possible to begin investigating how this information is passed on, and how it is used.

Genomes
We use the word “genome” to describe all of the genetic material of the cell. That is, a genome is the entire sequence of nucleotides
in the DNA that is in all of the chromosomes of a cell. When we use the term genome without further qualification, we are
generally referring to the chromosomes in the nucleus of a eukaryotic cell. As you know, eukaryotic cells have organelles like
mitochondria and chloroplasts that have their own DNA (Figure 7.1 & 7.2). These are referred to as the mitochondrial or
chloroplast genomes to distinguish them from the nuclear genome.
Starting in the 1980s, scientists began to determine the complete sequence of the genomes of many organisms, in the hope of better
understanding how the DNA sequence specifies cellular functions. Today, the complete genome sequences have been determined
for thousands of species from all domains of life, and many more are in the process of being worked out by groups of scientists
across the world.

Figure 7.1 - Mitochondria carry their own genome

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 Figure 7.2 - The human mitochondrial genome - Wikipedia
Global genome initiative
The Global Genome Initiative, a collaborative effort to sequence at least one species from each of the 9,500 described invertebrate,
vertebrate, and plant families is one of many such ventures. The information from these various efforts is collected in enormous
online repositories, so that it is freely available to scientists. As the sequence databases compile ever more information, the fields of
computational biology and bioinformatics have arisen, to analyze and organize the data in a way that helps biologists understand
what the information in DNA means in the cellular context.

Genes
It has been known for many years that phenotypic traits are controlled by specific regions of the DNA that were termed “genes”.
Thus, DNA was envisioned as a long string of nucleotides, in which certain regions, the genes, were separated by non-coding
regions that were simply referred to as intergenic sequences (inter=between; genic=of genes). Early experiments in molecular
biology suggested a simple relationship between the DNA sequence of a gene and its product, and led scientists to believe that each
gene carried the information for a single protein. Changes, or mutations in the base sequence of a gene would be reflected in
changes in the gene product, which in turn, would manifest itself in the phenotype or observable trait. This simple picture, while
still useful, has been modified by subsequent discoveries that demonstrated that the use of genetic information by cells is somewhat
more complicated. Our definition of a gene is also evolving to take new knowledge into consideration.

Figure 7.3 - From genomes to genes - Wikipedia

Figure 7.4 - Human genes sorted by class

Matters of size

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A common-sense assumption about genomes would be that if genes specify proteins, then the more proteins an organism made, the
more genes it would need to have, and thus, the larger its genome would be. Comparison of various genomes shows, surprisingly,
that there is not necessarily a direct relationship between the complexity of an organism and the size of its genome (Figure 7.5). To
understand how this could be true, it is necessary to recognize that while genes are made up of DNA, all DNA does not consist of
genes (for purposes of our discussion, we define a gene as a section of DNA that encodes an RNA or protein product). In the
human genome, less than 2% of the total DNA seems to be the sort of coding sequence that directs the synthesis of proteins. For
many years, non-coding DNA in genomes was believed to be useless, and was described as “junk DNA” although it was perplexing
that there seemed to be so much “useless” sequence. Recent discoveries have, however, demonstrated that much of this so-called
junk DNA may play important roles in evolution, as well as in regulation of gene expression.

Figure 7.5 - Sizes of various genomes - Wikipedia

Introns
So, what is all the non-coding DNA doing there? We know that even coding regions in our DNA are interrupted by non-coding
sequences called introns. This is true of most eukaryotic genomes. An examination of genes in eukaryotes shows that non-coding
intron sequences can be much longer than the coding sections of the gene, or exons. Most exons are relatively small, and code for
fewer than a hundred amino acids, while introns can vary in size from several hundred base-pairs to many kilobase-pairs
(thousands of base-pairs) in length. For many genes in humans, there is much more of intron sequence than coding (a.k.a. exon)
sequence. Intron sequences account for roughly a quarter of the genome in humans.

Other non-coding sequences

What other kinds of non-coding sequences are there? One function for some DNA sequences that do not encode RNA or proteins is
in specifying when and to what extent a gene is used, or expressed. Such regions of DNA are called regulatory regions and each
gene has one or more regulatory sequences that control its expression. However, regulatory sequences do not account for all the
rest of the DNA in our genomes, either.
Transposable sequences
Surprisingly, almost half of the human genome appears to consist of several kinds of repetitive sequences. Many of the repetitive
sequences are known to be transposable elements (transposons), sections of DNA that can move around within the genome.
Sometimes referred to as “jumping genes” these transposable elements can move from one chromosomal location to another, either
through a simple “cut and paste” mechanism that cuts the sequence out of one region of the DNA and inserts it into another
location, or through a process called retrotransposition involving an RNA intermediate.
LINES & SINES
There are millions of copies of each of two major classes of such transposable elements, the LINEs (Long Interspersed Elements)
and SINEs (Short Interspersed Elements) in our genomes.
LINEs and SINEs are both a kind of transposable element called retrotransposons, sequences that are copied into RNA, then
reverse transcribed back into DNA before being inserted into new locations. This movement is typically not sequence specific,
meaning that the transposons can be inserted randomly in the genome, in many cases within coding regions. As might be expected,

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this can disrupt the function of the gene. Transposons may also insert within regulatory regions, and change the expression of the
genes they control. As a major cause of mutation in genomes, transposons play an important role in evolution.
Finally, recent findings have shown that much of the genome is transcribed into RNAs, even though only about 2% encodes
proteins. What are the RNAs that do not encode proteins? Ribosomal RNAs (Figure 7.7) and transfer RNAs, together with the
small nuclear RNAs that function in splicing, account for some of these non-translated transcripts, but not all. The remaining RNAs
are regulatory RNAs, small molecules that play an important role in regulating gene expression. As we understand more about
genomes, it is becoming evident that the so-called “junk” DNA is anything but.

Figure 7.6 - Components of the human genome - Wikipedia

Figure 7.7 - 5S rRNA structure

This page titled 7.2: Genes and Genomes is shared under a CC BY-NC-SA license and was authored, remixed, and/or curated by Kevin Ahern,
Indira Rajagopal, & Taralyn Tan.

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7.3: DNA Replication
Source: BiochemFFA_7_2.pdf. The entire textbook is available for free from the authors at
http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy

Copying instructions
The only way to make new cells is by the division of pre-existing cells. Single-celled organisms undergo division to produce more
cells like themselves, while multicellular organisms arise through division of a single cell, generally the fertilized egg. Each time a
cell divides, all of its DNA must be copied faithfully so that a copy of this information can be passed on to the daughter cell. This
process is called DNA replication. It is the means by which genetic information can be transmitted down generations of cells, and it
ensures that every new cell has a complete copy of the genome. In the next section, we will examine the process by which the DNA
of a cell is completely and accurately copied.

Figure 7.8 - Watson & Crick’s DNA structure - Wikipedia

The structure of DNA elucidated by Watson and Crick in 1953 immediately suggested a mechanism by which double-stranded
DNA could be copied to give two identical copies of the DNA. They proposed that the two strands of the DNA molecule, which
are held together by hydrogen bonds between the base-paired nucleotides, would separate and each serve as a template on which a
complementary strand could be assembled (Figure 7.8). The base-pairing rules would ensure that this process would result in the
production of two identical DNA molecules. The beautiful simplicity of this scheme was shown to be correct in subsequent
experiments by Meselson and Stahl, that demonstrated that DNA replication was semi-conservative, i.e., that after replication, each
of the two resulting DNA molecules was made up of one old strand and one new strand that had been assembled across from it
(Figure 7.9).

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 Figure 7.9 - Semi-conservative replication - Image by Martha Baker

Building materials
What are the ingredients necessary for building a new DNA molecule? As noted above, the original, or parental DNA molecule
serves as the template. New DNA molecules are assembled across from each template by joining together free DNA nucleotides as
directed by the base pairing rules, with As across from Ts and Gs across from Cs.
The nucleotides used in DNA synthesis are deoxyribonucleoside triphosphates or dNTPs. As can be inferred from their name, such
nucleotides have a deoxyribose sugar and three phosphates, in addition to one of the four DNA bases, A, T, C or G (Figure 7.10).
When dNTPs are added into a growing DNA strand, two of those phosphates will be cleaved off, as described later, leaving the
nucleotides in a DNA molecule with only one phosphate per nucleotide. This reaction is catalyzed by enzymes known as DNA
polymerases, which create phosphodiester linkages between one nucleotide and the next.

Figure 7.10 - The building blocks of DNA

Challenges
Before examining the actual process of DNA replication, it is useful to think about what it takes to accomplish this task
successfully. Consider the challenges facing a cell in this process:
The sheer number of nucleotides to be copied is enormous: e.g., in human cells, on the order of several billions.
A double-helical parental DNA molecule must be unwound to expose single strands of DNA that can serve as templates for the
synthesis of new DNA strands.
Unwinding must be accomplished without introducing topological distortion into the molecule.
The unwound single strands of DNA must be kept from coming back together long enough for the new strands to be
synthesized.
DNA polymerases cannot begin synthesis of a new DNA strand de novo and require a free 3' OH to which they can add
deoxynucleotides.
DNA polymerases can only extend a strand in the 5' to 3' direction. The 5' to 3' growth of both new strands means that one of
the strands is made in pieces.
The use of RNA primers requires that the RNA nucleotides must be removed and replaced with DNA nucleotides and the
resulting DNA fragments must be joined.
The copying of all the parental DNA must be accurate, so that mutations are not introduced into the newly made DNA.

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 Figure 7.11 - Replication of DNA - Wikipedia

Figure 7.12 - Human chromosomes

Figure 7.13 - Bidirectional replication from an origin - Martha Baker

Figure 7.14 - Prokaryotic vs. eukaryotic DNA replication - Wikipedia

Addressing challenges
With this in mind, we can begin to examine how cells deal with each of these challenges. Our understanding of the process of DNA
replication is derived from studies using bacteria, yeast, and other systems. These investigations have revealed that DNA
replication is carried out by the action of a large number of proteins that act together as a complex protein machine. Numerous
proteins involved in replication have been identified and characterized, including multiple different DNA polymerases in both

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prokaryotes and eukaryotes. Although the specific proteins involved are different in bacteria and eukaryotes, it is useful to
understand the basic considerations that are relevant in all cells. A generalized account of the steps in DNA replication is presented
below, focused on the challenges mentioned above.
The sheer number of nucleotides to be copied is enormous: e.g., in human cells, on the order of several billions.
Cells, whether bacterial or eukaryotic, have to replicate all of their DNA before they can divide. In cells like our own, the vast
amount of DNA is broken up into many chromosomes, each of which is composed of a linear strand of DNA (Figure 7.12). In cells
like those of E. coli, there is a single circular chromosome.
In either situation, DNA replication is initiated at sites called origins of replication. These are regions of the DNA molecule that are
recognized by special proteins called initiator proteins that bind the DNA. In E.coli, origins have small regions of A-T-rich
sequences that are “melted” to separate the strands, when the initiator proteins bind to the origin or replication. As you may
remember, A-T base-pairs, which have two hydrogen bonds between them are more readily disrupted than G-C base-pairs which
have three apiece (Figure 7.15).

Figure 7.15 - A-T and G-C base pairs

How many origins of replication are there on a chromosome? In the case of E. coli, there is a single origin of replication on its
circular chromosome. In eukaryotic cells there may be many thousands of origins of replication, with each chromosome having
hundreds (Figure 7.16). DNA replication is, thus, initiated at multiple points along each chromosome in eukaryotes. Electron
micrographs of replicating DNA from eukaryotic cells show many replication bubbles on a single chromosome. This makes sense
in light of the large amount of DNA that there is to be copied in cells like our own, where beginning at one end of each
chromosome and replicating all the way through to the other end from a single origin would simply take too long. This is despite
the fact that the DNA polymerases in human cells are capable of building new DNA strands at the very respectable rate of about 50
nucleotides per second!
A double-helical parental molecule must be unwound to expose single strands of DNA that can serve as templates for the
synthesis of new DNA strands.

Unwinding
Once a small region of the DNA is opened up at each origin of replication, the DNA helix must be unwound to allow replication to
proceed. The unwinding of the DNA helix requires the action of an enzyme called helicase.

Figure 7.16 - Multiple replication bubble on a eukaryotic chromosome - Image by Martha Baker

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 Figure 7.17 - View of a eukaryotic DNA replication fork with helicase shown in blue - Wikipedia
Helicase uses the energy released when ATP is hydrolyzed, to break the hydrogen bonds between the bases in DNA and separate
the two strands (Figure 7.17). Note that a replication bubble is made up of two replication forks that "move" or open up, in opposite
directions. At each replication fork, the parental DNA strands must be unwound to expose new sections of single-stranded
template.
This unwinding must be accomplished without introducing topological distortion into the molecule.
What is the effect of unwinding one region of the double helix? Local unwinding of the double helix causes over-winding
(increased positive supercoiling) ahead of the unwound region.
The DNA ahead of the replication fork has to rotate, or it will get twisted on itself and halt replication. This is a major problem, not
only for circular bacterial chromosomes, but also for linear eukaryotic chromosomes, which, in principle, could rotate to relieve the
stress caused by the increased supercoiling.
Topoisomerases
The reason this is problematic is that it is not possible to rotate the entire length of a chromosome, with its millions of base-pairs, as
the DNA at the replication fork is unwound. How, then, is this problem solved? Enzymes called topoisomerases can relieve the
topological stress caused by local “unwinding” of the extra winds of the double helix. They do this by cutting one or both strands of
the DNA and allowing the strands to swivel around each other to release the tension before rejoining the ends. In E. coli, the
topoisomerase that performs this function is called gyrase.
The separated single strands of DNA must be kept from coming back together so the new strands to be synthesized.
Single-strand DNA binding protein
Once the two strands of the parental DNA molecule are separated, they must be prevented from going back together to form
double-stranded DNA. To ensure that unwound regions of the parental DNA remain single-stranded and available for copying, the
separated strands of the parental DNA are bound by many molecules of a protein called single-strand DNA binding protein (SSB -
Figure 7.18).

Figure 7.18 - Proteins at a prokaryotic DNA replication fork - Image by Martha Baker

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 DNA polymerases cannot begin synthesis of a new DNA strand de novo and require a free 3' OH to which they can add DNA
nucleotides.
Although single-stranded parental DNA is now available for copying, DNA polymerases cannot begin synthesis of a
complementary strand de novo. This simply means that DNA polymerases can only add new nucleotides on to the 3' end of a pre-
existing chain, and cannot start a chain of nucleotides on their own. Because of this limitation, some enzyme other than a DNA
polymerase must first make a small region of nucleic acid, complementary to the parental strand, that can provide a free 3' OH to
which DNA polymerase can add a deoxyribonucleotide. This task is accomplished by an enzyme called a primase, which
assembles a short stretch of RNA base-paired to the parental DNA template. This provides a short base-paired region, called the
RNA primer, with a free 3'OH group to which DNA polymerase can add the first new DNA nucleotide (Figure 7.12).
Sliding clamp
Once a primer provides a free 3'OH for extension, other proteins get into the act. These proteins are involved in loading the DNA
polymerase onto the primed template and keeping it associated with the DNA. The first of these is the clamp loader. As its name
suggests, the clamp loader helps to load a protein complex called the sliding clamp onto the DNA at the replication fork (Figure
7.19 and 7.20). The sliding clamp, a multi-subunit ring-shaped protein, is then joined by the DNA Polymerase. The function of the
sliding clamp is to keep the polymerase associated with the replication fork - in fact, it has been described as a seat-belt for the
DNA polymerase. The sliding clamp ensures that the DNA polymerase is able to synthesize long stretches of new DNA before it
dissociates from the template. The property of staying associated with the template for a long time before dissociating is known as
the processivity of the enzyme. In the presence of the sliding clamp, DNA polymerases are much more processive, making
replication faster and more efficient.

Figure 7.19 - Top view of the sliding clamp (outside) surrounding DNA strand (inside)

Figure 7.20 - Side view of the sliding clamp surrounding a DNA strand (in purple)

Extending the primer

The DNA polymerase is now poised to start synthesis of the new DNA strand (in E. coli, the primary replicative polymerase is
called DNA polymerase III). As you already know, the synthesis of new DNA is accomplished by the addition of new nucleotides
complementary to those on the parental strand. DNA polymerase catalyzes the reaction by which an incoming deoxyribonucleotide,
complementary to the template, is added onto the 3' end of the previous nucleotide, starting with the 3'OH on the end of the RNA
primer. The importance of the 3’OH group lies in the nature of the reaction that builds a chain of nucleotides.
The reaction catalyzed by the DNA polymerase occurs through the nucleophilic attack by the 3’OH group at the end of a nucleic
acid strand on the α phosphate of the incoming dNTP (Figure 7.21). The immediate hydrolysis of the pyrophosphate that is cleaved
off the incoming dNTP drives the reaction forward. The sequential addition of new nucleotides at the 3’ end of the growing chain
of DNA accounts for the fact that the strand grows in a 5’ to 3’ direction.

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 Figure 7.21 - Mechanism of DNA chain growth - Wikipedia
The 5' phosphate on each incoming nucleotide is joined by the DNA polymerase to the 3' OH on the end of the growing nucleic
acid chain, to make a phosphodiester bond. Each added nucleotide provides a new 3’OH, allowing the chain to be extended for as
long as the DNA polymerase continues to synthesize the new strand. As we already noted, the new DNA strands are synthesized by
the addition of DNA nucleotides to the end of an RNA primer. The new DNA molecule thus has a short piece of RNA at the
beginning.
DNA polymerases can only extend a strand in the 5' to 3' direction. The 5' to 3' growth of both new strands means that one of
the strands is made in pieces.
Leading strand
We know that DNA polymerases can only build a new DNA strand in the 5' to 3' direction. We also know that the two parental
strands of DNA are antiparallel. This means that at each replication fork, one new strand, called the leading strand can be
synthesized continuously in the 5' to 3' direction because it is being made in the same direction that the replication fork is opening
up.
Lagging strand
The synthesis of the other new strand, called the lagging strand, also proceeds in the 5’ to 3’ direction. But because the template
strands are running in opposite directions, the lagging strand is being extended in the direction opposite to the opening of the
replication fork (Figure 7.22). As the replication fork opens up, the region behind the original start point for the lagging strand will
need to be copied. This means another RNA primer must be laid down and extended. This process repeats itself as the replication
fork opens up, with multiple RNA primers laid down and extended, producing many short pieces that are later joined. These short
nucleic acid pieces, each composed of a small stretch of RNA primer and about 1000-2000 DNA nucleotides, are called Okazaki
fragments, for Reiji Okazaki, the scientist who first demonstrated their existence.

Figure 7.22 - Bidirectional DNA replication with template strand (a), leading strand (b), Okazaki fragments (c), replication fork (d),
RNA primer (e), and DNA extended RNA primers (f) - Wikipedia

Figure 7.23 - Removal of RNA primers and joining of Okazaki fragments - Wikipedia

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 The use of RNA primers requires that the RNA nucleotides must be removed and replaced with DNA nucleotides.
Primer removal
We have seen that each newly synthesized piece of DNA starts out with an RNA primer, effectively making a new nucleic acid
strand that is part RNA and part DNA. The newly made DNA strand cannot be allowed to have pieces of RNA attached. So, the
RNA nucleotides must be removed and the gaps filled in with DNA nucleotides (Figure 7.23). This is done by DNA polymerase I
in E. coli. This enzyme begins adding DNA nucleotides at the end of each Okazaki fragment. However, the end of one Okazaki
fragment is adjacent to the RNA primer at the beginning of the next Okazaki fragment. DNA polymerase I has an exonuclease
activity acting in the 5’ to 3’ direction that removes the RNA nucleotides ahead of it, while the polymerase activity replaces the
RNA nucleotides with dNTPs. Once all the RNA nucleotides have been removed, the lagging strand is made up of stretches of
DNA. The DNA pieces are then joined together by the enzyme DNA ligase.
The steps outlined above essentially complete the process of DNA replication. But one issue still remains.
Ensuring accuracy in the copying of so much information

Accuracy
How accurate is the copying of information by DNA polymerase? As you are aware, changes in DNA sequence (mutations) can
change the amino acid sequence of the encoded proteins and that this is often, though not always, deleterious to the functioning of
the organism. When billions of bases in DNA are copied during replication, how do cells ensure that the newly synthesized DNA is
a faithful copy of the original information?
DNA polymerases, as we have noted earlier work fast (averaging 50 bases a second in human cells and up to 200 times faster in E.
coli). Yet, both human and bacterial cells seem to replicate their DNA quite accurately. This is because replicative DNA
polymerases have a proofreading function that enables the polymerase to detect when the wrong base has been inserted across from
a template strand, back up and remove the mistakenly inserted base, before continuing with synthesis (Figure 7.24).

Figure 7.24 - Error corrected by DNA polymerases

Multiple activities
This is possible because most DNA polymerases are dual-function enzymes. They can extend a DNA chain by virtue of their 5' to
3' polymerase activity. Some polymerases like DNA polymerase I can also remove RNA primers in the 5’ to 3’ direction, though
that is not a common activity of polymerases. Many polymerases, however have an ability to backtrack and remove the last inserted
base because they possess a 3' to 5' exonuclease activity.

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The exonuclease activity of a DNA polymerase allows it to excise a wrongly inserted base, after which the polymerase activity
inserts the correct base and proceeds with extending the strand.
In other words, the DNA polymerase is monitoring its own accuracy (also termed its fidelity) as it makes new DNA, correcting
mistakes immediately before moving on to add the next base. This mechanism, which operates during DNA replication, corrects
many errors as they occur, reducing by about a 100-fold the mistakes made when DNA is copied.

DNA polymerases
As noted earlier, both prokaryotic and eukaryotic cells have multiple DNA polymerases. In E.coli, for example, DNA polymerase
III is the major replicative polymerase (a.k.a. replicase) while DNA polymerase I is responsible for DNA repair as well as removal
of RNA primers and their replacement with DNA nucleotides during replication. DNA polymerase II plays a role in restarting
replication after DNA damage stalls replication, while DNA polymerases IV and V are both required in trans-lesion, or bypass,
synthesis, which allows replication past sites of DNA damage.
Eukaryotic polymerases
In eukaryotes, there are over fifteen different DNA polymerases. The primary replicative polymerases in the nucleus are ∂ and ε.
DNA polymerase α is also important for replication because it has primase and repair activities. Replication is initiated in
eukaryotic cells by DNA polymerase α, which binds to the initiation complex at the origin and lays down an RNA primer, followed
by about 25 nucleotides of DNA. It is then replaced by another polymerase, in a step called the pol switch. DNA polymerase ∂ or ε
then continues synthesizing DNA, depending on the strand. The role of polymerase ε appears to be synthesis of the leading strand
due to its high processivity and accuracy, whereas polymerase ∂ extends Okazaki fragments on the lagging strands. Proteins
analogous to the clamp loader and sliding clamp are also present. The protein RFC plays the role of clamp loader, while another
protein, PCNA acts like the sliding clamp. Several other DNA polymerases like β, γ and μ function in repairing gaps. Yet others are
involved in trans-lesion synthesis following DNA damage and are associated with hypermutation.
Despite their diversity, DNA polymerases share some common structural features. X-ray crystallographic studies have shown that
these enzymes have a structure that has been compared to a human right hand (Figures 7.25 & 7.26). The «palm» of the hand forms
a cleft in which the DNA lies. The cleft is also the where the polymerase catalytic activity resides. This is where the incoming
nucleotide is added on to the growing chain. «The fingers» position the DNA in the active site, while the «thumb» holds the DNA
as it exits the polymerase. A separate domain contains the exonuclease (proofreading) activity of the enzyme.
The enzyme alternates between its polymerizing activity and its proofreading activity. When a mismatched base pair is in the
polymerase catalytic site, the 3’end of the growing strand is moved from the polymerase site to the exonuclease active site (Figure
7.26). The mispair at the end is removed by the exonuclease, followed by repositioning of the 3’ end in the polymerase active site
to continue synthesis.

Figure 7.25 DNA polymerase I with hand structure

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 Figure 7.26 - Proofreading by DNA polymerase -Image by Pehr Jacobson

Figure 7.27 - Mammalian DNA polymerases

Termination of replication
In circular bacterial chromosomes, there are specific sequences known as terminator or Ter sites. These are multiple short
sequences that serve as termination sites, allowing the replication forks traveling clockwise and anticlockwise across the circular
chromosome to meet at one of the sites.
The binding of a protein, Tus, at a Ter site prevents further movement of the replication fork and ends replication. The parental and
newly made circular DNA are, at this point topologically interlinked and must be separated with the help of topoisomerase.
The end-replication problem
There is no fixed site for termination in linear eukaryotic chromosomes. As the replication forks reach the ends of the chromosome,
the leading strand can be synthesized all the way to the end of the template strand. On the lagging strand, the need for an RNA
primer to start synthesis creates a challenge. When the RNA primer at the extreme end of the lagging strand is removed, there is a
small stretch of the template strand that cannot be copied. As a result, in each round of replication a short sequence at the ends of
the chromosome will be lost. Over time, with many cycles of replication, chromosomes would become noticeably shorter. This
shortening of chromosomes has been observed in vitro, in cultured mammalian somatic cells. It is also seen in intact organisms,
with increasing age.
Telomeres
What effect does the loss of sequence from the ends of the chromosomes have on cells? We know that the ends of chromosomes are
characterized by structures called telomeres (Figure 7.28). Telomeres are made up of many copies of short repeated sequences (in
humans, the repeat is TTAGGG) and special proteins that specifically bind to these sequences. This structure of telomeres is useful
in distinguishing the ends of chromosomes from double-strand breaks in DNA, thus preventing the DNA repair mechanisms in
cells from joining chromosomes end to end.

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The other advantage of the repeated sequences, which do not encode proteins, is that losing some of the repeats does not lead to
loss of important coding information. Thus, the repeats act as a sort of buffer zone, where the loss of sequence does not doom the
cell. However, the shortening of chromosomes cannot continue indefinitely. After a certain number of replication cycles, cells are
known to stop dividing and enter a state known as replicative senescence. This suggests that the shortening of the telomeres serves
as a sort of clock, with the extent of shrinkage of the chromosomes serving as a measure of aging. Eventually cells that enter
senescence will die.

Figure 7.28 - Chromosomes with telomeres marked in white

Problems with sequence loss
Even if our cells are able to function with shorter chromosomes during our lifetimes, this leaves us with another problem. If our
chromosomes grow shorter with age, then presumably our children, who inherit our chromosomes will be born with shorter
chromosomes than we started with. They, in turn, would have their chromosomes shrink as they grew older, and their children
would have even shorter chromosomes. Over the course of multiple generations, this would lead to the point where further
chromosome shrinkage would result in cells that would enter senescence very early in life and die soon after. This obviously does
not happen. Generation after generation of children are born with full-length chromosomes, so there is a mechanism that must
ensure that at least in the reproductive cells, chromosomes do not get shorter.
To understand this mechanism, it is necessary to first examine the end of a newly made DNA molecule (Figure 7.29). While the
leading strand, which grows in the same direction as the movement of the replication fork, can copy its template all the way to the
end, the lagging strand encounters a problem. RNA primers are, as we noted, needed to start each of the Okazaki fragments of the
lagging strand. The primers must be removed later, and the RNA nucleotides replaced with DNA nucleotides. When the RNA
primer across from the end of the parental strand is removed, the RNA nucleotides cannot be replaced by DNA nucleotides because
the DNA polymerase has no primer to start from. A short region of the template cannot, therefore be copied.

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 Figure 7.29 - Replication of a linear chromosome results in loss of sequences at the very ends with each round of replication
Telomerase
How can this problem be solved? It can be seen from Figure 7.29 that the end of the original template strand has a short 3’
overhang resulting from the removal of the RNA primer across from it. In order to fill in this region, another primer would be
needed, situated past the end of the template strand. But in order to build such a primer, it would be necessary for the template
overhang to be longer. If it were possible to make the template strand longer, then another primer could be placed across from its
end and the end of the strand could be copied. Such an extension of the template strand is exactly what happens in our reproductive
cells. The parental template strand is extended by the enzyme telomerase, which adds telomere repeats and lengthens the template.
We will see shortly how it accomplishes that feat.
RNA template
Telomerase is an unusual enzyme, in that it is made up of two components, an RNA and a reverse transcriptase. A reverse
transcriptase is an RNA-dependent DNA polymerase, an enzyme that copies an RNA template to make DNA. The RNA
component of the human telomerase, called hTERC, has a sequence that is complementary to the telomere repeat, TAGGG. As
seen in Figure 7.31, this RNA can base-pair with the last telomere repeat on the parental DNA strand, while a portion of the RNA
remains unpaired.

Figure 7.30 - Telomerase holding its RNA template (in center) - Wikipedia
Template for extension
The function of the unpaired region of the RNA is to serve as a template that can be used to extend the overhanging 3’ end of the
original DNA molecule. The protein component of telomerase has reverse transcriptase activity and can copy the RNA sequence

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into DNA. In human telomerase, the protein component is known as hTERT (telomerase reverse transcriptase). As seen in Figure
7.31 and 7.32, the reverse transcriptase extends the original 3’ overhang using the RNA component as its template. The telomerase
can then dissociate, and repeat the process multiple times to add many repeats of the telomere sequence.

Figure 7.31 - Telomerase extends the template strand using its own RNA as template - Wikipedia

Figure 7.32 - Growing telomeres with telomerase and DNA polymerase. First, the 3’ end of the template is extended by telomerase,
then DNA polymerase completes synthesis of the lagging strand
Once the overhang has been extended by the addition of at least several telomere repeats, there is now room for the synthesis of an
RNA primer complementary to the newly extended overhang (pointing back towards the rest of the chromosome). This primer can
then be extended to complete synthesis of the lagging strand all the way to the end of the original parental DNA strand. Thus, the
addition of telomere repeats on the parental DNA strands keeps the newly made DNA strands from becoming shorter with each
cycle of replication. The fact that this happens in germ cells (reproductive cells) explains why each generation does not have
shorter chromosomes than the parental generation.
The proofreading function of DNA polymerases monitors the accuracy of DNA replication while the enzyme telomerase keeps
chromosomes that will be passed on to offspring from shortening. Between them, these two activities ensure that the genetic
information is copied accurately, and that succeeding generations receive a full complement of the genetic information

Disassembly and reassembly of nucleosomes

The events of replication have an additional twist in eukaryotes. Recall that DNA is found in eukaryotic cells as chromatin, a
complex of the DNA with proteins. At its least condensed, chromatin looks like a string of beads, consisting of the DNA wrapped
around histone cores to make nucleosomes. The nucleosome structure must be disrupted to make DNA available for replication and
restored after replication is completed (Figure 7.33).

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Ahead of the replication fork, chromatin structure is disassembled by ATP-dependent chromatin remodeling complexes, allowing
access to the DNA template. Once the new strands of DNA have been synthesized, both the original nucleosomes and new
nucleosomes must be reassembled behind the replication fork. Since replication gives rise to two DNA molecules where there was
one, twice the amount of histones is needed to package the DNA. Preparation for DNA replication, therefore, involves the synthesis
of large amounts of histones to supply the need. Interestingly, it appears that newly synthesized DNA is packaged into nucleosomes
using the original histones that were displaced to allow the replication fork to pass, as well as newly synthesized histones.
We also know that post-translational modifications like acetylation, methylation or phosphorylation of the histones can regulate the
degree to which a given region of the genome is accessible for use. One question that remains the subject of intense research is how
these modifications are accurately passed on to the new nucleosomes.

Figure 7.33 - Disruption of nucleosomes during DNA replication - Wikipedia

This page titled 7.3: DNA Replication is shared under a CC BY-NC-SA license and was authored, remixed, and/or curated by Kevin Ahern, Indira
Rajagopal, & Taralyn Tan.

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7.4: DNA Repair
Source: BiochemFFA_7_3.pdf. The entire textbook is available for free from the authors at
http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy

Safeguarding the genome

In the last section we considered the ways in which cells deal with the challenges associated with replicating their DNA, a vital
process for all cells. It is evident that if DNA is the master copy of instructions for an organism, then it is important not to make
mistakes when copying the DNA to pass on to new cells. Although proofreading by DNA polymerases greatly increases the
accuracy of replication, there are additional mechanisms in cells to further ensure that newly replicated DNA is a faithful copy of
the original, and also to repair damage to DNA during the normal life of a cell.

DNA damage
All DNA suffers damage over time, from exposure to ultraviolet and other radiation, as well as from various chemicals in the
environment (Figures 7.34 & 7.35). Even chemical reactions naturally occurring within cells can give rise to compounds that can
damage DNA. As you already know, even minor changes in DNA sequence, such as point mutations can sometimes have far-
reaching consequences. Likewise, unrepaired damage caused by radiation, environmental chemicals or even normal cellular
chemistry can interfere with the accurate transmission of information in DNA. Maintaining the integrity of the cell's "blueprint" is
of vital importance and this is reflected in the numerous mechanisms that exist to repair mistakes and damage in DNA.

Figure 7.34 - Chromosome breaks - Wikipedia

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 Figure 7.35 - Single- (top) and double-strand (bottom) damaged DNA - Wikipedia

Post-replicative mismatch repair

We earlier discussed proofreading by DNA polymerases during replication. While proofreading significantly reduces the error rate,
not all mistakes are fixed on the fly by DNA polymerases.
What mechanisms exist to correct the replication errors that are missed by the proof-reading function of DNA polymerases? Errors
that slip by proofreading during replication can be corrected by a mechanism called mismatch repair. While the error rate of DNA
replication is about one in 107 nucleotides in the absence of mismatch repair, this is further reduced a hundred-fold to one in 109
nucleotides when mismatch repair is functional.
What are the tasks that a mismatch repair system faces?
It must:
Scan newly made DNA to see if there are any mispaired bases (e.g., a G paired to a T)
Identify and remove the region of the mismatch.
Correctly fill in the gap created by the excision of the mismatch region.
Distinguishing strands
Importantly, the mismatch repair system must have a means to distinguish the newly made DNA strand from the template strand, if
replication errors are to be fixed correctly. In other words, when the mismatch repair system encounters an A-G mispair, for
example, it must know whether the A should be removed and replaced with a C or if the G should be removed and replaced with a
T.
But how does the mismatch repair system distinguish between the original and the new strands of DNA? In bacteria, the existence
of a system that methylates the DNA at GATC sequences is the solution to this problem. E.coli has an enzyme, DNA adenine
methylase (Dam) that adds methyl groups on the to adenines in GATC sequences in DNA (Figure 7.36). Newly replicated DNA has
not yet undergone methylation and thus, can be distinguished from the template strand, which is methylated.
The mismatch repair proteins selectively replace the strand lacking methylation, thus ensuring that it is mistakes in the newly made
strand that are removed and replaced. Because methylation is the criterion that enables the mismatch repair system to choose the
strand that is repaired, the bacterial mismatch repair system is described as being methyl-directed.

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 Figure 7.36- Dam methylase adds methyl groups at GATC sequences
Mismatch repair genes
Mismatch repair has been well studied in bacteria, and the proteins involved have been identified. In E.coli, mismatch repair
proteins are encoded by a group of genes collectively known as the mut genes. Important components of the mismatch repair
machinery are the proteins MutS, MutL and MutH (Figure 7.37).

Figure 7.37 - Mismatch repair in E. coli - Image by Aleia Kim

MutS acts to recognize the mismatch, while MutL and MutH are recruited to the mismatch site by the binding of MutS. MutH is an
endonuclease that cuts the newly synthesized and, as yet, unmethylated DNA strand at a GATC. This activates a DNA helicase and
an exonuclease that help unwind and remove the region containing the mismatch. DNA polymerase III fills in the gap, using the
opposite strand as the template, and ligase joins the ends, to restore a continuous strand.

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Eukaryotes also have a mismatch repair system that repairs not only single base mismatches but also insertions and deletions.
Homologs to the E. coli MutS and MutL have been identified in other organisms, including humans: hMSH1 and hMSH2 (human
MutS homolog 1 and 2) are homologous to MutS, while hMLH 1 is homologous to MutL. These, together with additional proteins,
carry out mismatch repair in eukaryotic cells.
DNA methylation is not used by eukaryotic cells as a way to distinguish the new strand from the template, and it is not yet
completely understood how the mismatch repair system in eukaryotes "knows" which strand to repair. There is evidence that the
newly made DNA may be recognized by the fact that it is nicked, or discontinuous. This suggests that discontinuity resulting from
Okazaki fragments that have not yet been joined together may permit the new strand to be distinguished from the old, continuous
template strand.

Repairing damage to DNA

In the preceding section we looked at mistakes made when DNA is copied, where the wrong base is inserted during synthesis of the
new strand. But even DNA that is not being replicated can get damaged or mutated. These sorts of damage are not associated with
DNA replication, rather they can occur at any time.
What causes damage to DNA?
Some major causes of DNA damage are:
a. Radiation (e.g., UV rays in sunlight and in tanning booths, or ionizing radiation)
b. Exposure to damaging chemicals, such as nitrosamines or polycyclic aromatic hydrocarbons, in the environment (see Figure
7.38)
c. Chemical reactions within the cell (such as the deamination of cytosine to give uracil, or the methylation of guanine to produce
methylguanine).
This means the DNA in your cells is vulnerable to damage simply from normal sorts of actions, such as walking outdoors, being in
traffic, or from the chemical transformations occurring in every cell as part of its everyday activities. (Naturally, the damage is
much worse in situations where exposure to radiation or damaging chemicals is greater, such as when people use tanning beds, or
smoke, regularly.)

Figure 7.38 - DNA adducted to benz-o-pyrene (highlighted in orange)

Types of damage
What kinds of damage do these agents cause? Radiation can cause different kinds of damage to DNA.
Sometimes, as with much of the damage done by UV rays, two adjacent pyrimidine bases in the DNA will be cross-linked to form
cyclobutane pyrimidine dimers or CPDs (see Figure 7.39). Note that these are two neighboring pyrimidine bases on the same strand
of DNA. UV exposure can also lead to the formation of another type of lesion, known as a (6-4) photoproduct or 6-4PP (Figure
7.39). Ionizing radiation can cause breaks in the DNA backbone, in one or both strands.

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 Figure 7.39 - Possible chemical structures of a pyrimidine dimer - 6-4PP (left) and CPD (right) - Wikipedia
Molecules like benzopyrene, found in automobile exhaust, can attach themselves to bases, forming bulky DNA adducts in which
large chemical groups are linked to bases in the DNA. Damage like pyrimidine dimers, 6-4PPs or chemical adducts can physically
distort the DNA helix, causing DNA and RNA polymerase to stall when they attempt to copy those regions of DNA (Figure 7.40).

Figure 7.40- Thymine dimers distort the DNA helix -Aleia Kim
Chemical reactions occurring within cells can cause cytosines in DNA to be deaminated to uracil. Other sorts of damage in this
category include the formation of oxidized bases like 8-oxo-guanine or alkylated bases like O6-methylguanine. These do not
actually change the physical structure of the DNA helix, but they can cause problems because uracil and 8-oxo-guanine pair with
different bases than the original cytosine or guanine, leading to mutations on the next round of replication. O6-methylguanine
similarly can form base pairs with thymine instead of cytosine.

Removing damage
Cells have several ways to remove the sorts of damage described above. The first of these is described as direct reversal. Many
organisms (though, unfortunately for us, not humans) can repair UV damage like CPDs and 6-4PPs because they possess enzymes
called photolyases (photo=light; lyase=breakdown enzyme - Figure 7.41). Photolyases work through a process called
photoreactivation, and use blue light energy to catalyze a photochemical reaction that breaks the aberrant bonds in the damaged
DNA and returns the DNA to its original state.

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 Figure 7.41- Direct repair of thymine dimers by photolyase - Pehr Jacobsen
Suicide enzyme
O6-methylguanine in DNA can also be removed by direct reversal, with the help of the enzyme O6-methylguanine
methyltransferase. This is a very unusual enzyme that removes the methyl group from the guanine and transfers it onto a cysteine
residue in the enzyme. The addition of the methyl group to the cysteine renders the enzyme non-functional.
As you know, most enzymes are catalysts that remain unchanged over the course of the reaction, permitting a single enzyme
molecule to repeatedly catalyze a reaction. Because the O6-methylguanine methyltransferase does not fit this description, it is
sometimes not regarded as a true enzyme. It has also been called a suicide enzyme, because the enzyme “dies” as a result of its own
activity.

Excision repair
Excision repair is another common strategy. Excision repair is a general term for the cutting out and re-synthesizing of the damaged
region of a DNA. There are several different kinds of excision repair, but they all involve excising the portion of the DNA that is
damaged, followed by repair synthesis using the other strand as template, and finally, ligation to restore continuity to the repaired
strand. Cells possess several different kinds of excision repair, each geared to specific kinds of DNA damage. Between them, these
repair systems deal with the wide variety of insults to the genome.
Nucleotide excision repair

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 Figure 7.42 - Nucleotide excision repair - Aleia Kim
Nucleotide excision repair (NER) fixes damage such as the formation of chemical adducts, as well as UV damage. Both chemical
adducts and the formation of CPDs or 6,4 photoproducts can cause significant distortion of the DNA helix. NER proteins act to cut
the damaged strand on either side of the lesion. A short portion of the DNA strand containing the damage is then removed and a
DNA polymerase fills in the gap with the appropriate nucleotides. Nucleotide excision repair has been extensively studied in
bacteria.
In E. coli, recognition and excision of the damage is carried out by a group of proteins encoded by the uvrABC and uvrD genes.
The protein products of the uvrA, uvrB and uvrC genes function together as the so-called UvrABC excinuclease. The damage is
initially recognized and bound by a complex of the UvrA and UvrB proteins. Once the complex is bound, the UvrA dissociates,
leaving the UvrB attached to the DNA, where it is then joined by the UvrC protein.
Strand nicking
It is the complex consisting of UvrB and C that acts to cut the phosphodiester backbone on either side of the damage, creating nicks
in the strand about 12-13 nucleotides apart. A helicase encoded by uvrD then unwinds the region containing the damage, displacing
it from the double helix together with UvrBC. The gap in the DNA is filled in by DNA polymerase, which copies the undamaged
strand, and the nick is sealed with the help of DNA ligase.
Nucleotide excision repair is also an important pathway in eukaryotes. It is particularly important in the removal of UV damage in
humans, given that we lack photolyases. A number of proteins have been identified that function in ways similar to the Uvr
proteins.
The importance of these proteins is evident from the fact that mutations in the genes that encode them can lead to a number of
genetic diseases, like Xeroderma pigmentosum, or XP. People with XP are extremely sensitive to UV exposure, because the
damage caused by it cannot be repaired, leaving them at a much higher risk of developing skin cancer.
Two repair modes
Nucleotide excision repair operates in two modes, one known as global genomic repair and the other as transcription-coupled
repair. While the function of both is to remove helix-destabilizing damage like cyclobutane pyrimidine dimers or chemical adducts,
the way in which the lesions are detected differs.
In global genomic repair, damage is identified by surveillance of the entire genome for helix distorting lesions. In the case of
transcription-coupled repair, the stalling of the RNA polymerase at a site of DNA damage is the indicator that activates this mode
of nucleotide excision repair.
Base excision repair

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Base excision repair (BER) is a repair mechanism that deals with situations like the deamination of cytosine to uracil (Figure 7.43)
or the methylation of a purine base. These changes do not typically distort the structure of the DNA helix, unlike chemical adducts
or UV damage.
In base excision repair a single damaged base is first removed from the DNA, followed by removal of a region of the DNA
surrounding the missing base. The gap is then repaired.

Figure 7.43 - Spontaneous hydrolysis of cytosine to form uracil

Uracil-DNA glycosylase
The removal of uracil from DNA is accomplished by the enzyme uracil-DNA glycosylase that can recognize uracil in DNA and
break the glycosidic bond between the uracil and the sugar in the nucleotide (Figure 7.44). The removal of the base leaves a gap
called an apyrimidinic site (AP site) because, in this case, uracil, a pyrimidine was removed. It is important to remember that at this
point the backbone of the DNA is still intact, and the removal of a single base simply creates a gap like a tooth that has been
knocked out.
The formation of the AP site triggers the activity of an enzyme known as an AP endonuclease that cuts the DNA backbone 5’ to the
AP site. In the remaining steps, a DNA polymerase binds to the nick, then using its exonuclease and polymerase activities, replaces
the sequence in this region. Depending on the situation, a single nucleotide may be replaced (short patch BER) or a stretch of
several nucleotides may be removed and replaced (long patch BER). Finally, as always, DNA ligase acts to seal the nick in the
DNA.

Figure 7.44 - Base excision repair by uracil-DNA glycosylase. The yellow uracil base has been “flipped” out of the DNA helix for
excision

Repair of double-strand breaks

While all the repair mechanisms discussed so far fixed damage on one strand of DNA using the other, undamaged strand as a
template, these mechanisms cannot repair damage to both strands. What happens if both strands are damaged? Ionizing radiation,
exposure to certain chemicals, or reactive oxygen species generated in the cell can lead to double-strand breaks (DSBs) in DNA.
DSBs are a potentially lethal form of damage that, in addition to blocking replication and transcription, can also lead to
chromosomal translocations, where part of one chromosome gets attached to a piece of another chromosome. Two different cellular
mechanisms exist that help repair DSBs (Figure 7.45), homologous recombination (HR) and non-homologous end joining (NHEJ).

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 Figure 7.45 - Non-homologous end joining (left) versus homologous recombination (right) - Wikipedia
Homologous recombination repair commonly occurs in the late S and G2 phases of the cell, when each chromosome has been
replicated and information from a sister chromatid can be used as a template to achieve error-free repair. Note that in contrast to
excision repair, where the damaged strand was removed and the undamaged sister strand served as the template for filling in the
damaged region, HR must use the information from another DNA molecule, because both strands of the DNA are damaged in
DSBs.

Figure 7.46 - Result of homologous recombination - Wikipedia

Nuclease action
Detection of the double-strand break triggers nuclease activity that chews back one strand on each end of the break. This results in
the production of single-stranded 3’ overhangs on each end. These single-stranded ends are bound by several proteins, creating a
nucleoprotein filament that can then “search” for homologous (matching) sequences on a sister chromatid.
When such sequences are found, the nucleoprotein filament invades the undamaged sister chromatid, forming a crossover. This
creates heteroduplexes made up of DNA strands from different chromatids. Strand invasion (Figure 7.47) is followed by branch
migration, during which the Holliday junction moves along the DNA, extending the heteroduplex away from the original site of the
crossover (Figure 7.48). In E. coli, branch migration depends on the activity of two proteins, RuvA and RuvB. The resulting
recombination intermediate can be resolved, with the help of RuvC to give complete, error-free strands.

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 Figure 7.47 - Repair of DSBs by homologous recombination - Image by Pehr Jacobson

Figure 7.48 - Resolution of Holliday Junctions - Image by Pehr Jacobsen

Non-homologous end joining
In contrast to homologous recombination, Non-homologous end joining (NHEJ) is error-prone. It does not use or require a
homologous template to copy, and works by simply chewing back the broken ends of DSBs and ligating them together. Not
surprisingly, NHEJ introduces deletions in the DNA as a result.

Translesion DNA synthesis

As we have seen, cells have a variety of mechanisms to help safeguard the integrity of the information in DNA. One measure of
last resort is translesion DNA synthesis, also known as bypass synthesis. Translesion synthesis occurs when a DNA polymerase
encounters DNA damage on the template strand, but instead of stalling or skipping past the damage, replication switches to an
error-prone mode, ignoring the template and incorporating random nucleotides into the new strand. In E.coli, translesion synthesis
is dependent on the activities of proteins encoded by the umuC and umuD genes. Under the appropriate conditions (see SOS
response, below) UmuC and UmuD are activated to begin bypass synthesis. Being error-prone, translesion synthesis gives rise to
many mutations.

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The SOS response
Named for standard SOS distress signals, the term “SOS repair” refers to a cellular response to UV damage. When bacterial cells
suffer extensive damage to their DNA as a result of UV exposure, they turn on the coordinated expression of a large number of
genes that are necessary for DNA repair. These include the uvr genes needed for nucleotide excision repair and recA, which is
involved in homologous recombination. In addition to these mechanisms, which can carry out error-free repair, the SOS response
can also induce the expression of translesion polymerases encoded by the dinA, dinB and umuCD genes.
How are all these genes induced in a coordinated way following UV damage? All of the genes induced in the SOS response are
regulated by two components. The first is the presence of a short DNA sequence upstream of their coding region, called the SOS
box. The second is a protein, the LexA repressor (Figure 7.49), that binds to the SOS box and prevents transcription of the
downstream genes. Expression of the genes requires the removal of LexA from its binding site. How is this achieved?

Figure 7.49 -Lex A repressor, N-terminal domain

Figure 7.50 - Activation of SOS genes by lexA in E. coli

When exposure to radiation results in DNA breaks, the presence of single-stranded regions triggers the activation and binding of
RecA proteins to the single-stranded region, creating a nucleoprotein filament. The interaction of the RecA with the LexA repressor
leads to autocleavage of the repressor, allowing the downstream gene(s) to be expressed (Figure 7.50).
The genes controlled by the LexA repressor, as mentioned earlier, encode proteins that are necessary for accurate DNA repair as
well as error-prone translesion synthesis. The various genes involved in DNA repair are induced in a specific order. In the initial
stages, the repair genes that are derepressed are for nucleotide excision repair, followed by homologous recombination, both error-
free mechanisms for repair. If the damage is too extensive to be repaired by these systems, error-prone repair mechanisms may be
brought into play as a last resort.
SOS response and antibiotic resistance
The increased mutation rate in the SOS response may play a role in the acquisition of antibiotic resistance in bacteria (Figure 7.51).
An example is the development of resistance to topoisomerase poisons like the fluoroquinolone family of drugs. Fluoroquinolones
inhibit the ability of topoisomerases to religate the ends of their substrates after nicking them to allow overwound DNA to relax.
This results in accumulation of strand breaks that can trigger the SOS response. As a consequence of error-prone DNA synthesis by
low fidelity polymerases during the SOS response, there is a large increase in the number of mutations. While some mutations may
be lethal to the bacteria, others can contribute to the rapid development of drug resistance in the population.

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 Figure 7.51 - A proposed mechanism of evolved bacterial antibiotic resistance - Wikipedia

This page titled 7.4: DNA Repair is shared under a CC BY-NC-SA license and was authored, remixed, and/or curated by Kevin Ahern, Indira
Rajagopal, & Taralyn Tan.

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7.5: Transcription
Source: BiochemFFA_7_4.pdf. The entire textbook is available for free from the authors at
http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy
In the preceding sections, we have discussed the replication of the cell's DNA and the mechanisms by which the integrity of the
genetic information is carefully maintained. What do cells do with this information? How does the sequence in DNA control what
happens in a cell? If DNA is a giant instruction book containing all of the cell's "knowledge" that is copied and passed down from
generation to generation, what are the instructions for? And how do cells use these instructions to make what they need?

Information flow
You have learned in introductory biology courses that genes, which are instructions for making proteins, are made of DNA. You
also know that information in genes is copied into temporary instructions called messenger RNAs that direct the synthesis of
specific proteins. This description of flow of information from DNA to RNA to protein is often called the central dogma of
molecular biology and is a good starting point for an examination of how cells use the information in DNA.
Consider that all of the cells in a multicellular organism have arisen by division from a single fertilized egg and therefore, all have
the same DNA. Division of that original fertilized egg produces, in the case of humans, over a trillion cells, by the time a baby is
produced from that egg (that's a lot of DNA replication!). Yet, we also know that a baby is not a giant ball of a trillion identical
cells, but has the many different kinds of cells that make up tissues like skin and muscle and bone and nerves. How did cells that
have identical DNA turn out so different?
The answer lies in gene expression, which is the process by which the information in DNA is used. Although all the cells in a baby
have the same DNA, each different cell type uses a different subset of the genes in that DNA to direct the synthesis of a distinctive
set of RNAs and proteins. The first step in gene expression is transcription, which we will examine next (Figure 7.52).

Transcription
Transcription is the process of copying information from DNA sequences into RNA sequences. This process is also known as
DNA-dependent RNA synthesis. When a sequence of DNA is transcribed, only one of the two DNA strands is copied into RNA.
We will consider what determines which strand of DNA is copied into RNA, later on.
But, apart from copying one, rather than both strands of DNA, how is transcription different from replication of DNA? DNA
replication serves to copy all the genetic material of the cell and occurs before a cell divides, so that a full copy of the cell's genetic
information can be passed on to the daughter cell. Transcription, by contrast, copies short stretches of the coding regions of DNA to
make RNA. Different genes may be copied into RNA at different times in the cell's life cycle. RNAs are, essentially, temporary
copies of the information in DNA and different sets of instructions are copied for use at different times and in different cell types.
Cells make several different kinds of RNA:
mRNAs that code for proteins
rRNAS that form part of ribosomes
tRNAs that serve as adaptors between mRNA and amino acids during translation
Small RNAs that regulate gene expression, including miRNAs and siRNAs
Other small RNAs that have a variety of functions, including the small nuclear RNAs that are part of the splicing machinery.
Long noncoding RNAs (lnc RNAs - Figure 7.53)
Building an RNA strand is very similar to building a DNA strand. This is not surprising, knowing that DNA and RNA are very
similar molecules. Transcription is catalyzed by the enzyme RNA Polymerase. "RNA polymerase" is a general term for an enzyme
that makes RNA. There are several different kinds of RNA polymerases in eukaryotic cells, while in prokaryotes, a single type of
RNA polymerase is responsible for all transcription.

RNA synthesis
Like DNA polymerases, RNA polymerases synthesize new strands only in the 5' to 3' direction, but because they are making RNA,
they use ribonucleotides (i.e., RNA nucleotides - Figure 7.54) rather than deoxyribonucleotides. Ribonucleotides are joined in
exactly the same way as deoxyribonucleotides, i.e., the 3'OH of the last nucleotide on the growing chain is joined to the 5'
phosphate on the incoming nucleotide to make a phosphodiester bond.

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One important difference between DNA polymerases and RNA polymerases is that the latter do not require a primer to start
making RNA. Once RNA polymerases are in the right place to start copying DNA, they just begin making RNA by joining
together RNA nucleotides complementary to the DNA template.

Starting points
This, of course, brings us to an obvious question- how do RNA polymerases "know" where to start copying on the DNA?
Unlike the situation in replication, where every nucleotide of the parental DNA must eventually be copied, transcription, as we
have already noted, only copies selected portions of the DNA into RNA at any given time. Consider the challenge here: in a human
cell, there are approximately 6 billion base-pairs of DNA. Much of this is non-coding DNA, meaning that it will not need to be
transcribed. The small percentage of the genome that is made up of coding sequences still amounts to between 20,000 and 30,000
genes in each cell. Of these genes, only a small number will need to be expressed at any given time.What indicates to an RNA
polymerase where to start copying DNA to make a transcript?

Promoters
It turns out that patterns in the DNA sequence indicate where RNA polymerase should start and end transcription. These sequences
are recognized by the RNA polymerase or by proteins that help RNA polymerase determine where it should bind the DNA to start
transcription. A DNA sequence at which the RNA polymerase binds to start transcription is called a promoter. The DNA sequence
that indicates the endpoint of transcription, where the RNA polymerase should stop adding nucleotides and dissociate from the
template is known as a terminator sequence. The promoter and terminator, thus, bracket the region of the DNA that is to be
transcribed.
A promoter is described as being situated upstream of the gene that it controls (Figure 7.57). What this means is that on the DNA
strand that the gene is on, the promoter sequence is "before" the gene, or to put it differently, it is on the side of the gene opposite to
the direction of transcription. Also notice that the promoter is said to "control" the gene it is associated with. This is because
expression of the gene is dependent on the binding of RNA polymerase to the promoter sequence to begin transcription. If the RNA
polymerase and its helper proteins do not bind at the promoter, the gene cannot be transcribed and it will therefore, not be
expressed.
What is special about a promoter sequence? In an effort to answer this question, scientists examined many genes and their
surrounding sequences (Figure 7.57). Because the same RNA polymerase has to bind to many different promoters, it would be
predicted that promoters would have some similarities in their sequences. As expected, common sequence patterns were seen to be
present in many promoters.
We will first take a look at prokaryotic promoters.
When prokaryotic genes were examined, the following features commonly emerged:
A transcription start site (this the base in the DNA across from which the first RNA nucleotide is paired), which, by convention,
is denoted as +1.
A -10 sequence: this is a 6 bp region centered about 10 bp upstream of the start site. The consensus sequence at this position is
TATAAT. In other words, if you count back from the transcription start site, the sequence found at roughly -10 in the majority of
promoters studied is TATAAT.
A -35 sequence: this is a 6 bp sequence at about 35 basepairs upstream from the start of transcription. The consensus sequence
at this position is TTGACA.
It is important to understand that each nucleotide in a consensus sequence is simply the one that appeared at that position in the
majority of promoters examined, and does not mean that the entire consensus sequence is found in all promoters. In fact, few
promoters have -10 and -35 sequences that exactly match the consensus. The box at the left shows the -10 and -35 sequences by
percentage of occurrence of each base in the promoter.
What is the significance of these sequences? It turns out that the sequences at -10 and -35 are necessary for recognition of the
promoter region by RNA polymerase (Figure 7.58). The sequences at -10 and -35 may vary a little in individual promoters, as
mentioned above, but the extent to which they are different is limited. It is only when the RNA polymerase has stably bound at the
promoter that transcription can begin. The process by which the promoter is recognized and bound stably has been well studied for
the RNA polymerase of E. coli.

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Core polymerase and holoenzyme
The E. coli RNA polymerase is made up of a core enzyme of five subunits (α2ββ’and ω) and an additional subunit called the σ
(sigma) subunit. Together, the σ subunit and core polymerase make up what is termed the RNA polymerase holoenzyme. The core
polymerase is the part of the RNA polymerase that is responsible for the actual synthesis of the RNA, while the σ subunit is
necessary for binding of the enzyme at promoters to initiate transcription.
Loose association
The core polymerase and σ subunit are not always associated with each other. For the most part, the core polymerase is loosely
associated with DNA, although it does not discriminate between promoters and other sequences in DNA, and the DNA strands are
not opened up to allow transcription in this state. The role of the σ subunit is to reduce the affinity of the core polymerase for non-
specific DNA sequences and to help the enzyme specifically bind to promoter sequences.
Holoenzyme binding
It is when the σ subunit associates with the core polymerase that the holoenzyme is able to bind specifically to promoter sequences.
The initial binding of the holoenzyme at the promoter results in what is called a “closed” complex, meaning that the DNA template
is still double-stranded and has not opened up to allow transcription. This closed complex is then converted to an “open” complex
by the separation of the DNA strands to create a transcription bubble about 12-14 base-pairs long (Figure 7.60). The conversion of
the closed complex to the open complex also requires the presence of the σ subunit.
Open complex & initiation
Once the open complex has formed, the DNA template can begin to be copied, and the core polymerase adds nucleotides
complementary to one strand of the DNA. At this stage, known as initiation, the polymerase adds several nucleotides while still
bound to the promoter, and without moving along the DNA template. Initially, short pieces of RNA a few nucleotides long may be
made and released, without the polymerase leaving the promoter. Eventually, the enzyme makes the transition to the next stage,
elongation, when an RNA of 8-9 bases is made and the enzyme moves beyond the promoter region.
Elongation
Once elongation commences and the RNA polymerase is moving down the DNA template, the σ subunit is no longer necessary and
may dissociate from the core enzyme. The core polymerase can move along the template, unwinding the DNA ahead of it to
maintain a transcription bubble of 12-15 base-pairs and synthesizing RNA complementary to one of the strands of the DNA. As
already mentioned, an RNA chain, complementary to the DNA template, is built by the RNA polymerase by the joining of the 5'
phosphate of an incoming ribonucleotide to the 3'OH on the last nucleotide of the growing RNA strand. Behind the RNA
polymerase, the DNA template is rewound, displacing the newly made RNA from its template strand.
Termination
As mentioned earlier, a sequence of nucleotides called the terminator is the signal to the RNA polymerase to stop transcription and
dissociate from the template. Some terminator sequences, known as intrinsic terminators, allow termination by RNA polymerase
without the help of any additional factors, while others, called rho-dependent terminators, require the assistance of a protein factor
called rho (ρ).
How does the sequence of the terminator cause the RNA polymerase to stop adding nucleotides and release the transcript?
To understand this, it is useful to know that the terminator sequence precedes the last nucleotide of the transcript. In other words,
the terminator is part of the end of the sequence that is transcribed (Figure 7.61).
Intrinsic terminators
In intrinsic terminators, this sequence in the RNA has self-complementary regions that can base-pair with each other to form a
hairpin structure that contains a GC-rich run in the “stem” of the hairpin. This hairpin is followed by a single-stranded region that is
rich in U’s (Figure 7.62). The secondary structure formed by the folding of the end of the RNA into the hairpin causes the RNA
polymerase to pause. Meanwhile, the run of U’s at the end of the hairpin permits the RNA-DNA hybrid in this region to come
apart, because the base-pairing between A’s in the DNA template and the U’s in the RNA is relatively weak. This allows the
transcript to be released from the DNA template and from the RNA polymerase.
Rho-dependent termination

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In the case of rho-dependent termination, an additional protein factor, rho, is necessary. Rho is a helicase that can separate the
transcript from the template it is paired with. As in intrinsic termination, rho-dependent termination requires the formation of a
hairpin structure in the RNA that causes pausing of the RNA polymerase. Meanwhile, rho binds to a region of the transcript called
the rho utilization site (rut) and moves along the RNA till it reaches the paused RNA polymerase. It then acts on the RNA-DNA
hybrid, releasing the transcript from the template.
Coupled transcription and translation
In prokaryotes, which lack a nucleus, the DNA is not separated from the rest of the cell in a separate compartment, so the mRNA is
immediately available to the translation machinery, as the transcript is coming off the template DNA. Indeed, in prokaryotic cells,
translation of the mRNA can begin before the entire gene has been transcribed. Ribosomes can assemble at the 5’ end of the
transcript, as it is displaced from the template, while the 3’ end of the gene is still being copied. The lag time between transcription
and translation is thus, very short in prokaryotes.
Transcription in eukaryotes
Although the process of RNA synthesis is the same in eukaryotes as in prokaryotes, there are some additional considerations in
eukaryotes. One is that in eukaryotes, the DNA template exists as chromatin, where the DNA is tightly associated with histones and
other proteins. The "packaging" of the DNA must therefore be opened up to allow the RNA polymerase access to the template in
the region to be transcribed (Figure 7.63). The restructuring of chromatin to allow access to regions of DNA is thus an important
factor in determining which genes are expressed.
Multiple RNA polymerases
A second difference is that eukaryotes have multiple RNA polymerases, not just one as in bacterial cells. The different eukaryotic
polymerases transcribe different classes of genes. For example, RNA polymerase I transcribes the ribosomal RNA genes, while
RNA polymerase III copies tRNA genes. The RNA polymerase we will focus on most is RNA polymerase II, which transcribes
protein-coding genes to make mRNAs.
All three eukaryotic RNA polymerases need additional proteins to help them get transcription started. In prokaryotes, RNA
polymerase by itself can initiate transcription (the σ subunit is a subunit of the RNA polymerase, not an entirely separate protein).
The additional proteins needed by eukaryotic RNA polymerases are referred to as transcription factors. We will see below that
there are various categories of transcription factors.
Transcription and translation are de-coupled
Finally, in eukaryotic cells, transcription is separated in space and time from translation. Transcription happens in the nucleus, and
the RNAs produced are processed further before they are sent into the cytoplasm.
Protein synthesis (translation) happens in the cytoplasm. As noted earlier, in prokaryotic cells, mRNAs can be translated as they are
coming off the DNA template, and because there is no nuclear envelope, transcription and protein synthesis occur in a single
cellular compartment. A representative eukaryotic gene, depicted in Figure 7.64 shows that transcription starts some 25 bp
downstream of the TATA box, and creates a transcript that begins with a 5’ untranslated region (5’UTR) followed by the coding
region which may include multiple introns and ending in a 3’ untranslated region or 3‘UTR (Figure 7.64). As detailed below, the
initial transcript is further processed before it is used.
Eukaryotic promoters
Like genes in prokaryotes, eukaryotic genes also have promoters that determine where transcription will begin. As with
prokaryotes, there are specific sequences in the promoter regions that are recognized and bound by proteins involved in the
initiation of transcription. We will focus primarily on the genes encoding proteins that are transcribed by RNA polymerase II. Such
promoters commonly have a TATA box, a sequence similar to the -10 sequence in prokaryotic promoters. The TATA box is a
sequence about 25-35 basepairs upstream of the start of transcription (+1). (Some eukaryotic promoters lack TATA boxes, and
have, instead, other recognition sequences, known as DPE, or downstream promoter elements.) Interestingly, the TATA box is not
directly recognized and bound by RNA polymerase II. Instead, this sequence is bound by other proteins that, together with the
RNA polymerase, form the transcription initiation complex.
Eukaryotic promoters also have, in addition, several other short stretches of sequences, that affect transcription, within about 100 to
200 base-pairs upstream of the transcription start site. These sequences, which are sometimes called upstream elements or
promoter-proximal upstream elements, are bound by activator proteins that interact with the transcription complex that forms at the
TATA box. Examples of such upstream elements are the CAAT box and the GC box (Figure 7.64).

7.5.4 https://bio.libretexts.org/@go/page/7846
Making transcripts in eukaryotes
We noted earlier that all eukaryotic RNA polymerases need additional proteins to bind promoters and start transcription. The
proteins that help eukaryotic RNA polymerases find promoter sites and initiate RNA synthesis are termed general transcription
factors. We will focus on the transcription factors that assist RNA polymerase II, the enzyme that transcribes protein-coding genes.
These transcription factors are named TFIIA, TFIIB and so on (TF= transcription factor, II=RNA polymerase II, and the letters
distinguish individual transcription factors).
Transcription by RNA polymerase II requires the general transcription factors and the RNA polymerase to form a complex, at the
TATA box, called the basal transcription complex or transcription initiation complex (Figure 7.65).
This is the minimum requirement for any gene to be transcribed. The first step in the formation of this complex is the binding of the
TATA box by a transcription factor, TFIID. TFIID is made up of several proteins, one of which is called the TATA Binding Protein
or TBP. Binding of the TBP causes the DNA to bend at this spot and take on a structure that is suitable for the binding of additional
transcription factors and RNA polymerase.
Interestingly, the binding of the TBP is a necessary step in forming a transcription initiation complex even when the promoter lacks
a TATA box. The order of binding of additional proteins after binding of the TBP, as determined through in vitro experiments,
appears to be TFIIB, followed by TFIIF and RNA polymerase II, then TFIIE. The final step in the assembly of the basal
transcription complex is the binding of a general transcription factor called TFIIH. Some evidence suggests that following the
binding of the TBP to DNA, the rest of the proteins in the initiation complex may assemble as a very large complex that then binds
directly to the DNA. In any case, the presence of all of these general transcription factors and RNA Polymerase II bound at the
promoter is necessary for the initiation of transcription.
As in prokaryotic transcription, once the RNA polymerase binds, it can begin to assemble a short stretch of RNA. This must be
followed by promoter clearance, in order to move down the template and elongate the transcript. This requires the action of TFIIH.
TFIIH is a multifunctional protein that has helicase activity (i.e., it is capable of opening up a DNA double helix) as well as kinase
activity. The kinase activity of TFIIH adds phosphates onto the C-terminal domain (CTD) of the RNA polymerase II. This
phosphorylation appears to be the signal that releases the RNA polymerase from the basal transcription complex and allows it to
move forward on the template, building the new RNA as it goes (Figure 7.66).
Termination of transcription is not as well understood as it is in prokaryotes. Termination does not occur at a fixed distance from
the 3’ end of mature RNAs. Rather, it seems to occur hand in hand with the processing of the 3’ end of the primary transcript. The
polyadenylation signal in the 3’ untranslated region of the transcript appears to play a role in RNA polymerase pausing, and
subsequent release of the completed primary transcript. Recognition of the polyadenylation signal triggers the binding of proteins
involved in 3’end processing and termination.
Information Processing: Transcription
748
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749
Figure 7.52 - Overview of eukaryotic transcription
Wikipedia
750
Figure 7.53 - Transcripts may code for protein or may be functional as RNAs
Wikipedia
751
Figure 7.55 - RNA (green) being synthesized from DNA template (blue strand) by T7 RNA polymerase (purple). The non-template
DNA strand is in red.
Figure 7.54 - The four ribonucleotides for making RNA

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752
Figure 7.56 - Central dogma - DNA to RNA to protein
Wikipedia
753
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Figure 7.57 - Sequences upstream of transcription start site in several prokaryotic genes
Image by Martha Baker
754
-10 Sequence
TATAAT
77% 76% 60% 61% 56% 82%
-35 Sequence
TTGACA
69% 70% 61% 56% 54% 54%
Figure 7.58 - RNA polymerase promoter binding
Wikipedia
Figure 7.59 - A bacterial RNA polymerase (α2ββ’and ω)
755
Figure 7.60 - Synthesis of RNA in the transcription bubble
756
Figure 7.61 - Promoter and Terminator sequences determine where transcription starts and ends.
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Figure 7.62 Transcription termination by intrinsic (top) and rho-dependent (bottom) mechanisms
Wikipedia
758
Figure 7.63 - Eukaryotic DNA is complexed with proteins in chromatin
Wikipedia
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759
Figure 7.64 - Region surrounding the transcriptional start site in eukaryotic DNA
Figure 7.65 - Transcription pre-initiation complex in eukaryotes
Wikipedia
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7.5.7 https://bio.libretexts.org/@go/page/7846
7.6: RNA Processing
Source: BiochemFFA_7_5.pdf. The entire textbook is available for free from the authors at
http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy
So far, we have looked at the mechanism by which the information in genes (DNA) is transcribed into RNA. The newly made
RNA, also known as the primary transcript is further processed before it is functional. Both prokaryotes and eukaryotes process
their ribosomal and transfer RNAs. The major difference in RNA processing, however, between prokaryotes and eukaryotes, is in
the processing of messenger RNAs. We will focus on the processing of mRNAs in this section. You will recall that in bacterial
cells, the mRNA is translated directly as it comes off the DNA template. In eukaryotic cells, RNA synthesis, which occurs in the
nucleus, is separated from the protein synthesis machinery, which is in the cytoplasm. The initial product of transcription of an
mRNA is sometimes referred to as the pre-mRNA. After it has been processed and is ready to be exported from the nucleus, it is
called the mature mRNA. The three main processing steps for mRNAs are (Figure 7.67):
• Capping at the 5' end
• Splicing to remove introns
• Addition of a polyA tail at the 3' end.
Although this description suggests that these processing steps occur post-transcriptionally, after the entire gene has been
transcribed, there is evidence that processing occurs co-transcriptionally. That is, the steps of processing are occurring as the
mRNA is being made. Proteins involved in mRNA processing have been shown to be associated with the phosphorylated C-
terminal domain (CTD) of RNA polymerase II.

Capping
As might be expected, the addition of an mRNA cap at the 5’ end is the first step in mRNA processing, since the 5’end of the RNA
is the first to be made. Capping occurs once the first 20-30 nucleotides of the RNA have been synthesized. The addition of the cap
involves removal of a phosphate from the first nucleotide in the RNA to generate a diphosphate. This is then joined to a guanosine
monophosphate which is subsequently methylated at N7 of the guanine to form the 7mG cap structure (Figure 7.68). This cap is
recognized and bound by a complex of proteins that remain associated with the cap till the mRNA has been transported into the
cytoplasm. The cap protects the 5' end of the mRNA from degradation by nucleases and also helps to position the mRNA correctly
on the ribosomes during protein synthesis.

Splicing
Eukaryotic genes have introns, noncoding regions that interrupt the gene. The mRNA copied from genes containing introns will
also therefore have noncoding regions that interrupt the information in the gene. These noncoding regions must be removed (Figure
7.69) before the mRNA is sent out of the nucleus to be used to direct protein synthesis.

Intron removal
Introns are removed from the pre-mRNA by the activity of a complex called the spliceosome. The spliceosome is made up of
proteins and small RNAs that are associated to form protein-RNA enzymes called small nuclear ribonucleoproteins or snRNPs
(pronounced snurps).

Splice junctions
The splicing machinery must be able to recognize splice junctions (i.e., where each exon ends and its associated intron begins) in
order to correctly cut out the introns and join the exons to make the mature, spliced mRNA. What signals indicate exon-intron
boundaries? The junctions between exons and introns are indicated by specific base sequences. The consensus sequence at the 5’
exon-intron junction (also called the 5’ splice site) is AGGURAGU. In this sequence, the intron starts with the second G (R stands
for any purine). The 3' splice junction has the consensus sequence YAGRNNN, where YAG is within the intron, and RNNN is part
of the exon (Y stands for any pyrimidine, and N for any nucleotide).
There is also a third important sequence within the intron, about a hundred nucleotides from the 3’ splice site, called a branch point
or branch site, that is important for splicing. This site is defined by the presence of an A followed by a string of pyrimidines. The
importance of this site will be seen when we consider the steps of splicing.

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Splicing mechanism
There are two main steps in splicing. The first step is the nucleophilic attack by the 2’OH of the branch point A on the 5' splice site
(the junction of the 5' exon and the intron). As a result of a trans-esterification reaction, the 5' exon is released, and a lariat-shaped
molecule composed of the 3’ exon and the intron sequence is generated (Figure 7.70). In the second step, the 3' OH of the 5’ exon
attacks the 3’ splice site, and the two exons are joined together, and the lariat-shaped intron is released .

Spliceosome
As mentioned earlier, splicing is carried out by a complex consisting of small RNAs and proteins. The five small RNAs crucial to
this complex, U1, U2, U4, U5 and U6 are found associated with proteins, as snRNPs. These and many other proteins work together
to facilitate splicing. Although many details remain to be worked out, it appears that components of the splicing machinery
associate with the CTD of the RNA polymerase and that this association is important for efficient splicing. The assembly of the
spliceosome requires the stepwise interaction of the various snRNPs and other splicing factors (Figure 7.71). The initial step in this
process is the interaction of the U1 snRNP with the 5’ splice site. Additional proteins such as U2AF (AF = associated factor) are
also loaded onto the pre-mRNA near the branch site. This is followed by the binding of the U2 snRNA to the branch site.
Next, a complex of the U4/U6 and U5 snRNPs is recruited to the spliceosome to generate a pre-catalytic complex. This complex
undergoes rearrangements that alter RNA-RNA and protein-RNA interactions, resulting in displacement of the U4 and U1 snRNPs
and the formation of the catalytically active spliceosome. This complex then carries out the two splicing steps described earlier.

Alternative splicing
On average, human genes have about 9 exons each. However, the mature mRNAs from a gene containing nine exons may not
include all of them. This is because the exons in a pre-mRNA can be spliced together in different combinations to generate
different mature mRNAs. This is called alternative splicing, and allows the production of many different proteins using relatively
few genes, since a single RNA with many exons can, by combining different exons during splicing, create many different protein
coding messages. Because of alternative splicing, each gene in our DNA gives rise, on average, to three different proteins.
Alternative splicing allows the information in a single gene to be used to specify different proteins in different cell types or at
different developmental stages (Figure 7.72).

Polyadenylation
The 3' end of a processed eukaryotic mRNA typically has a “poly(A) tail” consisting of about 200 adenine-containing nucleotides.
These residues are added by a template-independent enzyme, poly(A)polymerase, following cleavage of the RNA at a site near the
3’ end of the new transcript. Components of the polyadenylation machinery have been shown to be associated with the CTD of the
RNA polymerase, showing that all three steps of pre-mRNA processing are tightly linked to transcription. There is evidence that
the polyA tail plays a role in efficient translation of the mRNA, as well as in the stability of the mRNA. Like alternative splice
sites, genes can have alternative polyA sites as well (Figure 7.73).
The cap and the polyA tail on an mRNA are also indications that the mRNA is complete (i.e., not defective). Once protein-coding
messages have been processed by capping, splicing and addition of a poly A tail, they are transported out of the nucleus to be
translated in the cytoplasm. Mature mRNAs are sent into the cytoplasm bound to export proteins that interact with the nuclear pore
complexes in the nuclear envelope (Figure 7.74). Once the mature mRNA has been translocated to the cytoplasm, it is ready to be
translated.

RNA editing
In addition to undergoing the three processing steps outlined above, many RNAs undergo further modification called RNA editing.
Editing has been observed in not only mRNAs but also in transfer RNAs and ribosomal RNAs. As the name suggests, RNA editing
is a process during which the sequence of the transcript is altered post-transcriptionally. A well-studied example of RNA editing is
the alteration of the sequence of the mRNA for apolipoprotein B (see also HERE). The editing results in the deamination of a
cytosine in the transcript to form a uracil, at a specific location in the mRNA. This change converts the codon at this position,
CAA, which encodes a glutamine, into UAA, a stop codon. The consequence of this is that a shorter version of the protein is made,
when the edited transcript is translated. It is interesting that the editing of this transcript occurs in intestinal cells but not in liver
cells. Thus, the protein product of the apolipoprotein B gene is longer in the liver than it is in the intestine.

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Insertion/deletion
Another kind of RNA editing involves the insertion or deletion of one or more nucleotides. One example of this sort of editing is
seen in the mitochondrial RNAs of trypanosomes. Small guide RNAs indicate the sites at which nucleotides are inserted or deleted
to produce the mRNA that is eventually translated (Figure 7.75).
The effect of either of these kinds of editing on the mRNA is that the encoded protein product is different, providing another point
at which the product of expression of a gene can be controlled.

tRNA synthesis & processing

tRNAs are synthesized by RNA polymerase III, which makes precursor molecules called pre-tRNA that then undergo processing to
generate mature tRNAs. The initial transcripts contain additional RNA sequences at both the 5’ and 3’ ends. Some pre-tRNAs also
contain introns. These additional sequences are removed from the transcript during processing.
The 5’ leader sequence of the pre-tRNA (the additional nucleotides at the 5’-end) is removed by an unusual endonuclease called
ribonuclease P (RNase P - Figure 7.76). RNase is a ribonucleoprotein complex composed of a catalytic RNA and numerous
proteins. The 3’ trailer sequence (extra nucleotides at the 3’ end of the pre-tRNA) is later removed by different nucleases. All
tRNAs must have a 3’ CCA sequence that is necessary for the charging of the tRNAs with amino acids. In bacteria, this CCA
sequence is encoded in the tRNA gene, but in eukaryotes, the CCA sequence is added post-transcriptionally by an enzyme called
tRNA nucleotidyl transferase (tRNT).

Introns
As mentioned earlier, some tRNA precursors contain an intron located in the anticodon arm. In eukaryotes, this intron is typically
found immediately 3’ to the anticodon. The introns is spliced out with the help of a tRNA splicing endonuclease and a ligase.

Base modifications
Mature tRNAs contain a high proportion of bases other than the usual adenine (A), guanine (G), cytidine (C) and uracil (U). These
unusual bases are produced by modifying the bases in the tRNA to form variants, such as pseudouridine (Figure 7.77) or
dihyrouridine. Modifications to the bases are introduced into the tRNA at the final processing step by a variety of specialized
enzymes. Different tRNAs have different subsets of modifications at specific locations, often the first base of the anti-codon (the
wobble position).

rRNA synthesis and processing

Cells contain many copies of rRNA genes (between 100 and 2000 copies are seen in mammalian cells). These genes are organized
in transcription units separated by non-transcribed spacers. Each transcription unit contains sequences coding for 18S, 5.8S and 28S
rRNA, and is transcribed by RNA polymerase I into a single long transcript (47S). The 5S rRNA is separately transcribed. The
sizes of ribosomal RNAs are, by convention, indicated by their sedimentation coefficients, which is a measure of their rate of
sedimentation during centrifugation. Sedimentation is expressed in Svedberg units (hence the S at the end of the number) with
larger numbers indicating greater mass.
The initial transcript contains 5’ and 3’ external transcribed spacers (ETS) as well as internal transcribed sequences (ITS). The
primary transcript is first trimmed at both ends by nucleases to give a 45S pre-rRNA. Further processing of the pre-rRNA through
cleavages guided by RNA-protein complexes containing snoRNAs (small nucleolar RNAs), gives rise to the mature 18S, 5.8S and
28S rRNAs (Figure 7.79). Ribosomal RNAs are also modified both on the ribose sugars and on the bases. Interestingly,
methylation of ribose sugars is the major modification in rRNA. The modified base pseudouridine is also common in rRNA. Other
modifications include base methylation, and acetylation. These modifications are thought to be important in modulating ribosome
function.
Information Processing: RNA Processing
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7.6.3 https://bio.libretexts.org/@go/page/7847
768
Figure 7.68 - 5’ capping of eukaryotic mRNAs
Wikipedia
Figure 7.67 - Steps in processing of pre-mRNA
769
Figure 7.69 - Removal of introns from the primary transcript
Interactive Learning
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770
Figure 7.70 - Splicing of introns
Wikipedia
771
Figure 7.71 - Assembly of the spliceosome complex
Wikipedia
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772
Figure 7.72 - Alternative splicing leads to different forms of a protein from the same gene sequence
Figure 7.73 - Alternative poly-adenylation sites for a gene
773
Figure 7.74 - Structure of a mature eukaryotic mRNA
Interactive Learning
Module
HERE
774
Figure 7.76 - Structure of the RNA component of ribonuclease P
Figure 7.75 - Template guided - one mechanism of RNA editing
775
Figure 7.78 - Sequence of a mature tRNA
Wikipedia
Figure 7.77 - Synthesis of pseudouridine from uridine
Wikipedia
776
Figure 7.79 - Processing of ribosomal RNA
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HERE & HERE
Graphic images in this book were products of the work of several talented students. Links to their Web pages are below
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778
The Codon Song
To the tune of “When I’m Sixty Four”
Metabolic Melodies Website HERE
Building of proteins, you oughta know
Needs amino A’s
Peptide bond catalysis in ribosomes
Triplet bases, three letter codes

Mixing and matching nucleotides

Who is keeping score?

7.6.5 https://bio.libretexts.org/@go/page/7847
Here is the low down
If you count codons
You'll get sixty four
Got - to - line - up - right
16-S R-N-A and
Shine Dalgarno site
You can make peptides, every size
With the proper code
Start codons positioned
In the P site place
Initiator t-RNAs

UGA stops and AUGs go

Who could ask for more?
You know the low down
Count up the codons
There are sixty four
Recording by Tim Karplus
Lyrics by Kevin Ahern
Recording by Tim Karplus Lyrics by Kevin Ahern

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7.7: Translation
Source: BiochemFFA_7_6.pdf. The entire textbook is available for free from the authors at
http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy
Translation is the process by which information in mRNAs is used to direct the synthesis of proteins. As you have learned in
introductory biology, in eukaryotic cells, this process is carried out in the cytoplasm of the cell, by large RNA-protein machines
called ribosomes. Ribosomes contain ribosomal RNA (rRNA) and proteins. The proteins and rRNA are organized into two
subunits, a large and a small. Ribosomes function by binding to mRNAs and holding them in a way that allows the amino acids
encoded by the RNA to be joined sequentially to form a polypeptide. Transfer RNAs are the carriers of the appropriate amino acids
to the ribosome.

The Genetic Code

We speak of genes (i.e., DNA) coding for proteins and the central dogma, which states that DNA makes RNA makes protein. What
does this actually mean? A code can be thought of as a system for storing or communicating information. A familiar example is the
use of letters to represent the names of airports (e.g., PDX for Portland, Oregon and ORD for Chicago’s O’Hare). When a tag on
your luggage shows PDX as the destination, it conveys information that your bag should be sent to Portland, Oregon. To function
well, such a set-up must have unique identifiers for each airport and people who can decode the identifiers correctly. That is, PDX
must stand only for Portland, Oregon and no other airport. Also, luggage handlers must be able to correctly recognize what PDX
stands for, so that your luggage doesn’t land in Phoenix, instead.
How does this relate to genes and the proteins they encode?
Genes are first transcribed into mRNA, as we have already discussed. The sequence of an mRNA, copied from a gene, directly
specifies the sequence of amino acids in the protein it encodes. Each amino acid in the protein is specified by a sequence of 3 bases
called a codon in the mRNA (Figure 7.81). For example, the amino acid tryptophan is encoded by the sequence UGG on an
mRNA. All of the twenty amino acids used to build proteins have, likewise, 3-base sequences that encode them.
Degeneracy
Given that there are 4 bases in RNA, the number of different 3-base combinations that are possible is 43, or 64. There are, however,
only 20 amino acids that are used in building proteins in cells. This discrepancy in the number of possible codons and the actual
number of amino acids they specify is explained by the fact that the same amino acid may be specified by more than one codon. In
fact, with the exception of the amino acids methionine and tryptophan, all the other amino acids are encoded by multiple codons.
Codons for the same amino acid are often related, with the first two bases the same and the third being variable. An example would
be the codons for alanine: GCU, GCA, GCC and GCG all stand for alanine. This sort of redundancy in the genetic code is termed
degeneracy.

Stop and start codons

Three of the 64 codons are what are known as termination or stop codons, and as their name suggests, indicate the end of a protein
coding sequence. The codon for methionine, AUG, is used as the initiation or start codon for the majority of proteins. This
ingenious system is used to direct the assembly of a protein in the same way that you might string together colored beads in a
particular order using instructions that used symbols like UGG for a red bead, followed by UUU for a green bead, CAC for yellow,
and so on, till you came to UGA, indicating that you should stop stringing beads.

Translating the code

While the ribosomes are literally the factories that join amino acids together using the instructions in mRNAs, another class of
RNA molecules, the transfer RNAs (tRNAs) are also needed for translation (Figure 7.83 and Interactive 7.1). Transfer RNAs are
small RNA molecules, about 75-90 nucleotides long, that function to 'interpret' the instructions in the mRNA during protein
synthesis. Transfer RNAs are extensively modified post-transcriptionally and contain a large number of unusual bases. The
sequences of tRNAs have several self complementary regions, where the single-stranded tRNA folds on itself and base-pairs to
form what is sometimes described as a clover leaf structure.
This structure is crucial to the function of the tRNA, providing both the sites for attachment of the appropriate amino acid and for
recognition of codons in the mRNA. In terms of the bead analogy above, someone or something has to be able to bring a red bead

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in when the instructions indicate UGG, and a green bead when the instructions say UUU. This, then, is the function of the tRNAs.
They must be able to bring the amino acid corresponding to the instructions to the ribosome.

t-RNA specificity
A given transfer RNA is specific for a particular amino acid. It is linked covalently at its 3' end to the appropriate amino acid by an
enzyme called aminoacyl tRNA synthetase. For example, there is a transfer RNA that is specific to the amino acid tryptophan, and
a corresponding aminoacyl tRNA synthetase, called a tryptophanyl tRNA synthetase, that can attach the tryptophan specifically to
this tRNA. Likewise, there is an aminoacyl tRNA synthetase specific for each amino acid. A tRNA with an amino acid attached to
it is said to be charged (Figure 7.84). A pool of charged tRNAs is necessary to carry out protein synthesis. How do these tRNAs,
carrying specific amino acids assist the ribosome in stringing together the correct amino acids, as specified by the sequence of the
mRNA?

Codon recognition
As we already know, the amino acid sequence of the protein is determined by the order of the codons in the mRNA. We also have
charged tRNAs carrying the various amino acids present.
How are the amino acids attached to each other in the order indicated by the base sequence of the mRNA? This requires
recognition of the codons on the mRNA by the appropriate charged tRNAs. The amino acid tryptophan, as we noted, is specified
by the codon UGG in the mRNA. This codon must be recognized by a tRNA charged with tryptophan. Every tRNA has a a
sequence of 3 bases, the anticodon, that is complementary to the codon for the amino acid it is carrying. When the tRNA
encounters the codon for its amino acid on the messenger RNA, the anticodon will base-pair with the codon. For the tryptophan
tRNA this is what it would look like:
Sequence of tryptophan codon in mRNA:
5’ UGG 3’
Anticodon sequence in tryptophan tRNA:
5’ CCA 3’
Note that the sequences are both written, by convention, in the 5’ to 3’ direction. To base pair, though, they must be oriented in
opposite directions (anti-parallel). The codon-anticodon basepair in the antiparallel orientation then would be:
5’ UGG 3’
3’ ACC 5’
The base-pairing of the anticodon on a charged tRNA with the codon on the mRNA is what brings the correct amino acids in to the
ribosome to be added on to the growing protein chain (Figure 7.85).

Making a Polypeptide
With an idea of the various components necessary for translation and how they work, we can now take a look at the process of
protein synthesis. The main steps in the process are similar in prokaryotes and eukaryotes. As we already noted, ribosomes bind to
mRNAs and facilitate the interaction between the codons in the mRNA and the anticodons on charged tRNAs.
Ribosomes have two sites (P-site and A-site) for binding and positioning charged tRNAs so each can form base pairs between their
anticodon and a codon from the mRNA. The start codon (AUG) is positioned to base pair with the tRNA in the P-site (peptidyl
site). Next, the charged tRNA complementary to the codon adjacent to the start codon binds and occupies the A-site (aminoacyl
site) in the ribosome (Figure 7.86).
At this point, the ribosome joins the amino acids carried on each tRNA by making a peptide bond. The bond between the amino
acid and the tRNA in the P-site is broken and the dipeptide is joined to the tRNA on the A-site.
The initiator tRNA without its amino acid is then released, moving into a site known as the Exit or E-site, while the second tRNA
carrying the dipeptide (and the codon it is base paired to) moves into the P-site. The A-site now is ready with a new codon for the
next incoming charged tRNA. This process is repeated, with the ribosome moving on the mRNA one codon at a time, until the stop
codon reaches the A-site. At this point, a release factor binds at the A-site, and helps to free the completed polypeptide from the
ribosome. The ribosome then dissociates into the small and large subunits, once more.

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Three steps
Having considered the steps of translation in broader terms, we can now look at them in greater detail. We will consider the three
steps of translation (bel0w) individually.
Initiation (binding of the ribosomal subunits to the transcript and initiator tRNA)
Elongation (repeated addition of amino acids to the growing polypeptide, based on the sequence of the mRNA - Figure 7.87)
Termination (release of the completed polypeptide and dissociation of the ribosome into its subunits).
We already know that processed mRNAs are sent from the nucleus to the cytoplasm in eukaryotic cells, while in prokaryotic cells,
transcription and translation occur in a single cellular compartment. The small and large subunits of ribosomes, each composed of
characteristic rRNAs and proteins, are found in the cytoplasm and assemble on mRNAs to form complete ribosomes that carry out
translation. Both prokaryotic and eukaryotic ribosomal subunits are made up of one or more major rRNAs together with a large
number of ribosomal proteins. The small subunits of prokaryotic cells are called the 30S ribosomal subunits, while their
counterparts in eukaryotes are the 40S subunits. The large ribosomal subunits in prokaryotes are the 50S subunits, while those in
eukaryotic cells are 60S. These differences reflect the larger mass of eukaryotic ribosomes. The rRNA components of ribosomes
are important for the recognition of the 5’ end of the mRNA, and also play a catalytic role in the formation of peptide bonds.

Initiation
Messenger RNAs have non-coding sequences both at their 5' and 3' ends, with the actual protein-coding region sandwiched in
between these untranslated regions (called the 5' UTR and 3' UTR, respectively). The ribosome must be able to recognize the 5' end
of the mRNA and bind to it, then determine where the start codon is located. It is important to note that both in prokaryotes and
eukaryotes, ribosomes assemble at the 5’ end of the transcript by the stepwise binding of the small and large subunits. The small
subunit first binds to the mRNA at specific sequences in the 5’ UTR. The large subunit then binds to the complex of the mRNA
and small subunit, to form the complete ribosome.
Initiator tRNA
Initiation also requires the binding of the first tRNA to the ribosome. As we have noted earlier, the initiation, or start codon is
usually AUG, which codes for the amino acid methionine. Thus, the initiator tRNA is one that carries methionine and is designated
as tRNAmet or methionyl tRNAmet. In bacteria, the methionine on the initiator tRNA is modified by the addition of a formyl
group, and is designated tRNAfmet. The initiator tRNA carrying methionine to the AUG is different from the tRNAs that carry
methionine intended for other positions in proteins. As such, the initiator tRNA is sometimes referred to as tRNAimet.
Prokaryotic initiation
In prokaryotes, the 5’ end of the mRNA is the only free end available, as transcription is tightly coupled to translation and the
entire mRNA is not transcribed before translation begins. Nevertheless, the ribosome must be correctly positioned at the 5’ end of
the messenger RNA in order to initiate translation. How does the ribosome “know” exactly where to bind in the 5’UTR of the
mRNA?
Shine-Dalgarno sequence
Examination of the sequences upstream of the start codon in prokaryotic mRNAs reveals that there is a short purine-rich sequence
ahead of the start codon that is crucial to recognition and binding by the small ribosomal subunit (Figure 7.89). This sequence,
called the Shine-Dalgarno sequence, is complementary to a stretch of pyrimidines at the 3’ end of the 16S rRNA component of the
small ribosomal subunit (Figure 7.90). Base-pairing between these complementary sequences positions the small ribosomal subunit
at the right spot on the mRNA, with the AUG start codon at the P-site.
Initiation factors
The binding of the small ribosomal subunit to the mRNA requires the assistance of three protein factors called Initiation Factors 1,
2 and 3 (IF1, IF2, IF3). These proteins, which are associated with the small ribosomal subunit, are necessary for its binding to
mRNA, but dissociate from it when the 50S ribosomal subunit binds. Of these proteins, IF3 is important for the binding of the
small subunit to the mRNA, while IF2 is involved in bringing the initiator tRNA to the partial P-site of the small ribosomal subunit.
IF1 occupies the A-site, preventing the binding of the initiator tRNA at that site.
Once the small ribosomal subunit is bound to the mRNA and the initiator tRNA is positioned at the P-site, the large ribosomal
subunit is recruited and the initiation complex is formed. Binding of the 50S ribosomal subunit is accompanied by the dissociation

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of all three initiation factors. The removal of IF1 from the A-site on the ribosome frees up the site for the binding of the charged
tRNA corresponding to the second codon (Figure 7.91).
Eukaryotic initiation
In eukaryotes, initiation follows a similar pattern, although the order of events and the specific initiation factors are different.
Eukaryotes have a large number of IFs that are known as eIFs (eukaryotic initiation factors). These initiation factors are involved in
the binding of the initiator tRNA to the small subunit, as well as in association of the small subunit with mRNA and subsequent
attachment of the large subunit.
Ribosome assembly
The assembly of the translation machinery in eukaryotes begins with the binding of the initiator tRNA to the 40S (small) subunit.
This step requires the assistance of eIF2 and eIF3. Next the small subunit with the initiator tRNA binds to the 7-methyl G cap on
the 5'end of the mRNA. The 40S subunit then moves along the mRNA, scanning for a a start codon. Binding of the ribosomal
subunit to the mRNA is dependent not just on finding an AUG, but on the sequences surrounding the codon.
Kozak sequences
Specific sequences surrounding the AUG, called Kozak sequences for the scientist who defined them, have been shown to be
necessary for the binding of the 40S subunit, with the bases at -4 and +1 relative to the AUG being especially important (Figure
7.92). Once the small subunit is properly positioned, the large ribosomal subunit (60S) binds, forming the initiation complex.
Elongation
After the ribosome is assembled with the initiator tRNA positioned at the AUG in the P-site, the addition of further amino acids can
begin. In both prokaryotes and eukaryotes, the elongation of the polypeptide chain requires the assistance of elongation factors. In
bacteria, the binding of the second charged tRNA at the A-site requires the elongation factor EF-Tu complexed with GTP (Figure
7.93). When the charged tRNA has been loaded at the A-site, EF-Tu hydrolyzes the GTP to GDP and dissociates from the
ribosome. The free EF-Tu can then work with another charged tRNA to help position it at the A-site (Figure 7.94), after exchanging
its GDP for a new GTP.
The corresponding step in eukaryotic cells is dependent on the elongation factor eEF1α.GTP. Once both P-site and A-site are
occupied, the methionine carried by the tRNA in the P-site is joined to the amino acid carried by tRNA in the A-site, forming a
peptide bond.
The reaction that joins the amino acids occurs in the ribosomal peptidyl transferase center, which is part of the large ribosomal
subunit (Figure 7.95).
Ribozyme
Interestingly, there is strong evidence that this reaction is catalyzed by rRNA components of the large subunit, making the
formation of peptide bonds an example of the activity of RNA enzymes, or ribozymes. The result of the peptidyl transferase
activity is that the tRNA in the A-site now has two amino acids attached to it, while the tRNA at the P-site has none. This “empty”
or deacylated tRNA is moved to the E-site on the ribosome, from which it can exit. The tRNA in the A-site, then moves to occupy
the vacated P-site, leaving the A-site open for the next incoming charged tRNA. Yet another elongation factor, EF-G complexed
with GTP, is required for the translocation of the ribosome along the mRNA in bacteria, while in eukaryotes, this role is played by
eEF2.GTP. Repeated cycles of these steps result in the elongation of the polypeptide by one amino acid per cycle, until a
termination, or stop codon is in the A-site.
Termination
When a stop codon is in the A-site, proteins called release factors (RFs) are needed to recognize the stop codon and cleave and
release the newly made polypeptide. In bacteria, RF1 is a release factor that can recognize the stop codon UAG, while RF2
recognizes UGA. Both RF1 and RF2 can recognize UAA. A third release factor, RF3, works with RF1 and RF2 to hydrolyze the
linkage between the polypeptide and the final tRNA, to release the newly synthesized protein. This is followed by the dissociation
of the ribosomal subunits from the mRNA, ending the process of translation .
Polypeptide processing
What happens to the newly synthesized polypeptide after it is released from the ribosome? As you know, functional proteins are not
simply strings of amino acids. The polypeptide must fold properly in order to perform its function in the cell. It may also undergo a

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variety of modifications such as the addition of phosphate groups, sugars, lipids, etc. Some proteins are produced as inactive
precursors that must be cleaved by proteases to be functional.
An additional challenge in eukaryotic cells is the presence of internal, membrane-bounded compartments. Each compartment
contains different proteins with different functions. But the vast majority of proteins in eukaryotic cells are made by ribosomes, free
or membrane-bound, in the cytoplasm of the cell (the exceptions are a handful of proteins made within mitochondria and
chloroplasts).
Delivery
Each of the thousands of proteins made in the cytoplasm must, therefore, be delivered to the appropriate cellular compartment in
which it functions. Some proteins are delivered to their destinations in an unfolded state, and are folded within the compartment in
which they function. Others are fully folded and may be post-translationally modified before they are sent to their cellular (or
extracellular) destinations.
Some proteins are delivered as they are being synthesized (co-translationally - see Movie 7.3) while others are sorted to their
compartments post-translationally. But, with the exception of cytosolic proteins, all proteins must cross membrane barriers, through
membrane channels or other "gates", or by transport within membrane vesicles that fuse with the membrane of the target organelle
to deliver their contents.
Folding and post-translational modifications
Proper folding of a protein into its 3-dimensional conformation is necessary for it to function effectively. As described in an earlier
chapter (HERE), the folding of a protein is largely influenced by hydrophobic interactions that result in folding of the protein in
such a way as to position hydrophobic residues in the interior, or core, of the protein, away from the aqueous environment of the
cell.
Proper folding may also involve the interaction of regions of the polypeptide that are distant from each other, so that portions of the
N-terminal region of the polypeptide may be in close proximity to parts of the C-terminus of the final folded molecule.
As a polypeptide emerges from the ribosome, however, the N-terminal region of the polypeptide may begin to fold on itself, with
adjacent parts of the chain interacting in inappropriate ways, before the entire protein has been made. This can result in misfolding
of the protein and consequent malfunction. To prevent misfolding, cells have protein chaperones, whose function is to bind to and
shield regions of polypeptides as they emerge from the ribosome, and keep them from improperly interacting with one another or
with other proteins in the vicinity, until they can fold into their correct final shape (Figure 7.98). In addition, there are classes of
chaperones that are able sequester proteins in such a way as to permit unfolding and refolding of misfolded polypeptides. These
proteins ensure that the vast majority of proteins in cells are folded into their correct, functional 3-dimensional shapes.
Protein sorting
The process by which proteins are identified as belonging to a particular compartment and then correctly delivered to that
destination is known as protein sorting. How does a cell know where a particular protein should be sent?
Proteins have "address labels" or sorting signals that indicate which cellular compartment they are destined for. Characteristic
sorting signals are found on proteins that are sent to the nucleus, the ER (Figure 7.99), the mitochondria, etc.
Signal sequences
What do these sorting signals look like?
Most sorting signals (also called signal sequences) are short stretches of amino acid sequence (that is, they are part of the amino
acid sequence of the protein). Different cellular compartments have different "address labels".
Signal sequences may be found at the N-terminal or C-terminal region of proteins, or they may be within the amino acid sequence
of the proteins. The location of the signal sequence for any given protein is fixed, however. Signal sequences for proteins to be
delivered to the endoplasmic reticulum (ER) are found at the N-terminus of the protein. Mitochondrial and chloroplast proteins
encoded by nuclear genes also have N-terminal signal sequences. Signal sequences for nuclear proteins, by contrast, are internal to
the polypeptide, and may consist of one or more stretches of amino acids that will be displayed on the surface of these proteins
once they are folded.
Free and membrane-bound ribosomes

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Proteins are synthesized by ribosomes in the cytoplasm or by those that associate with membranes temporarily (membrane-bound
ribosomes). The free ribosomes make proteins that are destined for the nucleus, as well as those going to chloroplasts, mitochondria
and peroxisomes. Nuclear proteins are delivered in their folded state, while chloroplast and mitochondrial proteins are threaded
through translocation channels in the membranes of these organelles, to be folded at their destination.
Proteins that are destined for the ER, the Golgi apparatus, lysosomes as well as those that are to be secreted from the cell are first
delivered to the ER by ribosomes that associate with the membrane of the rough ER and synthesize the protein directly into the ER.
Proteins delivered by this manner into the lumen of the ER undergo folding and modification in the ER. All proteins delivered to
the ER, regardless of their final destination, have an N-terminal ER signal sequence of 15-30 amino acids.

Protein delivery into the endoplasmic reticulum

The N-terminal part of a protein is the first part of a nascent polypeptide that emerges from the ribosome (Figure 7.100). The
sequence of amino acids in this region, if it is an ER signal, will be recognized and bound by a ribonucleoprotein complex called
the Signal Recognition Particle (SRP). Binding of the SRP to the N-terminal signal sequence causes translation to pause. The SRP,
in turn, is bound by an SRP receptor in the ER membrane (Movie 7.3 & Figure 7.101), effectively anchoring the ribosome to the
membrane.
The location of SRP receptors near membrane channels in the ER positions the ribosome over a translocation channel. Once the
ribosome is docked over the channel, the SRP releases the signal sequence, which is threaded through the channel, with its
hydrophobic residues interacting with the hydrophobic interior of the membrane. Translation resumes at this point and the rest of
the protein is delivered into the lumen of the ER as it is made. The ribosome remains associated with the ER membrane till
translation is completed, at which point it dissociates. The signal sequence, which is no longer needed once the protein has been
delivered, is cleaved off by a membrane associated signal peptidase, releasing the completed protein into the ER lumen.
While soluble proteins are delivered into the ER, integral membrane proteins do not pass all the way through, but, instead, are
anchored in the membrane of the ER by hydrophobic stop transfer sequences.
Folding and modification
Proteins in the lumen of the ER are folded with the help of numerous chaperones resident in the endoplasmic reticulum. The
environment within the ER lumen is also more oxidizing than the cytosol, and permits the formation of disulfide bonds to stabilize
the folded proteins. Protein disulfide isomerase, an enzyme active in the ER lumen both helps to make disulfide bonds and removes
bonds that were incorrectly made during the folding process. In addition, proteins in the ER undergo modifications such as
glycosylation and addition of glycolipids. Multimeric proteins are also assembled from their subunits in the ER.
Proteins that have been correctly folded and modified are transported from the ER, in membrane vesicles, to their final destinations.
Improperly folded proteins are recognized by a surveillance mechanism in the ER and are sent back to the cytoplasm to be
degraded in proteasomes.
Information Processing: Translation
779
YouTube Lectures
by Kevin
HERE & HERE
780
Figure 7.81 - The standard genetic code
Wikipedia
Figure 7.80 - The central dogma in a bacterial cell
Wikipedia
781
Figure 7.82 - Coding in DNA, transcribed to RNA, translated to protein
782

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Figure 7.83 - tRNA - 3D projection (left) and 2D projection (inset)
Wikipedia
783
Interactive 7.1 - Phenylalanyl-tRNA
PDB
Interactive 7.1 - Phenylalanyl-tRNA PDB
Figure 7.84 - Charging of a tRNA by aminoacyl tRNA synthetase
Wikipedia
784
Figure 7.85 - Codons in mRNA pair with anticodons on tRNA to bring the appropriate amino acid to the ribosome for polypeptide
assembly
YouTube Lectures
by Kevin
HERE & HERE
785
Figure 7.86 - The A, P, and E sites in a ribosome
Image by Martha Baker
Figure 7.87 - Overview of elongation
Wikipedia
Interactive Learning
Module
HERE
786
Movie 7.1 - 30S ribosomal subunit
Wikipedia
Movie 7.1 - 30S ribosomal subunit Wikipedia
Table 7.1 - Location and function of rRNAs.
rRNA Name
Prokaryotes
Eukaryotes
Function
5S
Large Subunit
Large Subunit
tRNA binding?
5.8S
Large Subunit
Translocation?
16S

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Small Subunit
mRNA alignment
18S
Large Subunit
mRNA alignment
23S
Large Subunit
Peptide bond formation
28S
Large Subunit
Peptide bond formation
787
Figure 7.89 - Conserved sequences adjacent to start codons for various bacterial genes
Image by Martha Baker
Figure 7.88 - Structure of 5S rRNA
788
Figure 7.90 - Base pairing between the Shine-Dalgarno sequence in the mRNA and the 16S rRNA
Image by Martha Baker
Movie 7.2 - Large ribosomal subunit
Wikipedia
Movie 7.2 - Large ribosomal subunit Wikipedia
789
YouTube Lectures
by Kevin
HERE & HERE
Figure 7.91 - Initiation - assembly of the ribosomal translation complex
Image by Martha Baker
790
Figure 7.92 - Kozak sequence plot showing relative abundance of bases surrounding the AUG (ATG) start codon of human genes
Figure 7.93 - EF-Tu (blue) bound to tRNA (red) and GTP (yellow)
791
Figure 7.94 - The process of elongation
Image by Martha Baker
792
Figure 7.95 - 50S ribosomal subunit. RNA in brown. Protein in blue. Peptidyl transferase site in red.
Wikipedia
Interactive Learning
Module

7.7.8 https://bio.libretexts.org/@go/page/7848
HERE
793
Figure 7.96 - The process of translation
Wikipedia
Movie 7.3 Translation of a protein secreted into the endoplasmic reticulum. Small subunit in yellow. Large subunit in green. tRNAs
in blue.
Wikipedia
Movie 7.3 Translation of a protein secreted into the endoplasmic reticulum. Small subunit in yellow. Large subunit in green. tRNAs
in blue. Wikipedia
794
Figure 7.97 - Another perspective of translation. The 3’ end of the mRNA is on the left and the ribosome is moving from right to
left
795
Figure 7.98 - Action of chaperone to facilitate proper folding of a protein (orange)
Image by Aleia Kim
YouTube Lectures
by Kevin
HERE & HERE
796
Figure 7.99 - Rough (ribosome bound) and smooth endoplasmic reticulum
Wikipedia
797
Figure 7.100 - N-terminal signal sequence (green) emerging from the ribosome.
798
Figure 7.101 - Translation of a protein into the endoplasmic reticulum
Image by Aleia Kim
799
Graphic images in this book were products of the work of several talented students. Links to their Web pages are below
Click HERE for
Martha Baker’s
Web Page
Click HERE for
Pehr Jacobson’s
Web Page
Click HERE for
Aleia Kim’s
Web Page
Click HERE for
Penelope Irving’s

7.7.9 https://bio.libretexts.org/@go/page/7848
Web Page
Problem set related to this section HERE
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Email Kevin Ahern / Indira Rajagopal / Taralyn Tan
801
Translation
To the tune of “Maria” (from “West Side Story”)
Metabolic Melodies Website HERE
Translation
The most intricate thing I ever saw
From five prime to three prime, translation, translation
The final step that we know about the central dog-ma
Amino, carboxyl, translation, translation. . . .
Translation, translation, translation . .
Translation!

I just learned the steps of translation

And all the things they say
About tRNA
Are true
Translation!
To form peptide bonds in translation
The ribosomal cleft
Must bind to an E-F
tee-you!
Translation!
A-U-G binds the f-met's cargo
16S lines up Shine and Dalgarno
Translation
I'll never stop needing translation

7.7.10 https://bio.libretexts.org/@go/page/7848
The most intricate thing I ever saw
Translationnnnnnnnnnnnnnnnnn
Recording by Tim Karplus
Lyrics by Kevin Ahern
Recording by Tim Karplus Lyrics by Kevin Ahern
802
Good Protein Synthesis
To the tune of “Good King Wenceslaus”
Metabolic Melodies Website HERE
Amino acids cannot join
By themselves together
They require ribosomes
To create the tether
All the protein chains get made
‘Cording to instruction
Carried by m-R-N-A
In peptide bond construction
Small subunit starts it all
With initiation
Pairing up two RNAs
At the docking station
Shine Dalgarno’s complement
In the 16 esses
Lines the A-U-G up so
Synthesis commences
Elongation happens in
Ribosomic insides
Where rRNA creates
Bonds for polypeptides
These depart the ribosome
Passing right straight through it
In the tiny channels there
Of the large subunit
Finally when the sequence of
One of the stop codons
Parks itself in the A site
Synthesis can’t go on
P-site RNA lets go
Of what it was holding

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So the polypeptide can
Get on with its folding
Recording by David Simmons
Lyrics by Kevin Ahern
Recording by David Simmons Lyrics by Kevin Ahern

This page titled 7.7: Translation is shared under a CC BY-NC-SA license and was authored, remixed, and/or curated by Kevin Ahern, Indira
Rajagopal, & Taralyn Tan.

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7.8: Gene Expression
Source: BiochemFFA_7_7.pdf. The entire textbook is available for free from the authors at
http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy
The processes of transcription and translation described so far tell us what steps are involved in the copying of information from a
gene (DNA) into RNA and the synthesis of a protein directed by the sequence of the transcript (Figure 7.102). These steps are
required for gene expression, the process by which information in DNA directs the production of the proteins needed by the cell.
But what determines whether a gene is expressed at a given time? Cells do not, as we know, express all of their genes all of the
time. Some genes are expressed in particular cell types but not others, while others may be expressed at specific stages of
development. Cells must also be able alter their patterns of gene expression in response to internal and external cues, controlling
the production of proteins as needed, to meet their changing needs. Regulating gene expression is, therefore, crucial. Given that
there are multiple steps involved in gene expression, there are several different points at which the process could be regulated. Not
surprisingly, many regulatory mechanisms are known, each acting at a different stage in the path from DNA to protein.

Regulation of Transcription
The first step in gene expression is transcription, so regulation of transcription is an obvious way to affect whether a gene is
expressed and to what extent.
What are the molecular switches that turn transcription on or off? Although there are additional factors that affect transcription,
such as the accessibility of a gene to the transcriptional machinery, the basic mechanism by which transcription is regulated
depends on highly specific interactions between transcription regulating proteins and regulatory sequences on DNA.
What are these regulatory sequences and what proteins bind them? In addition to the promoter sequences required for transcription
initiation, genes have additional cis regulatory sequences (sequences of DNA on the same DNA molecule as the gene) that control
when a gene is transcribed. Regulatory sequences are bound tightly and specifically by transcriptional regulators, proteins that can
recognize DNA sequences and bind to them. The binding of such proteins to the DNA can regulate transcription by preventing or
increasing transcription from a particular promoter.

Transcriptional regulation in prokaryotes

Let us first consider some examples from prokaryotes. In bacteria, genes are often clustered in groups, such that genes that need to
be expressed at the same time are next to each other and all of them are controlled as a single unit by the same promoter. Groups of
genes that are coordinately regulated by a single promoter are referred to as operons. The entire set of genes in an operon can be
controlled through the action of DNA binding proteins that act as either repressors (preventing transcription of the genes) or
activators (increasing transcription of the genes). The binding of these proteins to their DNA targets is allosterically controlled by
the binding of specific small molecules that signal the state of the cell.

Induction of the lac operon

The lac operon is one such group of coordinately regulated genes that encode proteins needed for the uptake and breakdown of the
sugar lactose. E.coli cells preferentially use glucose for their energy needs, but if glucose is unavailable, and lactose is present, the
bacteria will take up lactose and break it down for energy. Since the proteins for taking up and breaking down lactose are only
needed when glucose is absent and lactose is available, the bacterial cells need a way to express the genes of the lac operon only
under those conditions. The default state of the lac operon is OFF.

Removing a repressor
Transcription of the lac cluster of genes is primarily controlled by a repressor protein that binds to a region of the DNA just
downstream of the -10 sequence of the lac promoter (Figure 7.104). Recall that the promoter is where the RNA polymerase must
bind to begin transcription. The location on the DNA where the lac repressor is bound is called the operator (Figure 7.105). When
the repressor is bound at this position, it physically blocks the RNA polymerase from transcribing the genes, just as a vehicle
blocking your driveway would prevent you from pulling out. Obviously, if you want to leave, the vehicle that is blocking your path
must be removed. Likewise, in order for transcription to occur, the repressor must be removed from the operator to clear the path
for RNA polymerase (Figure 7.106).

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How is the repressor removed? When the sugar lactose is present, a small amount of it is taken up by the cells and converted to an
isomeric form, allolactose (Figure 7.107). Allolactose binds to the repressor, changing its conformation so that it no longer binds to
the operator. When the repressor is no longer bound to the operator, the "road-block" in front of the RNA polymerase is removed,
permitting the transcription of the genes of the lac operon
What makes this an especially effective control system is that the genes of the lac operon encode proteins that enable the break
down of lactose. Turning on these genes requires lactose to be present. Once the lactose has been broken down, the lac repressor
binds to the operator once more and the lac genes are no longer expressed. This allows the genes to be expressed only when they
are needed.
Recruiting RNA polymerase
But how do glucose levels affect the expression of the lac genes? We noted earlier that if glucose was present, lactose would not be
used. A second level of control is exerted by a protein called Catabolite Activator Protein (CAP - Figure 7.108)). CAP (also
sometimes called CBP or cAMP binding protein) binds to a site adjacent to the promoter and is necessary to recruit RNA
polymerase to bind the lac promoter.
cAMP binding
CAP binds to its site only when glucose levels are low. Low glucose levels are linked to the activation of an enzyme, adenylate
cyclase, that makes the molecule cyclic AMP (cAMP). The binding of cAMP to the CAP causes a conformational change in CAP
that allows it to bind to the CAP-binding site. When CAP is bound at this site, it is able to recruit RNA polymerase to bind at the
promoter, and begin transcription.
The combination of CAP binding and the lac repressor dissociating from the operator when lactose levels are high ensures
transcription of the lac operon just when it is most needed. The binding of CAP may be thought of as a green light for the RNA
polymerase, while the removal of lac repressor is like the lifting of a barricade in front of it. When both conditions are met, the
RNA polymerase transcribes the downstream genes.
Control of the trp operon by repression
The lac operon we have just described is a set of genes that are expressed only under the specific conditions of glucose depletion
and lactose availability. Other genes may be expressed unless a particular condition is met. For these genes, the default state is ON.
An example of this is the trp operon, which encodes enzymes necessary for the synthesis of the amino acid tryptophan. These genes
are constitutively expressed (always on), except when tryptophan is available from the cell's surroundings, making its synthesis
unnecessary. Under conditions where tryptophan is abundant in the environment, the trp genes can be turned off. This is achieved
by a repressor protein that will bind to the operator only in the presence of tryptophan (Figure 7.110). Binding of tryptophan to the
repressor causes binding of the repressor to the operator. Because it acts together with the repressor to turn off the trp genes,
tryptophan is called a co-repressor.
Attenuation
Another mechanism that regulates the expression of the trp operon is attenuation. Attenuation is a process by which the expression
of an operon is controlled by termination of transcription before the first gene of the operon (Figure 7.111).
In the trp operon, this functions as follows: Transcription begins some distance upstream of the first gene in the operon, producing
what is termed a 5’ leader sequence. This leader sequence contains an intrinsic terminator that can form a hairpin structure that
stops transcription when high levels of tryptophan are available to the cells. It can also form a different structure that permits
continued transcription of the genes in the operon when tryptophan levels are low. How does the level of tryptophan influence
which of these two structures are formed?
Recall that the 5’ end of the RNA is the first part of the transcript to be made and that in bacteria translation is linked to
transcription, so the 5’ end of the RNA begins to be translated before the entire transcript is made. It turns out that the 5’ leader
sequence of the trp operon mRNA encodes a short peptide that contains two tryptophan codons. If there is plenty of tryptophan
available, the leader sequence will be easily translated. Under these conditions, the leader sequence is able to form the termination
hairpin, preventing the transcription of the downstream trp genes.
If, however, levels of tryptophan are low, then the ribosome stalls as it attempts to translate the leader sequence. Under these
conditions, the leader sequence adopts a different conformation that permits continued transcription of the genes of the trp operon.
Riboswitches

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Similar in concept to the attenuation of the trp operon described above, but not dependent on translation, is a control mechanism
called a riboswitch (Figure 7.113). Riboswitches are typically found in the 5'UTR of messenger RNAs (i.e., they are part of the
sequence of the RNA). These sequences can control transcription of the downstream genes based on the conformation they adopt.
One conformation allows continued transcription, while the other terminates it. So, what determines which conformation they
adopt?
Features
Riboswitches have two characteristic features that are important for their function. One is a region of the sequence called an
aptamer, which folds into a three-dimensional shape that can bind a small effector molecule. The other is an adjacent region of the
RNA, called the expression platform, that can fold into different conformations depending on whether or not the aptamer is bound
to the effector.
An example of a riboswitch found in bacteria is the guanine riboswitch, which controls the expression of genes required for purine
biosynthesis. The aptamer region of this riboswitch binds to the effector, guanine, when levels of the base are high. The binding of
the guanine triggers a change in the folding of the downstream expression platform, causing it to adopt a conformation that
terminates transcription of the genes needed for the synthesis of guanine. In the absence of guanine, the expression platform
assumes a different conformation that allows transcription of the purine biosynthesis genes. Thus, levels of guanine can be sensed
and the genes needed for its synthesis can be expressed as needed.
Regulation of transcription in eukaryotes
Transcription in eukaryotes is also regulated by the binding of proteins to specific DNA sequences, but with some differences from
the simple schemes outlined above.
For most eukaryotic genes, general transcription factors and RNA polymerase (i.e., the transcription initiation complex) are
necessary but not sufficient for high levels of transcription. Promoter-proximal DNA sequences like the CAAT box and GC box
bind proteins that interact with the transcription initiation complex, influencing its formation (Figure 7.114).
Distant regulatory sequences
Additional regulatory sequences called enhancers and the proteins that bind to them are needed to achieve high levels of
transcription. Enhancers are short DNA sequences that regulate the transcription of genes, but may be located at a distance from the
gene they control (although they are on the same DNA molecule as the gene). Often enhancers are many kilobases away on the
DNA, either upstream or downstream of the gene. As the name suggests, enhancers can enhance (increase) transcription of a
particular gene. How can a DNA sequence far from the gene being transcribed affect the level of transcription?
Transcriptional activators
Enhancers work by binding proteins (transcriptional activators) that can, in turn, interact with the proteins bound at the promoter.
The enhancer region of the DNA, with its associated transcriptional activator(s) can come in contact with the transcription initiation
complex that is bound at a distant site by looping of the DNA (Figure 7.115). This allows the protein bound at the enhancer to
make contact with the proteins in the basal transcription complex. The interaction of the activator with the transcription initiation
complex may be direct, or it may be through a “middle-man”, a protein complex called mediator.
One effect of this interaction is to assist in recruiting proteins necessary for transcription, like the general transcription factors and
RNA polymerase to the promoter, increasing the frequency and efficiency of formation of the transcription initiation complex.
There is also evidence that at some promoters, following assembly of the transcription initiation complex, the RNA polymerase
remains stalled at the promoter. In such cases, the interaction with the transcription initiation complex of an activator bound to an
enhancer could play a role in facilitating the transition of the RNA polymerase to the elongation phase of transcription.
Chromatin remodeling proteins
Another mechanism by which activators bound at the enhancer can affect transcription is by recruiting to the promoter proteins that
can modify the structure of that region of the chromosome. In eukaryotes, DNA is packaged with proteins to form chromatin.
When the DNA is tightly associated with these proteins, it is difficult to access for transcription. So proteins that can make the
DNA more accessible to the transcription machinery can also play a role in the extent to which transcription occurs.
Silencers
In addition to enhancers, there are also negative regulatory sequences called silencers. Such regulatory sequences bind to
transcriptional repressor proteins. Like the transcriptional activators, these repressors work by interacting with the transcription

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initiation complex. In the case of repressors, the effect they have on the transcription initiation complex is to reduce transcription.
DNA binding proteins
Transcriptional activators and repressors are modular proteins- they have a part that binds DNA and a part that activates or
represses transcription by interacting with the transcription initiation complex (Figure 7.118). The DNA binding domain is the part
of the protein that confers specificity for determining which gene(s) will be activated or repressed. The activation domain is the
part of the protein that stimulates or represses transcription. The DNA binding domains of transcriptional activators form
characteristic structures that recognize their target DNA sequences by making contacts with bases, usually in the major groove of
the DNA helix. It is possible to engineer hybrid transcription factors that combine the DNA binding domain of one activator with
the activation domain of another. Such proteins retain the specificity dictated by the DNA binding domain. Truncated transcription
factors can also be generated that have their DNA binding domain but lack the activation domain. Such transcription factors can be
useful tools in studying transcriptional regulation because their DNA binding domains can compete with the endogenous
transcription factors for regulatory binding sites without increasing transcription from the target promoters.
Multiple factors
The description above may suggest that each gene in eukaryotes is controlled by the binding of a single transcriptional activator or
repressor to a particular enhancer or silencer site. However, it turns out that the transcription of any given gene may be
simultaneously regulated by a combination of proteins, both activators and repressors, bound at multiple regulatory sites on the
DNA, all of which interact with the transcription initiation complex. The combinatorial nature of such regulation provides great
versatility, with different combinations of regulatory elements and proteins working together in response to a wide variety of
conditions and signals.
The mechanisms described so far have focused on the sequence elements in DNA that regulate transcription through the activator
and repressor proteins bound to them. Following transcription, alternative splicing (see HERE) and editing of the transcripts can
also modify the proteins that are produced by the cell. We will now examine some of the other ways in which gene expression is
modulated in cells.
First, we will consider some so-called epigenetic mechanisms that affect gene expression. The term epigenetics derives from epi
(above, or on top of) and genetic (of genes) and refers to the fact that these mechanisms act in addition to, or overlaid on, the
information in the gene sequences. Two such epigenetic mechanisms are the covalent modifications of histones in chromatin and
the methylation of DNA sequences.
Histone modification
As noted earlier, transcription in eukaryotes is complicated by the fact that the DNA is packaged with histones to make chromatin.
This means that for a gene to be transcribed, the relevant regions of the chromatin must be opened up to allow access to the RNA
polymerase and transcription factors. This provides another potential point of control of gene expression. Chromatin remodeling
factors, mentioned earlier, assist in reorganizing the nucleosome structure at regions that need to be made accessible.
But what determines that a given region of the chromatin will be acted upon by the remodeling complexes? Transcriptional
activator proteins bound at enhancers, sometimes work by recruiting histone modifying enzymes to the promoter region. An
example of such a modifying enzyme is histone acetyl transferase (HAT) that works to acetylate specific amino acid residues in the
tails of the histones forming the nucleosome core (Figures 7.119 & 7.120). Acetylation of histones is thought to be responsible for
loosening the interaction between histones and the DNA in nucleosomes and helps to make the DNA more readily accessible for
transcription. The opposite effect may be achieved if the enzymes recruited are histone deacetylases (HDAC) which remove acetyl
groups from the tails of the histones in the nucleosome, and lead to tighter packing of the chromatin.
Writers, readers and erasers
In addition to the histone acetyl transferases and the deacetylases, other enzymes may add or remove methyl groups, phosphate
groups, and other chemical moieties to specific amino acid side chains on the histone tails. The patterns of these covalent
modifications, sometimes called the histone code, are established by the so-called "writers", or enzymes, such as histone
methyltransferases, that add the chemical groups on to the histone tails. Yet other enzymes, like the histone demethylases, may act
as "erasers," removing the chemical groups added by the "writers." The histone code is interpreted by "readers," proteins that bind
to specific combinations of the modifications and assist in either silencing the expression of genes in the vicinity or making the
region more transcriptionally active.
DNA methylation

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Gene expression can also be regulated by methylation of the other component of chromatin - DNA. Enzymes called DNA
methyltransferases (DNMTs) catalyze the covalent addition of a methyl group to C5 of cytosines in DNA. Patterns of cytosine
methylation vary in different organisms, with methylation concentrated in some parts of the genome in some groups and scattered
throughout the genome in others. In vertebrates, the cytosines that are methylated are generally next to a guanine (the CG
dinucleotide is commonly abbreviated as CpG). Methylation of DNA seems to correlate with gene silencing while demethylation is
associated with increased transcription (Figure 7.121).
How does methylation of the DNA at CpG sites regulate gene expression? Although the extent of DNA methylation near promoters
has been observed to correlate with gene silencing, it is not clear how exactly methylation brings about this effect. It has been
suggested that methylation could block the binding of proteins necessary for transcription. Methylation at enhancer sites might also
prevent the binding of transcriptional activators to them.
Another interesting observation is that certain proteins that bind to methylated CpG sites also seem to interact with histone
deacetylases. As noted above, histones deacetylases remove acetyl groups from histones, and promote tighter packing of chromatin
and transcriptional silencing. Thus, methylation on DNA likely works in combination with histone modification to affect gene
expression.
Regulatory RNAs
One of the most unexpected discoveries in the past few decades has been the role that RNAs play in regulating gene expression.
The classic view that RNA either encoded proteins (mRNA) or assisted in their synthesis (rRNA and tRNA) is now known to be a
vast underestimate of the various ways in which RNAs function in gene expression. It is now clear that regulatory RNAs have
widespread and significant effects on gene expression, a realization that has revolutionized our understanding of gene regulation.
What are some of the ways in which regulatory RNAs function to modulate the expression of genes?
Small regulatory RNAs
MicroRNAs (miRNAs) and Short Interfering RNAs (siRNAs) are small, non-coding RNAs that act at the post-transcriptional level
to regulate gene expression (Figure 7.123 & 7.124). These RNAs appear to silence genes by base-pairing with target mRNAs and
marking them for degradation, or by blocking their translation. The functional forms of both miRNAs and siRNAs are from 20-30
nucleotides long and are derived by processing from longer primary transcripts. Mature miRNAs and siRNAs work in association
with a class of proteins called Argonaute proteins to form a gene silencing complex.
MicroRNAs are transcribed from specific genes by RNA polymerase II. The primary transcript, known as a pri-miRNA folds on
itself to form double-stranded hairpin structures that are cleaved by an RNase in the nucleus called Drosha. The products of Drosha
cleavage, double-stranded RNAs of roughly 60-70 nucleotides known as pre-miRNAs, are exported to the cytoplasm, where they
are further processed into the small 20-30 nucleotide lengths of mature double-stranded miRNAs by an enzyme known as Dicer.
The RNA duplexes of miRNAs are not perfectly matched, and have loops and mismatches (Figure 7.124).
siRNAs also derive from double-stranded RNAs, but these may arise from either endogenous or exogenous sources (such as
viruses). These double-stranded RNAs are processed in the cytoplasm by the same enzyme, Dicer, that generates the mature
miRNAs, to produce the small, 20-30 nucleotide double-stranded RNAs.
In contrast to miRNAs, the mature siRNAs are perfectly base-paired along their lengths.
RISC assembly
Both miRNAs and siRNAs then are assembled with Argonaute proteins to form a silencing complex called RISC (RNA-induced
silencing complex). Recall that both miRNAs and siRNAs are, at this point double-stranded. One strand of the RNA is referred to
as the guide RNA, while the other is called the passenger RNA.
During the process of loading the RNA onto the Argonaute protein, the guide strand of the RNA remains associated with the
protein, while the passenger strand is removed. The guide RNA associated with the Argonaute protein is the functional gene
silencing complex (Figure 7.125).
Sequence specific base-pairing of the guide RNA with an mRNA leads to either the degradation of the mRNA by the Argonaute
protein (in the case of the siRNAs) or in suppression of translation of the mRNA (for miRNAs). The extent to which these
processes play a role in regulating gene expression is impressive. The expression of at least a third of all human genes has already
been shown to be modulated by miRNAs, demonstrating clearly that these RNAs play a major role in gene regulation.
Long noncoding RNAs

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Long noncoding RNAs (lncRNAs) are RNAs of greater than 200 nucleotides that do not code for proteins. Some of these RNAs are
derived from intron sequences, while others, transcribed from intergenic regions form a subset of lncRNAs called lincRNAs (long
intergenic noncoding RNAs). Yet other lncRNAs are produced as antisense transcripts of coding genes. An astounding 30,000
transcripts in humans are thought to be lncRNAs, but little is known of their function. From the few lncRNAs that have been
intensively studied, it is evident that they do not all function in the same way. However, they appear to affect gene expression in a
variety of ways including modification of chromatin structure, regulation of splicing, or serving as structural scaffolds for the
assembly of nucleoprotein complexes. Additional mechanisms will doubtless be uncovered as these fascinating RNAs are
investigated in years to come.
Regulation of translation
The synthesis of proteins is dependent on the availability of the mRNAs encoding them. If an mRNA is blocked at its 5' end, it
cannot be translated. The rate of degradation of an mRNA will influence how long it is around to direct the synthesis of the protein
it codes for. Gene expression can also, therefore, be regulated by mechanisms that alter the rate of mRNA degradation. Regulation
of translation is used to control the production of many proteins. Two examples, ferritin and the transferrin receptor, are important
for iron storage and transport in cells. Ferritin is an iron-binding protein that sequesters iron atoms in cells to keep them from
reacting. When iron levels are high, there is a need for more ferritin than when iron levels are low. How are ferritin levels
regulated? The 5'UTR of the ferritin mRNA contains a 28-nucleotide sequence called the Iron Response Element, or IRE (Figure
7.127). When iron levels are low, the IRE is bound by a protein. The presence of the IRE-binding protein at the 5'UTR blocks
translation of the ferritin mRNA. However, if iron levels are high, the iron binds to the IRE-binding protein, which undergoes a
conformational change and dissociates from the IRE. This frees up the 5' end of the ferritin mRNA for ribosome assembly and
translation, producing more ferritin.
The other protein involved in iron transport, the transferrin receptor, is required for uptake of iron into cells, when intracellular iron
levels are low. In the case of the transferrin receptor, it is when iron levels are low that more of it is needed. When iron levels are
high, there is no need to make more transferrin receptor. The mRNA encoding the transferrin receptor also has IRE sequences, but
in this case, the IRE is situated in the 3'UTR of the transcript (Figure 7.128). The IRE is, as in the case of ferritin, bound by the
IRE-binding protein. When iron levels in the cell are high, the iron binds the IRE-binding protein, which dissociates from the IRE.
This leaves the 3'UTR susceptible to attack by RNases, leading to degradation of the transferrin receptor mRNA. At times when
iron levels are low, the IRE-binding protein remains bound to the 3' UTR of the mRNA, stabilizing it and permitting more
transferrin receptor to be made by translation.
Gene expression is controlled at many steps
As can be seen from the examples in this section, regulation of gene expression in eukaryotic cells is a function of multiple
mechanisms that act at different stages in the flow of information from DNA to protein, responding to the internal state of the cell
as well as external conditions and signals.
Information Processing: Gene Expression
803
YouTube Lectures
by Kevin
HERE & HERE
804
Figure 7.102 - Multiple levels of control of gene expression
Wikipedia
805
Figure 7.103 - Prokaryotic genes organized in an operon
Wikipedia
Figure 7.104 - Protein binding sites in the lac regulatory region
Image by Martha Baker

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Interactive Learning
Module
HERE
806
Figure 7.105 - Lac operon structure and products
Image by Martha Baker
Figure 7.106 - Lac operon in the absence (middle) and presence (bottom) of inducer
Image by Martha Baker
807
Figure 7.107 - Allolactose (top) and lactose (bottom)
Figure 7.108 - CAP (blue) bound to the DNA adjacent to the lac promoter (orange). cAMP shown in pink.
Wikipedia
Figure 7.109 - Lac operon in the presence (top) and absence (bottom) of glucose
Image by Martha Baker
808
Figure 7.110 - Structure and regulation of the trp operon
Wikipedia
YouTube Lectures
by Kevin
HERE & HERE
809
Figure 7.111 - Attenuation in regulation of the trp operon
Wikipedia
Figure 7.112 - Sequence of the leader region of the trp operon
XX AUG AAA GCA AUU UUC GUA CUG AAA GGU UGG UGG CGC ACU UCC UGA -XX
MET LYS ALA ILE PHE VAL LEU LYS GLY TRP TRP ARG THR SER STOP
810
Figure 7.113 - Riboswitch features
811
Figure 7.114 - Regulatory sequences for a eukaryotic gene
Wikipedia
812
Figure 7.115 - DNA looping allows contact between activator bound at a distant enhancer and the basal transcription complex
Image by Martha Baker
813
Figure 7.116 - Transcription factors in regulation of eukaryotic transcription
Wikipedia
YouTube Lectures

7.8.7 https://bio.libretexts.org/@go/page/8565
by Kevin
HERE & HERE
814
Figure 7.117 - Binding of c-myc protein to its target DNA sequence
Wikipedia
Figure 7.118 Activators bound at multiple sites can regulate transcription from a given promoter
OpenStax
815
Figure 7.119 - Transcriptional activation (right) and deactivation (left) by histone modification
Wikipedia
816
Figure 7.120 - Chromatin configuration affects transcription
Wikipedia
Interactive Learning
Module
HERE
817
Figure 7.121 - Inactivation of transcription by CpG methylation
Image by Indira Rajagopal
YouTube Lectures
by Kevin
HERE & HERE
818
Figure 7.122 - Epigenetic changes through histone and DNA modification
819
Figure 7.123 - miRNAs function in the regulation of gene expression
Wikipedia
Figure 7.124 Pre-miRNA hairpin structures with the mature guide miRNAs shown in red
Wikipedia
820
Figure 7.125 - Gene silencing by siRNA
Image by Pehr Jacobson
821
Figure 7.126 - Processed siRNA duplex with perfect base-pairing, 5’ phosphates and two bases overhanging at each 3’ end
822
Figure 7.127 -Regulation of ferritin mRNA translation
Image by Aleia Kim
YouTube Lectures

7.8.8 https://bio.libretexts.org/@go/page/8565
by Kevin
HERE & HERE
823
Figure 7.128 -Regulation of transferrin receptor mRNA translation
Image by Aleia Kim
Graphic images in this book were products of the work of several talented students. Links to their Web pages are below
Click HERE for
Martha Baker’s
Web Page
Click HERE for
Pehr Jacobson’s
Web Page
Click HERE for
Aleia Kim’s
Web Page
Click HERE for
Penelope Irving’s
Web Page
Problem set related to this section HERE
Point by Point summary of this section HERE
To get a certificate for mastering this section of the book, click HERE
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Email Kevin Ahern / Indira Rajagopal / Taralyn Tan
God Bless These Complexes
To the tune of “God Bless America”
Metabolic Melodies Website HERE
All information in
Cells’ DNA

7.8.9 https://bio.libretexts.org/@go/page/8565
Just increases
With pieces
Mixed and matched in the mRNAs
Linking exons
All together
Using snurps in
Complex-ES
God bless the spliceosomes
And trans-crip-tomes
(slow and loud) God bless the spliceosomes
And my ge-nome
Your blueprint info is
In DNA
Since you need it
Proofread it
Or you’ll mutate the mRNA
You can translate
All the codons
With the cells’ gen-
et-ic code
God bless the ribosomes
They translate code
(slow and loud) God bless the ribosomes
And proteomes
Recording by David Simmons
Lyrics by Kevin Ahern
Recording by David Simmons Lyrics by Kevin Ahern
The Book of Life
To the tune of “The Look of Love”
Metabolic Melodies Website HERE
The book of life - the stuff of dreams
Is everywhere, it seems
The book of life, is biochemistry and
Its words fill every day
Just what it says is written in the DNA
I just want to get to know it
How the info’s coded
What are all the secrets?

7.8.10 https://bio.libretexts.org/@go/page/8565
Ribosomes can read it
Goodness knows it’s needed
And so its alphabet’s
In codon forms
For ribosome bookworms
They read it right
A protein’s function to its sequence corresponds
It’s not just randomly created peptide bonds
What a marvel of creation, how they do translation
Of m-R-N-A chains,
Using bits of glycine
Proline and some lysine
Translate the code
Instrumental
I just marvel at the knowledge
That I got in college
To learn all the secrets
Double helix spaces
Complementary bases
Pyrimidines
Paired to purines
The book of life
Recording by Carol Adriane Smith
Lyrics by Kevin Ahern
Recording by Carol Adriane Smith Lyrics by Kevin Ahern

This page titled 7.8: Gene Expression is shared under a CC BY-NC-SA license and was authored, remixed, and/or curated by Kevin Ahern, Indira
Rajagopal, & Taralyn Tan.

7.8.11 https://bio.libretexts.org/@go/page/8565
7.9: Signaling
Source: BiochemFFA_7_8.pdf. The entire textbook is available for free from the authors at
http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy
Up to this point we have considered how cells carry out biochemical reactions and how they regulate the expression of the genes in
response to their internal and external environments. It is intuitively obvious that even unicellular organisms must be able to sense
features of their environment, such as the presence of nutrients, if they are to survive. In addition to being able to receive and
respond to information from the environment, multicellular organisms must also find ways by which their cells can communicate
among themselves.

Coordination
Since different cells take on specialized functions in a multicellular organism, they must be able to coordinate activities. Cells
grow, divide, or differentiate in response to specific signals. They may change shape or migrate to another location. At the
physiological level, cells in a multicellular organism, must respond to everything from a meal just eaten to injury, threat, or the
availability of a mate. They must know when to divide, when to undergo apoptosis (programmed cell death), when to store food,
and when to break it down. A variety of mechanisms have arisen to ensure that cell-cell communication is not only possible, but
astonishingly swift, accurate and reliable.
How are signals sent between cells? Like pretty much everything that happens in cells, signaling is dependent on molecular
recognition. The basic principle of cell-cell signaling is simple. A particular kind of molecule, sent by a signaling cell, is
recognized and bound by a receptor protein in (or on the surface of) the target cell. The signal molecules are chemically varied-
they may be proteins, short peptides, lipids, nucleotides or catecholamines, to name a few.

Signal properties
The chemical properties of the signal determine whether its receptors are on the cell surface or intracellular. If the signal is small
and hydrophobic it can cross the cell membrane and bind a receptor inside the cell. If, on the other hand, the signal is charged, or
very large, it would not be able to diffuse through the plasma membrane. Such signals need receptors on the cell surface, typically
transmembrane proteins that have an extracellular portion that binds the signal and an intracellular part that passes on the message
within the cell (Figure 7.130).
Receptors are specific for each type of signal, so each cell has many different kinds of receptors that can recognize and bind the
many signals it receives. Because different cells have different sets of receptors, they respond to different signals or combinations
of signals. The binding of a signal molecule to a receptor sets off a chain of events in the target cell. These events could cause
change in various ways, including, but not limited to, alterations in metabolic pathways or gene expression in the target cell.
How the binding of a signal to a receptor brings about change in cells is the topic of this section. We will examine a few of the
major receptor types and the consequences of signal binding to these receptors. Although the specific molecular components of the
various signal transduction pathways differ, they all have some features in common (Figure 7.131):
• The binding of a signal to its receptor is usually followed by the generation of a new signal(s) within the cell. The process by
which the original signal is converted to a different form and passed on within the cell to bring about change is called signal
transduction.
• Most signaling pathways have multiple signal transduction steps by which the signal is relayed through a series of molecular
messengers that can amplify and distribute the message to various parts of the cell.
• The last of these messengers usually interacts with a target protein(s) and changes its activity, often by phosphorylation.
• When a signal sets a particular pathway in motion, it is acting like an ON switch. This means that once the desired result has been
obtained, the cell must have a mechanism that acts as an OFF switch.
Understanding this underlying similarity is helpful, because learning the details of the different pathways becomes merely a matter
of identifying which molecular component performs a particular function in each individual case. We will consider several different
signal transduction pathways, each mediated by a different kind of receptor.

Ligand-gated ion channel receptors

The simplest and fastest of signal pathways is seen in the case of signals whose receptors are gated ion channels (Figure 7.132).
Gated ion channels are made up of multiple transmembrane proteins that create a pore, or channel, in the cell membrane.

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Depending upon its type, each ion channel is specific to the passage of a particular ionic species. The term "gated" refers to the fact
that the ion channel is controlled by a "gate" which must be opened to allow the ions through. The gates are opened by the binding
of an incoming signal (ligand) to the receptor, allowing the almost instantaneous passage of millions of ions from one side of the
membrane to the other. Changes in the interior environment of the cell are thus brought about in microseconds and in a single step.

Swift response
This type of swift response is seen, for example, in neuromuscular junctions, where muscle cells respond to a message from the
neighboring nerve cell (Figure 7.133). The nerve cell releases a neurotransmitter signal into the synaptic cleft, which is the space
between the nerve cell and the muscle cell it is "talking to". An example of such a neurotransmitter signal is acetylcholine. When
the acetylcholine molecules are released into the synaptic cleft, they diffuse rapidly till they reach their receptors on the membrane
of the muscle cell. The binding of the acetylcholine to its receptor, an ion channel on the membrane of the muscle cell, causes the
gate in the ion channel to open. The resulting ion flow through the channel can immediately change the membrane potential of the
cell. This, in turn, can trigger other changes in the cell.
The speed with which changes are brought about in neurotransmitter signaling is evident when you think about how quickly you
remove your hand from a hot surface. Sensory neurons carry information to the brain from your hand on the hot surface and motor
neurons signal to your muscles to move the hand, in less time than it took you to read this sentence!

Nuclear hormone receptors

The receptors for signals like steroid hormones are part of a large group of proteins known as the nuclear hormone receptor
superfamily. These receptors recognize and bind not only steroid hormones, but also retinoic acid, thyroid hormone, vitamin D and
other signals. The subset of the nuclear hormone receptors that bind steroid hormones are intracellular proteins. Steroid hormones
(Figure 7.135), as you are aware, are related to cholesterol, and as hydrophobic molecules, they are able to cross the cell membrane
by themselves. This is unusual, as most signals coming to cells are incapable of crossing the plasma membrane, and thus, must
have cell surface receptors.
Once within the cell, steroid hormones bind to their receptors, which may reside in the cytoplasm or in the nucleus. Steroid
hormone receptors are proteins with a double life: they are actually dormant transcription regulators that are inactive till a steroid
hormone binds and causes a conformational change in them. When this happens, the receptors, with the hormone bound, bind to
regulatory sequences in the DNA and modulate gene expression. Because steroid hormone receptors act by modulating gene
expression, the responses to steroid hormones are relatively slow. (There are also some effects of steroid hormones that do not
involve transcriptional regulation, but the majority work through changing gene expression.) Like other transcriptional activators,
steroid receptors have a DNA-binding domain (DBD) and an activation domain. They also have a ligand-binding domain (LBD)
that binds the hormone.

Glucocorticoid receptor
Examples of such signaling pathways are those mediated by the glucocorticoid receptor (Figures 7.136 & 7.137). Glucocorticoids,
sometimes described as stress hormones, are made and secreted by the adrenal cortex. Physiologically, they serve to maintain
homeostasis in the face of stress and exhibit strong anti-inflammatory and immunosuppressive properties. Because of these effects,
synthetic glucocorticoids are used in the treatment of a number of diseases from asthma and rheumatoid arthritis to multiple
sclerosis. All of these effects are mediated through the signaling pathway which starts with the binding of a glucocorticoid hormone
to its receptor. Recall that steroids can cross the plasma membrane, so glucocorticoids can diffuse into the cell and bind their
receptors which are in the cytoplasm.
In the absence of the signal, glucocorticoid receptors are found bound to a protein chaperone, Hsp90 (Figure 7.137). This keeps the
receptors from being transported to the nucleus. When a glucocorticoid molecule binds the receptor, the receptor undergoes a
conformational change and dissociates from the Hsp90. The receptor, then, with the hormone bound, translocates into the nucleus.
In the nucleus, it can increase the transcription of target genes by binding to specific regulatory sequences (labeled HRE for
hormone-response elements). The binding of the hormone-receptor complex to the regulatory elements of hormone-responsive
genes modulates their expression. Many of these genes encode anti-inflammatory proteins, and their increased production accounts
for the physiological effect of corticosteroid therapies.
The steroid receptor pathways are relatively simple and have only a couple of steps (Figure 7.138). Most other signaling pathways
involve multiple steps in which the original signal is passed on and amplified through a number of intermediate steps, before the

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cell responds to the signal.

Cell surface receptors

We will now take a look at two signaling pathways, each mediated by a major class of cell surface receptor- the G-protein coupled
receptors (GPCRs) and the receptor tyrosine kinases (RTKs). While the specific details of the signaling pathways that follow the
binding of signals to each of these receptor types are different, it is easier to learn them when you can see what the pathways have
in common, namely, interaction of the signal with a receptor, followed by relaying and amplification of the signal through a
variable number of intermediate molecules, with the last of these molecules interacting with a target or target proteins and
modifying their activity in the cell.

G-protein coupled receptors

G-protein coupled receptors (GPCRs) are involved in responses of cells to many different kinds of signals, from epinephrine, to
odors, to light. In fact, a variety of physiological phenomena including vision, taste, smell, and the fight-or-flight response are
mediated by GPCRs. What are G-protein coupled receptors?
G-protein coupled receptors are cell surface receptors that pass on the signals that they receive with the help of guanine nucleotide
binding proteins (a.k.a. G-proteins). Before thinking any further about the signaling pathways downstream of GPCRs, it is
necessary to know a few important facts about these receptors and the G-proteins that assist them.
Though there are hundreds of different G-protein coupled receptors, they all have the same basic structure (Figure 7.139):
They all consist of a single polypeptide chain that threads back and forth seven times through the lipid bilayer of the plasma
membrane. For this reason, they are sometimes called seven-pass transmembrane (7TM) receptors. One end of the polypeptide
forms the extracellular domain that binds the signal while the other end is in the cytosol of the cell.
When a ligand (signal) binds the extracellular domain of a GPCR, the receptor undergoes a conformational change, on its
cytoplasmic side, that allows it to interact with a G-protein that will then pass the signal on to other intermediates in the signaling
pathway.
G-proteins
What is a G-protein? As noted above, a G-protein is a guanine nucleotide-binding protein that can interact with a G-protein linked
receptor. G-proteins are associated with the cytosolic side of the plasma membrane, where they are ideally situated to interact with
the tail of the GPCR, when a signal binds to the GPCR. There are many different G-proteins, all of which share a characteristic
structure- they are composed of three subunits called α, β and γ (Figure 7.140). Because of this, they are sometimes called
heterotrimeric G proteins (hetero=different, trimeric= having three parts).
Ligand binding
The guanine nucleotide binding site is on the α subunit of the G-protein. This site can bind GDP or GTP. The α subunit also has a
GTPase activity, i.e., it is capable of hydrolyzing a GTP molecule bound to it into GDP.
In the unstimulated state of the cell, that is, in the absence of a signal bound to the GPCR, the G-proteins are found in the trimeric
form (α-β-γ bound together) and the α subunit has a GDP molecule bound to it. In this form, the α subunit is inactive. With this
background on the structure and general properties of the GPCRs and the G-proteins, we can now look at what happens when a
signal arrives at the cell surface and binds to a GPCR (Figure 7.141).

The signaling pathway

The binding of a signal molecule by the extracellular part of the G-protein linked receptor causes the cytosolic tail of the receptor to
interact with, and alter the conformation of, a G-protein associated with the inner face of the plasma membrane.
This has two consequences. First, the α subunit of the G-protein loses its GDP and binds a GTP, instead. Second, the G-protein
breaks up into the GTP-bound α part and the β-γ part.
The binding of GTP to the α subunit and its dissociation from the β-γ subunits activate the α subunit. The activated α subunit can
diffuse freely along the cytosolic face of the plasma membrane and act upon its targets. (The β-γ unit is also capable of activating
its own targets.)
What happens when G-proteins interact with their target proteins? That depends on what the target is. G-proteins interact with
different kinds of target proteins, of which we will examine two major categories:

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Ion channels
We have earlier seen that some gated ion channels can be opened or closed by the direct binding of neurotransmitters to a receptor
that is an ion-channel protein. In other cases, ion channels are regulated by the binding of G-proteins. That is, instead of the signal
directly binding to the ion channel, it binds to a GPCR, which activates a G-protein that then may cause opening of the ion channel,
either directly, by binding to the channel, or indirectly, through activating other proteins that can bind to the channel. The change in
the distribution of ions across the plasma membrane causes a change in the membrane potential.
Enzyme activation
The interaction of G-proteins with their target enzymes can regulate the activity of the enzyme, either increasing or decreasing its
activity. The change in activity of the target enzyme, in turn, results in downstream changes in other proteins in the cell, and alters
the metabolic state of the cell. This is best understood by examining the well-studied response of cells to epinephrine, mediated
through the β-adrenergic receptor, a type of G-protein coupled receptor.
Epinephrine (Figure 7.142), also known as adrenaline, is a catecholamine that plays an important role in the body's 'fight or flight'
response. In response to stressful stimuli, epinephrine is secreted into the blood, to be carried to target organs whose cells will
respond to this signal. If you were walking down a dark alley in an iffy neighborhood, and you heard footsteps behind you, your
brain would respond to potential danger by sending signals that ultimately cause the adrenal cortex to secrete epinephrine into the
blood stream. The epinephrine circulating in your system has many effects, including increasing your heart rate, but among its
prime targets are your muscle cells. The reason for this is that your muscle cells store energy in the form of glycogen, a polymer of
glucose. If you need to run or fight off an assailant, your cells will need energy in the form of glucose.
But how does epinephrine get your cells to break down the glycogen into glucose? Binding of epinephrine to the β-adrenergic
receptor on the surface of the cells causes the receptor to activate a G-protein associated with its cytoplasmic tail. As described
above, this leads to the α subunit exchanging its GDP for GTP and dissociating from the β-γ subunits. The activated α subunit then
interacts with the enzyme adenylate cyclase (also known as adenylyl cyclase) stimulating it to produce cyclic AMP (cAMP) from
ATP. Cyclic AMP is often described as a "second messenger", in that it serves to spread the signal received by the cell. How does
cAMP accomplish this?
cAMP molecules bind to, and activate an enzyme, protein kinase A (PKA - Figure 7.145). PKA is composed of two catalytic and
two regulatory subunits that are bound tightly together. Upon binding of cAMP, the catalytic subunits are released from the
regulatory subunits, allowing the enzyme to carry out its function, namely phosphorylating other proteins. Thus, cAMP can
regulate the activity of PKA, which in turn, by phosphorylating other proteins can change their activity. In this case, the relevant
protein that is activated is an enzyme, phosphorylase kinase. This enzyme can then phosphorylate and activate glycogen
phosphorylase, the enzyme ultimately responsible for breaking glycogen down into glucose-1-phosphate - readily converted to
glucose. The activation of glycogen phosphorylase supplies the cells with the glucose they need, allowing you to fight or flee, as
you might see fit. Simultaneously, PKA also phosphorylates another enzyme, glycogen synthase. In the case of glycogen synthase,
phosphorylation inactivates it, and prevents free glucose from being used up for glycogen synthesis, ensuring that your cells are
amply supplied with glucose (Figure 7.146).
Common pattern
Although the steps described above seem complicated, they follow the simple pattern outlined at the beginning of this section:
• Binding of signal to receptor
• Several steps where the signal is passed on through intermediate molecules (G-proteins, adenylate cyclase, cAMP, and finally,
PKA)
• Phosphorylation of target proteins by the kinase, leading to changes in the cell. The specific changes depend on the proteins that
are phosphorylated by the PKA.
Why so many steps? If you need to activate glycogen phosphorylase to break down glucose in a hurry, why not have a system in
which binding of a signal to the receptor directly activated the target enzyme?
The answer to this puzzle is simple: there is amplification of the signal at every step of the pathway. A single signal molecule
binding to a receptor sets in motion a cascade of reactions, with the signal getting larger at each step, so that binding of one
epinephrine molecule to its receptor results in the activation of a million glycogen phosphorylase enzyme molecules!
Turning signals off

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If the signal binding to the receptor serves as a switch that sets these events in motion, there must be mechanisms to turn the
pathway off. The first is at the level of the receptor itself. A kinase called G-protein receptor kinase (GRK) phosphorylates the
cytoplasmic tail of the receptor. The phosphorylated tail is then bound by a protein called arrestin, preventing further interaction
with a G-protein.
The next point of control is at the G-protein. Recall that the α subunit of the G-protein is in its free and activated state when it has
GTP bound, and that it associates with the β-γ subunits and has a GDP bound when it is inactive. We also know that the α subunit
has an activity that enables it to hydrolyze GTP to GDP. This GTP-hydrolyzing activity makes it possible for the α subunit, once it
has completed its task, to return to its GDP bound state, re-associate with the β-γ part and become inactive again.
A third "off switch" is further down the signaling pathway, and controls the level of cAMP. We just noted that cAMP levels
increase when adenylate cyclase is activated. When its job is done, cAMP is broken down by an enzyme called phosphodiesterase
(Figure 7.147). When cAMP levels drop, PKA returns to its inactive state, putting a halt to the changes brought about by the
activation of adenylate cyclase by an activated G-protein.
Yet another way that the effects of this pathway can be turned off is at the level of the phosphorylated target proteins. These
proteins, which are activated by phosphorylation, can be returned to their inactive state by the removal of the phosphates by
phosphatases.
Receptor tyrosine kinases
Another major class of cell surface receptors are the receptor tyrosine kinases or RTKs. Like the GPCRs, receptor tyrosine kinases
bind a signal, then pass the message on through a series of intracellular molecules, the last of which acts on target proteins to
change the state of the cell.
As the name suggests, a receptor tyrosine kinase is a cell surface receptor that also has a tyrosine kinase activity. The signal binding
domain of the receptor tyrosine kinase is on the cell surface, while the tyrosine kinase enzymatic activity resides in the cytoplasmic
part of the protein (Figure 7.148). A transmembrane α helix connects these two regions of the receptor.
What happens when signal molecules bind to receptor tyrosine kinases? Binding of signal molecules to the extracellular domains of
receptor tyrosine kinase proteins causes two receptor molecules to dimerize (come together and associate - Figure 7.149). This
brings the cytoplasmic tails of the receptors close to each other and causes the tyrosine kinase activity of these tails to be turned on.
The activated tails then phosphorylate each other on several tyrosine residues (Figure 7.150). This is called autophosphorylation.
The phosphorylation of tyrosines on the receptor tails triggers the assembly of an intracellular signaling complex on the tails. The
newly phosphorylated tyrosines serve as binding sites for a variety of signaling proteins that then pass the message on to yet other
proteins to bring about changes in the cell. Receptor tyrosine kinases mediate responses to a large number of signals, including
peptide hormones like insulin and growth factors like epidermal growth factor (EGF). We will examine how insulin and EGF act
on cells by binding to receptor tyrosine kinases.
Insulin receptor
Insulin plays a central role in the uptake of glucose from the bloodstream. It increases glucose uptake by stimulating the movement
of glucose receptor GLUT4 to the plasma membrane of cells.
How does insulin increase GLUT4 concentrations in the cell membrane? The binding of insulin to the insulin receptor (IR - Figure
7.151), results in dimerization of the receptor monomers and subsequent autophosphorylation of the cytosolic kinase domains. The
activated tyrosine kinase domains also phosphorylate intracellular proteins called Insulin Receptor Substrates or IRS proteins.
These proteins interact with, and activate another kinase called the PI3-kinase. PI3-kinase then catalyzes the formation of the lipid
molecule PIP3, which serves to activate yet another kinase, PDK1, which in turn, activates the Akt group of kinases. It is this group
of enzymes that appears to increase the translocation of the GLUT4 to the plasma membrane (Figure 7.152), as cells that lack
functional Akts exhibit poor glucose uptake and insulin resistance.
EGFR pathway
Epidermal growth factor, EGF, is an important signaling molecule involved in growth, proliferation and differentiation in
mammalian cells. EGF acts through the EGF receptor, EGFR, a receptor tyrosine kinase (Figure 7.153). Because of its role in
stimulating cell proliferation and because overexpression of EGFR is associated with some kinds of cancers, EGFR is the target for
many anti-cancer therapies. We can trace the signal transduction pathway from the binding of EGF to its receptor to the stimulation
of cell division.

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EGF binding to the EGFR is followed by receptor dimerization and stimulation of the tyrosine kinase activity of the cytosolic
domains of the EGFR. Autophosphorylation of the receptor tails is followed by the assembly of a signaling complex nucleated by
the binding of proteins that recognize phosphotyrosine residues. An important protein that is subsequently activated by the
signaling complexes on the receptor tyrosine kinases is called Ras (Figure 7.154). The Ras protein is a monomeric guanine
nucleotide binding protein that is associated with the cytosolic face of the plasma membrane
(in fact, it is a lot like the α subunit of trimeric G-proteins). Just like the α subunit of a G-protein, Ras is active when GTP is bound
to it and inactive when GDP is bound to it. Also, like the α subunit, Ras can hydrolyze the GTP to GDP.
Ras activation
Activation of Ras accompanies the exchange of the GDP bound to the inactive Ras for a GTP. Activated Ras triggers a
phosphorylation cascade of three protein kinases, which relay and distribute the signal. These protein kinases are members of a
group called the MAP kinases (Mitogen Activated Protein Kinases). The final kinase in this cascade phosphorylates various target
proteins, including enzymes and transcriptional activators that regulate gene expression.
The phosphorylation of various enzymes can alter their activities, and set off new chemical reactions in the cell, while the
phosphorylation of transcriptional activators can change which genes are expressed. The combined effect of changes in gene
expression and protein activity alter the cell's physiological state and promote cell division.
Once again, in following the path of signal transduction mediated by RTKs, it is possible to discern the same basic pattern of
events: a signal is bound by the extracellular domains of receptor tyrosine kinases, resulting in receptor dimerization and
autophosphorylation of the cytosolic tails, thus conveying the message to the interior of the cell.
The message is then passed on via a signaling complex to proteins that stimulate a series of kinases. The terminal kinase in the
cascade acts on target proteins and brings about in changes in protein activities.
What is the OFF switch for RTKs? It turns out that RTKs with the signal bound can be endocytosed into the cell and broken down.
That is, the region of the plasma membrane that the RTK is on can be internally pinched off into a vesicle containing the ligand-
bound receptor which is then targeted for degradation.
Ras, which is activated by GTP binding, can also be deactivated by hydrolysis of the GTP to GDP. The importance of this
mechanism for shutting down the pathway is evident in cells that have a mutant ras gene encoding a Ras protein with defective
GTPase activity. Unable to shut off Ras, the cells continue to receive a signal to proliferate. The National Cancer Institute estimates
that more than 30% of human cancers are driven by mutations in ras genes.
The descriptions above provide a very simple sketch of some of the major classes of receptors and deal primarily with the
mechanistic details of the steps by which signals received by various types of receptors bring about changes in cells. A major take-
home lesson is the essential similarity of the different pathways. Another point to keep in mind is that while we have looked at each
individual pathway in isolation, a cell, at any given time receives multiple signals that set off a variety of different responses at
once (Figure 7.155). The pathways described above show a considerable degree of "cross-talk" and the response to any given
signal is affected by the other signals that the cell receives simultaneously. The multitude of different receptors, signals, and the
combinations thereof are the means by which cells are able to respond to an enormous variety of different circumstances.
RTKs, cancer and cancer therapies
As described above, binding of EGF to its receptor triggers a signaling pathway that results in the activation of a series of Mitogen
Activated Protein Kinases (MAP kinases). These kinases are so-called because they are activated by a mitogen, a molecule, like
EGF and other growth factors, that stimulates mitosis or cell division. The final kinase in the MAP kinase cascade phosphorylates a
number of target proteins, many of them transcription factors, that when activated, increase the expression of genes associated with
cell proliferation.
Given that the EGF-receptor pathway normally functions to stimulate cell division, it is not surprising that malfunctions in the
pathway could lead to uncontrolled cell proliferation, or cancer. Next, we will take a brief look at some examples of such defects.
HER2
The human EGF receptor (HER) family has four members, HER1, HER2, HER3 and HER4. These are all receptor tyrosine
kinases, cell surface receptors that bind EGF (Figure 7.157) and stimulate cell proliferation.
A crucial step in the signal transduction pathway is the dimerization of the receptors following binding of the signal, EGF, to the
receptor. While HER1, HER3 and HER4 must bind the signal to dimerize, the structure of the HER2 receptor can, apparently,

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allow the receptor monomers to dimerize independently of EGF binding.
This means that the downstream events of the signaling pathway can be triggered even in the absence of a growth signal. In normal
cells, only a few HER2 receptors are expressed at the cell surface, so this property of HER2 plays a relatively minor role in
stimulating cell division. However, in about a quarter of breast cancer patients, HER2 receptors are overexpressed, leading to
increased dimerization and subsequent uncontrolled cell proliferation.
Breast cancers that are HER2-positive can be more aggressive with a greater tendency to metastasize (spread) so therapy that
blocks HER2 signaling is key in successful treatment of such cancers. Herceptin, a monoclonal antibody against the HER2
receptor, has been shown to be an effective treatment against Her2-positive breast cancers. Herceptin works by binding specifically
to the extracellular domain of the HER2 receptor (Figure 7.158). This prevents dimerization of the receptor and thus blocks
downstream signaling. Additionally, the binding of the Herceptin antibody to the receptor signals the immune system to destroy the
HER2-positive cells.
Bcr-abl
Another example of a cancer caused by defects in an RTK signaling pathway is chronic myeloid leukemia (CML). Patients with
CML have an abnormal receptor tyrosine kinase that is the product of a hybrid gene called bcr-abl, formed by the breakage and
rejoining of chromosomes 9 and 22. This abnormal tyrosine kinase is constitutively dimerized, even when no signal is bound. As a
result, it continuously signals cells to divide, leading to the massive proliferation of a type of blood cells called granulocytes.
As with HER2, the problem in CML is a receptor tyrosine kinase that dimerizes in the absence of a growth signal. The approach in
this case was to target the next step in the signaling pathway. As you know, dimerization of RTKs activates the tyrosine kinase
domain of the receptor, which results in the autophosphorylation of the cytoplasmic domains of both monomers. The
phosphorylated tyrosines serve to recruit a number of other signaling proteins that pass the signal on within the cell.
In the case of the bcr-abl RTK, the drug Gleevec (imatinib) was designed to bind near the ATP-binding site of the tyrosine kinase
domain. This "locks" the site in a conformation that inhibits the enzymatic activity of the tyrosine kinase and thus blocks
downstream signaling. With no "grow" signal passed on, cells stop proliferating.
Information Processing: Signaling
827
YouTube Lectures
by Kevin
HERE & HERE
828
Figure 7.130 - Schematic representation of a transmembrane receptor protein. E = extracellular; P = plasma membrane; I =
intracellular
Wikipedia
Figure 7.129 - Some examples of signal molecules
829
Figure 7.132 - Ligand-gated ion channel receptor opening in response to a signal (ligand)
Wikipedia
Figure 7.131 -General features of signal transduction pathways
830
Figure 7.133 - Neuromuscular signaling - A = motor neuron axon; B = axon terminal; C = synaptic cleft; D = muscle cell; E =
myofibril . Steps in the process - 1) action potential reaches the axon terminal; 2) voltage-dependent calcium gates open; (3)
neurotransmitter vesicles fuse with the presynaptic membrane and acetylcholine (ACh) released into the synaptic cleft; (4) ACh
binds to postsynaptic receptors on the sarcolemma; (5) ACh binding causes ion channels to open and allows sodium ions to flow
across the membrane into the muscle cell; 6) flow of sodium ions across the membrane into the muscle cell generates action
potential which travels to the myofibril and results in muscle contraction.

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Wikipedia
831
Figure 7.134 - Nerve systems
Wikipedia
832
Figure 7.135 - Steroid hormones structures, with the names of their receptors
Wikipedia
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by Kevin
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833
Figure 7.136 - Glucocorticoid receptor with its three domains - DNA binding (left), activator domain (top), and ligand binding
domain (boxed).
Wikipedia
Figure 7.137 - Glucocorticoid signaling pathway
Wikipedia
834
Figure 7.138 - Steroid hormone signaling
Image by Aleia Kim
835
Figure 7.139 - Structure of a G-protein linked receptor
Wikipedia
836
Figure 7.140- A heterotrimeric G-protein: α subunit in blue, βγ subunits red and green
Interactive Learning
Module
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837
Figure 7.141 - Cycle of G-protein activation - 1) binding of ligand; 2) change of receptor structure; 3) stimulation of α-subunit; 4)
binding of GTP, release of GDP; 5) separation of α-subunit from β-γ; 6) hydrolysis of GTP by α-subunit and return to inactive
state.
Wikipedia
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838
Figure 7.142 - β2-adrenergic receptor embedded in membrane (gray)
Wikipedia
Figure 7.143 - Epinephrine

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Wikipedia
839
Figure 7.144 - G-protein coupled receptor. Signal starts with ligand binding (orange circle). Gs = G-protein; AC = adenylate
cyclase.
Wikipedia
Figure 7.145 - Activation of Protein Kinase A by cAMP
Image by Martha Baker
840
Figure 7.146 - Simultaneous activation of glycogen breakdown and inhibition of glycogen synthesis by epinephrine’s binding of b-
adrenergic receptor. Red enzyme names = activated forms; black enzyme names = inactivated forms; GPb = glycogen
phosphorylase b; GPa = glycogen phosphorylase a.
Image by Penelope Irving
841
β-Adrenergic Signaling Off Switches
1. GRK Phosphorylates Receptor Tail
Receptor Tail Bound by Arrestin
2. α Subunit G-protein Cleaves GTP to GDP
β-γ subunits Reassociate with α Subunit
3. cAMP Hydrolyzed by Phosphodiesterase
PKA Becomes Inactive
4. Dephosphorylation of Phosphorylated Proteins by Phosphoprotein Phosphatase
β-Adrenergic Signaling On Switches
1. Binding of Signal Molecule to Receptor
2. Passage of Signal Through Several Molecules (G-proteins, Adenylate Cyclase, cAMP, PKA)
3. Phosphorylation of Target Proteins
842
Figure 7.147 - Cyclic AMP is broken down by phosphodiesterase
Figure 7.148 - Structure of a receptor tyrosine kinase
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843
Figure 7.149 - Signal binding results in receptor dimerization and activation of tyrosine kinase activity
Figure 7.150 - Activated tyrosine kinases phosphorylate tyrosines on the receptor tails.
Figure 7.151 -The insulin receptor, a receptor tyrosine kinase
Wikipedia
Figure 7.152 - Effects of insulin binding to its receptor tyrosine kinase: 1) insulin binding; 2) activation of protein activation
cascades. These include: 3) translocation of Glut-4 transporter to plasma membrane and influx of glucose; 4) glycogen synthesis; 5)
glycolysis; and 6) fatty acid synthesis.
Wikipedia

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844
Interactive Learning
Module
HERE
845
Figure 7.153 - EGFR signaling beginning at top with binding of EGF, dimerization of receptor, transmission of signal through
proteins, activation of kinases, phosphorylation of transcription factors and effects on transcription
Image by Aleia Kim
Figure 7.154 - Ras with GTP bound
Wikipedia

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 CHAPTER OVERVIEW

8: Basic Techniques
The environment of a cell is very complex, making it difficult to study individual reactions, enzymes, or pathways in situ. The
traditional approach used by biochemists for the study of these things is to isolate molecules, enzymes, DNAs, RNAs, and other
items of interest so they can be analyzed independently of the millions of other processes occurring simultaneously. Today, these
approaches are used side by side with newer methods that allow us to understand events inside cells on a larger scale- for example,
determining all the genes that are being expressed at a given time in specific cells. In this section we take a brief look at some
commonly used methods used to study biological molecules and their interactions.
8.1: Cell Lysis
8.2: Fractionation and Chromatography Techniques
8.3: Electrophoresis
8.4: Detection, identification and quantitation of specific nucleic acids and proteins
8.5: Transcriptomics
8.6: Isolating Genes
8.7: Polymerase Chain Reaction (PCR)
8.8: Reverse Transcription
8.9: FRET
8.10: Genome Editing (CRISPR)
8.11: Protein Cleavage
8.12: Membrane Dynamics (FRAP)

Thumbnail: A western blot. Image used with permission (CC BY-SA 3.0; Magnus Manske).

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1
8.1: Cell Lysis
Source: BiochemFFA_8_1.pdf. The entire textbook is available for free from the authors at
http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy
To separate compounds from cellular environments, one must first break open (lyse) the cells. Cells are broken open, in buffered
solutions, to obtain a lysate. There are several ways of accomplishing this.
Osmotic shock and enzymes: One way to lyse cells is by lowering the ionic strength of the medium the cells are in. This can
cause cells to swell and burst. Mild surfactants may be used to disrupt membranes. Most bacteria, yeast, and plant tissues are
resistant to osmotic shocks, because of the presence of cell walls, and stronger disruption techniques are usually required.
Enzymes may be useful in helping to degrade the cell walls. Lysozyme, for example, is very useful for breaking down bacterial
walls. Other enzymes commonly employed include cellulase (plants), proteases, mannases, and others.
Mechanical disruption: Mechanical agitation may be employed in the form of beads that are shaken with a mixture of cells. In
this method, cells are bombarded with tiny, glass beads that break the cells open. Sonication (20-50 kHz sound waves) provides
an alternative type of agitation that can be effective. The method is noisy, however, and generates heat that can be problematic
for heat-sensitive compounds.
Pressure disruption: Another means of disrupting cells involves using a “cell bomb”. In this method, cells are placed under
very high pressure (up to 25,000 psi) and then the pressure is rapidly released. The rapid pressure change causes dissolved gases
in cells to be released as bubbles which, in turn, break open cells.
Cryopulverization: Cryopulverization is often employed for samples having a tough extracellular matrix, such as connective
tissue, seed, and cartilage. In this technique, tissues are frozen using liquid nitrogen and then impact pulverization (typically,
grinding, using a mortar and pestle or a powerful electric grinder) is performed. The powder so obtained is then suspended in
the appropriate buffer.

Figure 8.1 - A sonicator in use. Wikipedia

Whatever method is employed to create a lysate, crude fractions obtained from it must be further processed via fractionation.

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8.2: Fractionation and Chromatography Techniques
Fractionation of samples, as the name suggests, is a process of separating out the components or fractions of the lysate.
Fractionation typically begins with centrifugation of the lysate. Using low-speed centrifugation, one can remove cell debris, leaving
a supernatant containing the contents of the cell. By using successively higher centrifugation speeds (and resulting g forces) it is
possible to separate out different cellular components, like nuclei, mitochondria, etc., from the cytoplasm. These may then be
separately lysed to release molecules that are specific to the particular cellular compartment. The soluble fraction of any lysate can,
then, be further separated into its constituents using various methods.

Figure 8.2.1 : A high-speed centrifuge can be used to obtain different cell fractions from a crude lysate. Wikipedia

Column Chromatography
One powerful method used for this purpose is chromatography. We will consider several chromatographic approaches.
Chromatography is used to separate out the components of a mixture based on differences in their size, charge or other
characteristics. During chromatography, the mobile phase (buffer or other solvent) moves through the stationary phase (usually a
solid matrix) carrying the components of the mixture. Separation of the components is achieved, because the different components
move at different rates, for reasons that vary, depending on the type of chromatography used. We will consider several different
kinds of chromatography to illustrate this process.
Ion exchange chromatography
Gel exclusion chromatography
Affinity chromatography
HPLC

Figure 8.2.2 : An ion exchange column apparatus.Wikipedia

These variations on chromatography are performed with the stationary phase held within so-called columns (Figure ). These
8.2.1

are tubes containing the stationary phase (also called the “support” or solid phase).
Supports are composed of tiny beads suspended in buffer (Figure 8.2.3) and are designed to exploit the chemistry or size
differences of the components of the samples and thus provide a means of separation. Columns are “packed” or filled with the
support, and a buffer or solvent carries the mixture of compounds to be separated through the support. Molecules in the sample
interact differentially with the support and, consequently, travel through it at different speeds, thus enabling separation.

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 Figure 8.2.3 : Ion exchange beads.Wikipedia

Ion exchange chromatography

In ion exchange chromatography, the support consists of tiny beads to which are attached chemicals possessing a charge. Before
use, the beads are equilibrated in a solution containing an appropriate counter-ion to the charged molecule on the bead. Figure 8.2.5
shows the repeating unit of polystyrolsulfonate, a compound used as a cation exchange resin. As you can see, this molecule is
negatively charged, and thus the beads would be equilibrated in a buffer containing a positively charged ion, say sodium. In the
suspension, the negatively charged polystyrolsulfonate is unable to leave the beads, due to its covalent attachment, but the counter-
ions (sodium) can be “exchanged” for molecules of the same charge.

Figure 8.2.4 : Polystyrolsulfonate, a cation exchange resin. Wikipedia

Exchanges
Thus, a cation exchange column will have positively charged counter-ions and negatively charged molecules covalently attached to
the beads. Positively charged compounds from a cell lysate passed through the column will exchange with the counter-ions and
“stick” to the negatively charged compounds covalently attached to the beads. Molecules in the sample that are neutral in charge or
negatively charged will pass quickly through the column. At this point, only positively charged molecules from the original sample
would be bound to the column. These may then be washed off, or eluted, by using buffers containing high concentrations of salt.
Under these conditions, the interaction between the positively charged molecules and the polystyrosulfonate would be disrupted,
allowing the molecules that were bound to the column to be recovered.
Anion exchange
On the other hand, in anion exchange chromatography, the chemicals attached to the beads are positively charged and the
counterions are negatively charged (chloride, for example). Negatively charged molecules in the cell lysate will “stick” and other
molecules will pass through quickly. To remove the molecules “stuck” to a column, one simply needs to add a high concentration
of counter-ions to release them.
Uses
Ion exchange resins are useful for separating charged from uncharged, or oppositely charged, biomolecules in solution. The resins
have a variety of other applications, including water purification and softening. Figure 8.2.5 shows use of a polystyrolsulfonate
polymer in removing calcium for water softening.

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 Figure 8.2.5 : Removal of calcium ions by an ion exchanger. Wikipedia

Size Exclusion Chromatography

Size exclusion chromatography (also called molecular exclusion chromatography, gel exclusion chromatography, or gel filtration
chromatography) is a low resolution separation method that employs beads with tiny “tunnels” in them that each have a precise
opening. The size of the opening is referred to as an “exclusion limit,” which means that molecules above a certain molecular
weight will not be able to pass through the tunnels. Molecules with physical sizes larger than the exclusion limit do not enter the
tunnels and pass through the column relatively quickly, in the spaces outside the beads. Smaller molecules, which can enter the
tunnels, do so, and thus, have a longer path that they take in passing through the column and elute last (Figure 8.2.6).

Figure 8.2.6 : Separation of molecules by size in a size-exclusion (aka gel filtration) column
Figure 8.2.7 shows a profile of a group of proteins separated by size exclusion chromatography using beads with an exclusion limit
of about 30,000 Daltons. Proteins 30,000 in molecular weight or larger elute in the void volume (left) while smaller proteins elute
later (middle and right).

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 Figure 8.2.7 : Result of a size exclusion separation. Wikipedia

Affinity chromatography
Affinity chromatography is a very powerful and selective technique that exploits the binding affinities of sample molecules
(typically proteins) for molecules covalently linked to the support beads. In contrast to ion-exchange chromatography, where all
molecules of a given charge would bind to the column, affinity chromatography exploits the specific binding of a protein or
proteins to a ligand that is immobilized on the beads in the column.
For example, if one wanted to separate all of the proteins in a cell lysate that bind to ATP from proteins that do not bind ATP, one
could use a column that has ATP attached to the support beads and pass the sample through the column. All proteins that bind ATP
will “stick” to the column, whereas those that do not bind ATP will pass quickly through it. The bound proteins may then be
released from the column by adding a solution of ATP that will displace the bound proteins by competing, for the proteins, with the
ATP attached to the column matrix.
Histidine tagging
Histidine tagging (His-tagging) is a special kind of affinity chromatography and is a powerful tool for isolating a recombinant
protein from a cell lysate. His-tagging relies on altering the DNA coding region for a protein to add a series of at least six histidine
residues to the amino or carboxyl terminal of the encoded protein. This “His-Tag” is useful in purifying the tagged protein because
histidine side chains can bind to nickel or cobalt ions. Separation of His-tagged proteins from a cell lysate is relatively easy (Figure
8.2.8).Passing the crude cell lysate through a column with nickel or cobalt attached to beads allows the His-tagged proteins to

“stick,” while the remaining cell proteins all pass quickly through. The His-tagged proteins are then eluted by addition of imidazole
to the column. Imidazole, which resembles the side chain of histidine, competes with the His-tagged proteins and displaces them
from the column. Although non-tagged proteins in the lysate may also contain histidine as part of their sequence, they will not bind
to the column as strongly as the His-tagged protein and will, thus, be displaced at lower imidazole concentrations than needed to
elute the His-tagged protein. Surprisingly, many His-tagged proteins appear to function normally despite the added histidines, but if
needed, the histidine tags may be cleaved from the purified protein by treatment with a protease that excises the added histidines,
allowing the recovery of the desired protein with its native sequence.

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 Figure 8.2.8 : Affinity chromatographic purification of a protein by histidine tagging.Image by Aleia Kim

HPLC
High performance liquid chromatography (HPLC) is a powerful tool for separating a variety of molecules based on their
differential polarities (Figure 8.2.9). A more efficient form of column chromatography, it employs columns with tightly packed
supports and very tiny beads such that flow of solvents/buffers through the columns requires high pressures. The supports used may
be polar (normal phase separation) or non-polar (reverse phase separation). In normal phase separations, non-polar molecules elute
first followed by the more polar compounds. This order is switched in reverse phase chromatography. Of the two, reverse phase is
much more commonly employed due to more reproducible chromatographic profiles (separations) that it typically produces.

Figure 8.2.9 : HPLC: Pumps on left/Column in center/Detector on the right. Wikipedia

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8.3: Electrophoresis
Electrophoresis uses an electric field applied across a gel matrix to separate large molecules such as DNA, RNA, and proteins by
charge and size. Samples are loaded into the wells of a gel matrix that can separate molecules by size and an electrical field is
applied across the gel. This field causes negatively charged molecules to move towards the positive electrode. The gel matrix,
itself, acts as a sieve, through which the smallest molecules pass rapidly, while longer molecules are slower-moving.
For DNA and RNA, sorting molecules by size in this way is trivial, because of the uniform negative charge on the phosphate
backbone. For proteins, which vary in their charges, a clever trick must be employed to make them mimic nucleic acids - see
polyacrylamide gel electrophoresis (PAGE) below. Different kinds of gels have different pore sizes. Like sieves with finer or
coarser meshes, some gels do a better job of separating smaller molecules while others work better for larger ones. Gel
electrophoresis may be used as a preparative technique (that is, when purifying proteins or nucleic acids), but most often it is used
as an analytical tool.

Agarose Gel Electrophoresis

Agarose gel electrophoresis is a technique used to separate nucleic acids primarily by size. Agarose is a polysaccharide obtained
from seaweeds (Figure 8.11). It can be dissolved in boiling buffer and poured into a tray, where it sets up as it cools (Figure 8.12) to
form a slab. Agarose gels are poured with a comb in place to make wells into which DNA or RNA samples are placed after the gel
has solidified. The gel is immersed in a buffer and a current is applied across the slab. Double-stranded DNA has a uniform
negative charge that is independent of the sequence composition of the molecule. Therefore, if DNA fragments are placed in an
electric field they will migrate from the cathode (-) towards the anode (+). The rate of migration is directly dependent on the ability
of each DNA molecule to worm or wiggle its way through the sieving gel. The agarose matrix provides openings for
macromolecules to move through. The largest macromolecules have the most difficult time navigating through the gel, whereas the
smallest macromolecules slip through it the fastest.

Figure 8.11 - Structure of the agarose polysaccharide. Wikipedia

Because electrophoresis uses an electric current as a force to drive the molecules through the matrix, the molecules being separated
must be charged. Since the size to charge ratio for DNA and RNA is constant for all sizes of these nucleic acids, the molecules
simply sort on the basis of their size - the smallest move fastest and the largest move slowest.
All fragments of a given size will migrate the same distance on the gel, forming the so-called “bands” on the gel. Visualization of
the DNA fragments in the gel is made possible by addition of a dye, such as ethidium bromide, which intercalates between the
bases and fluoresces when viewed under ultraviolet light (Figure 8.13) By running reference DNAs of known sizes alongside the
samples, it is possible to determine the sizes of the DNA fragments in the sample. It is useful to note that, by convention, DNA
fragments are not described by their molecular weights (unlike proteins), but by their length in base-pairs( bp) or kilobases (kb).

Figure 8.12 - Agarose gel electrophoresis separation of DNA - orange bands are DNA fragments. Wikipedia

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 Figure 8.13 - DNA bands visualized with ethidium bromide staining. Wikipedia

Polyacrylamide gel electrophoresis (PAGE)

Like DNA and RNA, proteins are large macromolecules, but unlike nucleic acids, proteins are not necessarily negatively charged.
The charge on each protein depends on its unique amino acid sequence. Thus, the proteins in a mixture will not necessarily all
move towards the anode.
Additionally, whereas double-stranded DNA is rod-shaped, most proteins are globular (folded). Further, proteins are considerably
smaller than nucleic acids, so the openings of the matrix of the agarose gel are simply too large to effectively provide separation.
Consequently, unaltered (native) proteins are not very good prospects for electrophoresis on agarose gels. To separate proteins by
mass using electrophoresis, one must make several modifications.

Gel matrix
First, a matrix made by polymerizing and cross-linking acrylamide units is employed. A monomeric acrylamide (Figure 8.14) is
polymerized and the polymers are cross-linked using N,N’-Methylene-bisacrylamide (Figure 8.15) to create a mesh-like structure.
One can adjust the size of the openings of the matrix/mesh readily by changing the percentage of acrylamide in the reaction. Higher
percentages of acrylamide give smaller openings and are more effective for separating smaller molecules, whereas lower
percentages of acrylamide are used when resolving mixtures of larger molecules. (Note: polyacrylamide gels are also used to
separate small nucleic acid fragments, with some acrylamide gels capable of separating pieces of DNA that differ in length by just
one nucleotide.)

Figure 8.14 - Acrylamide monomer. Wikipedia

Figure 8.15 - N,N’-Methylenebisacrylamide - acrylamide crosslinking reagent. Wikipedia

Charge alteration by SDS

A second consideration is that proteins must be physically altered to “present” themselves to the matrix like the negatively charged
rods of DNA. This is accomplished by treating the proteins with the anionic detergent, SDS (sodium dodecyl sulfate). SDS
denatures the proteins so they assume a rod-like shape and the SDS molecules coat the proteins such that the exterior surface is
loaded with negative charges, masking the original charges on the proteins and making the charge on the proteins more
proportional to their mass, like the backbone of DNA.

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Since proteins typically have disulfide bonds that prevent them from completely unfolding in detergent, samples are boiled with
mercaptoethanol to break the disulfide bonds and ensure the proteins are as rod-like as possible in the SDS. Reagents like
mercaptoethanol (and also dithiothreitol) are sulfhydryl-containing reagents that become oxidized as they reduce disulfide bonds in
other molecules (see Figure 8.16)

Figure 8.16 - Reduction of disulfide bonds by dithiothreitol. Wikipedia

Stacking Gel
A third consideration is that a “stacking gel” may be employed at the top of a polyacrylamide gel to provide a way of compressing
the samples into a tight band before they enter the main polyacrylamide gel (called the resolving gel). Just like DNA fragments in
agarose gel electrophoresis get sorted on the basis of size (largest move slowest and smallest move fastest), the proteins migrate
through the gel matrix at velocities inversely related to their size. Upon completion of the electrophoresis, proteins may be
visualized by staining with compounds that bind to proteins, like Coomassie Brilliant Blue (Figure 8.17) or silver nitrate.

Figure 8.17 - Two SDS-PAGE gels - Proteins are the blue bands (stained with Coomassie Blue). Wikipedia
Non-denaturing gel electrophoresis
The SDS_PAGE technique described above is the commonest method used for electrophoretic separation of proteins. In some
situations, however, proteins may be resolved on so-called “native” gels, in the absence of SDS. Under these conditions, the
movement of proteins through the gel will be affected not simply by their mass, but by their charge at the pH of the gel, as well.
Proteins complexed with other molecules may move as single entity, allowing the isolation of the binding partners of proteins of
interest.

Isoelectric focusing
Proteins vary considerably in their charges and, consequently, in their pI values (pH at which their charge is zero). This can be
exploited to separate proteins in a mixture. Separating proteins by isoelectric focusing requires establishment of a pH gradient in a

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tube containing an acrylamide gel matrix. The pore size of the gel is adjusted to be large, to reduce the effect of sieving based on
size. Molecules to be separated are applied to the gel containing the pH gradient and an electric field is applied. Under these
conditions, proteins will move according to their charge.
Positively charged molecules, for example, move towards the negative electrode, but since they are traveling through a pH
gradient, as they pass through it, they reach a region where their charge is zero and, at that point, they stop moving. They are at that
point attracted to neither the positive nor the negative electrode and are thus “focused” at their pI (Figure 8.18). Using isoelectric
focusing, it is possible to separate proteins whose pI values differ by as little as 0.01 units.

Figure 8.18 - Isoelectric focusing: A. At the start of the run; B. at the end of the run

2D gel electrophoresis
Both SDS-PAGE and isoelectric focusing are powerful techniques, but a clever combination of the two is a powerful tool of
proteomics - the science of studying all of the proteins of a cell/tissue simultaneously. In 2-D gel electrophoresis, a lysate is first
prepared from the cells of interest. The proteins in the lysate are separated first by their pI, through isoelectric focusing and then by
size by SDS-PAGE.

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 Figure 8.19 - Scheme for performing 2-D gel analysis. Image by Aleia Kim
The mixture of proteins is first applied to a tube or strip (Figure 8.19, Step 1) where isoelectric focusing is performed to separate
the proteins by their pI values (Step 2). Next, as shown in the figure, the gel containing the proteins separated by their pIs is turned
on its side and applied along the top of a polyacrylamide slab for SDS-PAGE to separate on the basis of size (Step 3). The proteins
in the isoelectric focusing matrix are electrophoresed into the polyacrylamide gel and separated on the basis of size. The product of
this analysis is a 2-D gel as shown in Figure 8.20.The power of 2-D gel electrophoresis is that virtually every protein in a cell can
be separated and appear on the gel as a spot defined by its unique size and pI. In the figure, spots in the upper left correspond to
large positively charged proteins, whereas those in the lower right are small negatively charged ones. Every spot on a 2-D gel can
be eluted and identified by using high throughput mass spectrometry. This is particularly powerful when one compares protein
profiles between different tissues or between control and treated samples of the same tissue.

Figure 8.20 - Result of 2-D gel electrophoresis separation. Wikipedia

Protein profiles comparison
Comparison of 2-D gels of proteins from non-cancerous tissue and proteins from a cancerous tissue of the same type provides a
quick identification of proteins whose level of expression differs between the two. Information such as this can be useful in
designing treatments or in understanding the mechanism(s) by which the cancer develops.

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8.4: Detection, identification and quantitation of specific nucleic acids and proteins
While gel electrophoresis can be used to resolve molecules in a mixture, by itself, the technique does not permit the detection and
identification of specific nucleic acid sequences or proteins. For example, the 2-D gel shown above clearly separates a large
number of proteins in a sample into individual spots. However, if we wanted to know whether a specific protein was present, we
could not tell by simply looking at the gel. Likewise, in an agarose gel, while bands of DNA could be assigned a size, one could not
distinguish between two DNAs of different sequence if they were both the same length in base-pairs. One way to detect the
presence of a particular nucleic acid or protein is dependent on transferring the separated molecules from the gels onto a membrane
made of nitrocellulose or nylon to create a “blot” and probing for the molecule(s) of interest using reagents that specifically bind to
those molecules. The next section will discuss how this can be done for nucleic acids as well as for proteins.

Southern and Northern Blots

The Southern blot is named for its inventor, Oxford professor, Edwin Southern, who came up with a protocol for transferring DNA
fragments from a gel onto a nitrocellulose sheet and detecting a specific DNA sequence among the bands on the blot. As shown in
Figure 8.21, the method works as follows. A mixture of DNA molecules (often DNA that has been cut into smaller fragments using
restriction endonucleases) is loaded on an agarose gel, as usual. After the gel run is complete, the DNA bands are transferred from
the gel onto a membrane. This can be achieved by capillary transfer, where the gel is placed in contact with a piece of membrane
and buffer is pulled through the gel by wicking it up into a stack of absorbent paper placed above the membrane. As the buffer
moves, it carries with it the DNA fragments. The DNA binds to the membrane leaving a “print” of DNA fragments that exactly
mirrors their positions in the gel. The blotting membrane may be treated with UV light, heat, or chemicals to firmly attach the DNA
to the membrane.
Next, a probe, or visualizing agent specific for the molecule of interest is added to the membrane. In Figure 8.21, this is called a
labeled probe. The probes in a Southern blot are pieces of DNA designed to be complementary to the desired target sequence. If the
sequence of interest is present on the blot, the probe, which is complementary to it, can base-pair (hybridize) with it. The blot is
then washed to remove all unbound probe. Probes are labeled with radioactivity or with other chemical reagents that allow them to
be easily detected when bound to the blot, so it is possible to visually determine whether the probe has bound to any of the DNA
bands on the blot. Given that the Southern blot relies on specific base-pairing between the probe and the target sequence, it is easy
to adapt the technique to detect specific RNA molecules, as well. The modification of this method to detect RNAs was jokingly
named a “northern” blot.

Figure 8.21 - Northern or Southern blot scheme. Southern blotting adds strand denaturation between steps 4 and 5. Wikipedia

Western Blots
Proteins cannot, for obvious reasons, be detected through base-pairing with a DNA probe, but protein blots, made by transferring
proteins, separated on a gel, onto a membrane, can be probed using specific antibodies against a particular protein of interest.
Protein detection usually employs two antibodies, the first of which is not labeled. The label is on the second antibody, which is
designed to recognize only the first antibody in a piggyback fashion. The first antibody specifically binds to the protein of interest
on the blot and the second antibody recognizes and binds the first antibody. The second antibody commonly carries an enzyme or

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reagent which can cause a reaction to produce a color upon further treatment. In the end, if the molecule of interest is in the original
mixture, it will “light” up and reveal itself on the blot. This variation on the blotting theme was dubbed a western blot (Figure
8.22).

Figure 8.22 - Result of a western blot analysis. Wikipedia

In each of the blots described above, binding of the probe to the target molecule allows one to determine whether the target
sequence or protein was in the sample. Although blots are designed to be used for detection, rather than for precise quantitation, it
is possible to obtain estimates of the abundance of the target molecule from densitometry measurements of signal intensity.

Microarrays
2-D gels are a way of surveying a broad spectrum of protein molecules simultaneously. One approach to doing something similar
with DNA or RNA involves what are called microarrays. Microarrays are especially useful for monitoring the expressions of
thousands of genes, simultaneously. Where a northern blot would allow the identification of a single mRNA from a mixture of
mRNAs, a microarray experiment can allow the simultaneous identification of thousands of mRNAs that may be made by a cell at
a given time. It is also possible to perform quantitation much more reliably than with a blot.
Microarrays employ a glass slide, or chip, to which are attached short sequences of single-stranded DNA, arranged in a grid, or
matrix (Figure 8.23) Each position in the grid corresponds to a unique gene. That is, the DNA sequence at this spot is part of the
sequence of a specific gene. Each spot on the grid has multiple identical copies of the same sequence. The gene sequence
immobilized at each position in the grid is recorded.

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 Figure 8.23 - Microarray design. Image by Taralyn Tan
To the slide are added a mixture of sample molecules, some of which will recognize and bind specifically to the sequences on the
slide. Binding between the sample molecules and the sequences attached to the slide occurs by base pairing, in the case of DNA
microarrays. The slide is then washed to remove sample molecules that are not specifically bound to the sequences in the grid.
Sample molecules are tagged with a fluorescent dye, allowing the spots where they bind to be identified. The grid is analyzed spot
by spot for binding of the sample molecules to the immobilized sequences. The more sample molecules are bound at a spot, the
greater the intensity of dye fluorescence that will be observed. Information from this analysis can give information about the
presence/absence/abundance of molecules in the sample that bind to the sequences in the grid.

Figure 8.24 - Large scale microarray analysis of mouse transcriptome. Wikipedia

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8.5: Transcriptomics
Consider a matrix containing all of the known gene sequences in a genome. To make such a matrix for analysis, one would need to
make copies of every gene, either by chemical synthesis or by using the polymerase chain reaction. The strands of the resulting
DNAs would then be separated to obtain single-stranded sequences that could be attached to the chip. Each box of the grid would
contain sequence from one gene. With this grid, one could analyze the transcriptome - all of the mRNAs being made in selected
cells at a given time. For a simple analysis, one could take a tissue (say liver) and extract all the mRNAs from it. This mRNA
population represents all the genes that were being expressed in the liver cells at the time the RNA was extracted. These RNAs
should be able to hybridize (base-pair) with their corresponding genes on the microarray. Genes that were not being expressed
would have no mRNAs to bind to their corresponding genes on the grid.

Figure 8.25 - Copying and labeling of transcriptome. Image by Taralyn Tan

In practice, the mRNAs are not used directly, but are copied into single-stranded DNA copies called cDNAs. The cDNAs are
tagged with a fluorescent dye and added to the microarray under conditions that allow base pairing so that the cDNAs can find and
base pair with complementary sequences on the matrix (Figure 8.26). The matrix is then washed to remove unhybridized cDNAs.
The presence/absence/abundance of each mRNA is then readily determined by measuring the amount of dye at each box of the
grid.
Figure 8.26 - Add labeled cDNAs to microarray plate. Image by Taralyn Tan
In Figure 8.27, a fluorescent cDNA has bound to the spot on the far right in the third row of the grid. This means that the sequence
of the cDNA was complementary to the sequence of the gene sequence immobilized at that spot. Because the identity of the genes
at each position on the grid is known, we then know that the sample contained mRNA that corresponded to that particular gene. In
other words, that gene was being expressed in the cells from which the mRNAs were obtained.

Figure 8.27 - Binding of a fluorescent cDNA copy of a specific mRNA to DNA immobilized on one spot in a microarray.
Wikipedia
A more powerful analysis could be performed with two sets of mRNAs simultaneously. . One set of cDNAs could come from a
cancerous tissue and the other from a non-cancerous tissue, for example. The cDNAs derived from each sample is marked with a

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different color (say green for normal and red for cancerous) (Figure 8.25). The cDNAs are mixed and then added to the matrix and
complementary sequences are once again allowed to form duplexes (Figure 8.27).

Figure 8.28 - Microarray analysis comparing gene expression in normal and cancer cells. Wikipedia
Unhybridized cDNAs are washed away and then the plate is analyzed. Red grid boxes correspond to an mRNA present in the
cancerous tissue, but not in the non-cancerous tissue. Green grid boxes correspond to an mRNA present in the non-cancerous
tissue, but not in the cancerous tissue. Yellow would correspond to mRNAs present in equal abundance in the two tissues (Figure
8.28). The intensity of each spot also gives information about the relative amounts of each mRNA in each tissue.

Figure 8.29 - Automated high throughput sequencer. Wikipedia

The same principle used for nucleic acid microarrays can be adapted for analyzing other molecules. For example, polypeptides
could be bonded to the glass slide instead of DNA to create a protein chip. Protein chips are useful for studying the interactions of
proteins with other molecules as well as for diagnostics.

RNA-Seq Technique
Like microarrays, a newer method called RNA-Seq, is a tool for simultaneously detecting and quantitating all of the transcripts in a
given sample. This method relies on recently developed sequencing technologies called next-generation sequencing, or deep
sequencing. These techniques allow for rapid, parallel sequencing of millions of DNA fragments and can, thus, be used not only for
genomic DNA, but also to sequence all of the reverse-transcribed RNAs from a given sample.
To determine all the protein-coding genes that were being expressed in a particular set of cells under specific physiological
conditions, all of the mRNA would first be extracted and reverse-transcribed into cDNA. This step is similar to the preparation of
samples for microarrays. However, at this point, the cDNAs are fragmented into smaller pieces, and have small sequencing
adapters attached at either end. The fragments are then subjected to high-throughput sequencing, to obtain short sequences from all
of the fragments. These data are aligned against the genome sequence and used to measure the level of expression of different
genes. RNA-Seq offers some advantages over microarrays. With microarrays, an RNA can only be detected if the gene sequence

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corresponding to it is present on the grid. In RNA-Seq every RNA present in the sample is sequenced, so detection of RNAs is not
limited by the probes on a chip. RNA-Seq is more sensitive than microarrays and offers a much larger range over which gene
expression can be measured accurately.

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8.6: Isolating Genes
Earlier in this chapter, we discussed methods such as column chromatography that are used to purify proteins of interest. Using
combinations of these methods, it is possible to isolate a protein to a high degree of purity, thus enabling us to study the protein’s
activity and properties. This problem is harder to solve for nucleic acids. Genomic DNA can be readily obtained from cells, but is
too complex to be analyzed as a whole. Individual genes are the units of DNA that correspond to proteins, and thus, it makes more
sense to isolate specific genes for study. Methods to isolate genes were not available till the 1970s, when the discovery of
restriction enzymes and the invention of molecular cloning provided, for the first time, ways to obtain large quantities of specific
DNA fragments, for study. Although, for purposes of obtaining large amounts of a specific DNA fragment, molecular cloning has
been largely replaced by direct amplification using the polymerase chain reaction described later, cloned DNAs are still very useful
for a variety of reasons. The development of molecular cloning was dependent on the discovery of restriction endonucleases,
described below.

Restriction enzymes
Restriction enzymes, or restriction endonucleases, are enzymes made by bacteria. These enzymes protect bacteria by degrading
foreign DNA molecules that are carried into their cells by, for example, an invading bacteriophage. Each restriction enzyme
recognizes a specific sequence, usually of four or six nucleotides in the DNA. These sequences, when they occur in the bacterium's
own DNA, are chemically modified by methylation, so that they are not recognized and degraded. Where these sequences occur in
foreign DNA, they are cut by the restriction enzyme.
The utility and importance of restriction enzymes lies in their ability to recognize specific sequences in DNA and cut near or
(usually) at the site they recognize. Over 3000 such enzymes are known. Sequences recognized by these enzymes are typically 4-8
base pairs long and the most commonly used enzymes recognize sequences described as palindromic.

Figure 8.6.1 : -A restriction enzyme bound to its recognition sequence on DNA. Wikipedia

Palindrome
In molecular biology, the term palindrome means that the sequence of the recognition site when read in the 5‘ to 3‘ direction for the
top strand is exactly the same as that of the bottom strand. Consider the sequence recognized by the restriction enzyme known as
Hind III (pronounced hin-dee-three). It is
5’ -A-A-G-C-T-T-3’
3’ -T-T-C-G-A-A-5’
On the top strand, the recognition sequence is
5’ AAGCTT 3’
which is the same as the bottom strand (read in the same 5’ to 3’ direction).
While all restriction enzymes must recognize and bind to particular DNA sequences, the exact spot at which they cut the DNA
varies. Some enzymes leave a staggered sequence after cutting that has an overhang at the 5’ end of one strand of the duplex; some

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leave a staggered sequence after cutting that has an overhang at the 3’ end; and some cut both strands in the same place, leaving no
overhanging sequence - called blunt end cutters.
Consider cutting a DNA sequence that contains the Hind III recognition site, which is
5’ -A-A-G-C-T-T-3’
3’ -T-T-C-G-A-A-5’
Embedded within a DNA sequence, the Hind III sequence would look like this (Ns correspond to any base and represent all of the
DNA around the recognition site).
5’ -N-N-N-A-A-G-C-T-T-N-N-N-3’
3’ -N-N-N-T-T-C-G-A-A-N-N-N-5’
After cutting with Hind III, it would look as follows:
5’ -N-N-N-A 3‘ 5’A-G-C-T-T-N-N-N-N-3’
3’ -N-N-N-T-T-C-G-A-5‘ 3’ A-N-N-N-N-5’
where gaps have been inserted to illustrate where cutting has occurred. Hind III cuts between the two ‘A’ containing nucleotides
near the 5’ end of the recognition sequence and thus leaves 5’ overhangs (Figure 8.6.2).

Figure 8.6.2 : Result of cutting DNA with Hind III. Wikipedia

The restriction enzyme Pst I, on the other hand, recognizes the following sequence
5’ -N-N-N-C-T-G-C-A-G-N-N-N-N-3’
3’ -N-N-N-G-A-C-G-T-C-N-N-N-N-5’
and cuts between the A and the G near the 3’ end of the recognition sequence.
5’ -N-N-N-C-T-G-C-A 3‘ 5’G-N-N-N-N 3’
3’ -N-N-N-G 5‘ 3’ A-C-G-T-C-N-N-N-N 5’
As you can see, cutting a DNA with Pst I leaves 3’ overhangs of the recognition sequence. The ends left after cutting by a
restriction enzyme that overhang either at the 5’ end or the 3’ end are referred to as being “sticky” because they can form proper
base pairs and more readily be joined to a similarly “sticky end”. This means that you can take two unrelated pieces of DNA, cut
them with the same restriction enzyme so that they have compatible sticky ends, and then "paste" them together using DNA ligase
to form a new hybrid molecule, or recombinant.

Making Recombinant DNAs

Joining together of DNA fragments from different sources creates recombinant DNA. The ability to cut and paste DNA might seem
like purely a technical feat, but one key application that arose out of this is molecular cloning. In molecular cloning a gene of
interest can be inserted into a vector, usually a plasmid, by cutting both the vector and the gene (called the insert) with the same
enzyme to generate sticky ends and joining the two pieces together to generate a recombinant (Figure 8.6.3). A plasmid is a type of

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autonomously replicating, extrachromosomal DNA. It is quite simple to extract plasmids from the cells, engineer them to contain
the gene of interest and re-introduce the recombinant plasmid into the bacteria. The idea was that when the plasmid DNA was
replicated, the extra inserted gene would also be copied. Thus, by growing up a lot of the bacteria carrying the plasmid, many
copies of the gene of interest could be obtained, to provide sufficient amounts of the gene to use in experiments. While we now
have easier methods to accomplish this goal, cloned DNAs remain very useful. For example, it is possible to clone a gene that
encodes a protein of interest so that it can be expressed at high levels in the cells into which the recombinant plasmid is introduced.

Figure 8.6.3 : Recombinant DNA construction. Wikipedia

Whatever the purpose for which the recombinant plasmid is made, it typically carries an antibiotic resistance gene (or genes),
called a selectable marker. Cells that take up the plasmid will be able to grow in the presence of the antibiotic. If bacterial cells to
which the plasmid has been added are plated on agar containing the antibiotic, the cells which took up the plasmid will be able to
grow, while the others will not.

Figure 8.6.4 : Restriction site map for the pUC 18/19 plasmids, a classic plasmid vector. Genes identified by arrows. Numbers
correspond to the pUC 18/19 numbering convention

Expression cloning
As mentioned above, a gene of interest may be inserted into a vector and the recombinant plasmid be placed into a cell where the
gene can be expressed. For instance, one might desire to clone the gene coding for human growth hormone or insulin or other
medically important proteins and have a bacterium or yeast make large quantities of it very cheaply. Remember that these are
human proteins, and thus it is not feasible to extract the proteins in any quantity from human subjects.

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To clone a gene so that it can be expressed, one needs to set up the proper conditions in order for the human protein to be made in
the bacterial cells. This typically involves the use of specially designed plasmids. These plasmids have been engineered to 1)
replicate in high numbers; 2) carry markers that allow researchers to identify cells carrying them (antibiotic resistance, for
example) and 3) contain sequences (such as a promoter and Shine Dalgarno sequence) necessary for expression of the desired
protein, with convenient sites for insertion of the gene of interest in the appropriate place relative to the control sequences. A
plasmid which has all of these features is referred to as an expression vector. In addition to plasmids that can be used for expression
in bacterial cells, expression vectors are also available that allow protein expression in a variety of eukaryotic cells.
Many sophisticated variations on such vectors have been created that have made it easy to produce and purify large amounts of any
protein of interest for which the gene has been cloned. A handy feature in some expression vectors is a sequence encoding an
affinity tag either up- or downstream of the gene being expressed. This sequence allows a short affinity tag (such as a run of
histidine residues) to be fused onto the encoded protein. The tag can be used to readily purify the protein, as described in the
section on affinity chromatography.

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8.7: Polymerase Chain Reaction (PCR)
Molecular cloning was the first method available to isolate a gene of interest and make many copies of it to obtain sufficient
amounts of the DNA to study. Today, there is a faster and easier way to obtain large amounts of a DNA sequence of interest -the
polymerase chain reaction (PCR). PCR allows one to use the power of DNA replication to amplify DNA enormously in a short
period of time. As you know, cells replicate their DNA before they divide, and in doing so, double the amount of the cell’s DNA.
PCR essentially mimics cellular DNA replication in the test tube, repeatedly copying the target DNA over and over, to produce
large quantities of the desired DNA.

Selective Replication
In contrast to cellular DNA replication, which amplifies all of a cell’s DNA during a replication cycle, PCR does targeted
amplification to replicate only a segment of DNA bounded by the two primers that determine where DNA polymerase begins
replication. Figure 8.34 illustrates the process. Each cycle of PCR involves three steps, denaturing, annealing and extension, each
of which occurs at a different temperature.

Figure 8.34 - Steps in the polymerase chain reaction. Image by Aleia Kim

The Starting Materials

Since PCR is, basically, replication of DNA in a test-tube, all the usual ingredients needed for DNA replication are required:
A template (the DNA containing the target sequence that is being copied)
Primers (to initiate the synthesis of the new DNA strands)
Thermostable DNA polymerase (to carry out the synthesis). The polymerase needs to be heat stable, because heat is used to
separate the template DNA strands in each cycle.
dNTPs (DNA nucleotides to build the new DNA strands).
The template is the DNA that contains the target you want to amplify (the "target" is the specific region of the DNA you want to
amplify).
The primers are short synthetic single-stranded DNA molecules whose sequence matches a region flanking the target sequence. It
is possible to chemically synthesize DNA molecules of any given base sequence, to use as primers. To make primers of the correct
sequence that will bind to the template DNA, it is necessary to know a little bit of the template sequence on either side of the region
of DNA to be amplified. DNA polymerases and dNTPs are commercially available from biotechnology supply companies.

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 Figure 8.35 - PCR tubes with DNA samples ready for reaction. Wikipedia
First, all of the reagents are mixed together. Primers are present in millions of fold excess over the template. This is important
because each newly made DNA strand starts from a primer. The first step of the process involves separating the strands of the
target DNA by heating to near boiling.
Next, the solution is cooled to a temperature that favors complementary DNA sequences finding each other and making base pairs,
a process called annealing. Since the primers are present in great excess, the complementary sequences they target are readily found
and base-paired to the primers. These primers direct the synthesis of DNA. Only where a primer anneals to a DNA strand will
replication occur, since DNA polymerases require a primer to begin synthesis of a new strand.

Figure 8.36 - A PCR thermocycler system. Wikipedia

Extension
In the third step in the process, the DNA polymerase replicates DNA by extension from the 3’ end of the primer, making a new
DNA strand. At the end of the first cycle, there are twice as many DNA molecules, just as in cellular replication. But in PCR, the
process is repeated, usually for between 25 and 30 cycles. At the end of the process, there is a theoretical yield of 230 (over 1
billion times) more DNA than there was to start. (This enormous amplification power is the reason that PCR is so useful for
forensic investigations, where very tiny amounts of DNA may be available at a crime scene.)
The temperature cycles are controlled in a thermocycler, which repeatedly raises and lowers temperatures according to the set
program. Since each cycle can be completed in a couple of minutes, the entire amplification can be completed very rapidly. The

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resulting DNA is analyzed on a gel to ensure that it is of the expected size, and depending on what it is to be used for, may also be
sequenced, to be certain that it is the desired fragment.

Mutagenesis
PCR is frequently used to obtain gene sequences to be cloned into vectors for protein expression, for example. Besides simplicity
and speed, PCR also has other advantages. Because primers can be synthesized that differ from the template sequence at any given
position, it is possible to use PCR for site-directed mutagenesis. That is, PCR can be used to mutate a gene at a desired position in
the sequence. This allows the proteins encoded by the normal and mutant genes to be expressed, purified and compared.

Analysis of gene expression

PCR can also be used to measure gene expression. Where in PCR the amount of amplified product is not determined till the end of
all the cycles, a variation called quantitative real-time PCR is used, in which the accumulation of product is measured at each cycle.
This is possible because real-time PCR machines have a detector module that can measure the levels of a fluorescent marker in the
reaction, with the amount of fluorescence proportional to the amount of amplified product. By following the accumulation of
product over the cycles it is possible to calculate the amount of starting template. To measure gene expression, the template used is
mRNA reverse-transcribed into cDNA (see below). This coupling of reverse transcription with quantitative real-time PCR is called
qRT-PCR.

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8.8: Reverse Transcription
In the central dogma, DNA codes for mRNA, which codes for protein. One known exception to the central dogma is exhibited by
retroviruses. These RNA-encoded viruses have a phase in their life cycle in which their genomic RNA is converted back to DNA
by a virally-encoded enzyme known as reverse transcriptase. The ability to convert RNA to DNA is a method that is desirable in
the laboratory for numerous reasons. For example, converting RNAs of interest to cDNA is used in RT-PCR as well as in other
applications like microarray analysis.
Process
First, one creates a DNA oligonucleotide to serve as a primer for reverse transcriptase to use on a target RNA. The primer must, of
course, be complementary to a segment (near the 3’ end) of the RNA to be amplified. The RNA, reverse transcriptase, the primer,
and four dNTPs are mixed. With one round of replication, the RNA is converted to a single strand of DNA. Denaturation frees the
single stranded cDNA, which can be used as is, or converted to double-stranded cDNA, depending on the application.

Figure 8.37 - Reverse transcriptase of HIV. The nuclease function is needed for the viral life cycle, but not for lab use. Wikipedia

Detecting molecular interactions

The study of biochemistry is basically the study of the interactions of cellular molecules. Methods for detecting interactions among
biomolecules are, for this reason, very useful to biochemists. We will now discuss a couple of very different methods for detecting
these inter-molecular interactions.

Yeast two-hybrid system (Y2H)

Figure 8.38 - Structure of a eukaryotic transcriptional activator, showing the DNA-binding domain (DBD) and transcriptional
activation domain (TAD). Wikipedia
Yeast two-hybrid screening is a sophisticated technique for identifying which protein(s), out of a collection of all of a cell’s
proteins, interacts with a specific protein of interest. The method relies on the interaction between two proteins to reconstitute a
functional transcriptional activator within yeast cells. You may remember that many transcriptional activators are modular proteins
that have a domain that binds to DNA and another domain that activates transcription (Figure 8.38).

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If the transcription factor is split, so that the binding domain is attached to one protein, and the activation domain to another
protein, a functional transcriptional activator can only be re-created if the two “carrier”proteins come into close proximity - that is,
they interact. The presence of this functional activator can be detected by the expression of a reporter gene.
A simple way to understand this idea is by thinking of a transcriptional activator as a device, like a flashlight, that has two parts, the
battery and the lamp, that must be together in order to function. Neither a person who has just a battery nor one who has only the
lamp will be able to see in a dark room. But if the two interact by coming close enough to insert the battery in the flashlight, their
interaction can be detected by the fact that the flashlight will now be functional as evidenced by the light produced.
It takes two to tango
Figure 8.39 (A) shows the normal yeast transcriptional activator, GAL4, with both the DNA-binding (DBD) and Activation
domains (AD). It is able to stimulate transcription of the downstream reporter gene, lac z. Panels B and C show constructs that
produce the GAL4 DBD and AD, respectively, fused to other proteins, one of which is termed the “bait” and the other as “prey”.
Neither of these fusion proteins can stimulate transcription of the lac z gene. When constructs encoding both the bait and prey are
in the same yeast cell, if the bait protein interacts with the prey, the DBD and AD of the GAL4 will be brought together to
reconstitute a functional GAL4. The presence of functional GAL4 is readily detectable because it will stimulate expression of the
lac z reporter gene. If the bait and prey proteins do not interact, then there will be no lac z expression. When interaction is detected
through expression of the reporter gene, the specific prey protein can then be identified.
The yeast two-hybrid system allows for simultaneous screening of many prey proteins, by constructing large collections of fusion
constructs, with each potential protein partner of the bait protein fused to the GAL4 activation domain.

Figure 8.39 - Four scenarios for the yeast two-hybrid system. UAS = Upstream Activator Sequences - acts like a promoter.

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 Scenario A shows that the two transcription factors start out as one protein. Wikipedia

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8.9: FRET
Another method for detecting molecular interactions is Fluorescence resonance energy transfer (FRET) - also called Förster
resonance energy transfer, resonance energy transfer (RET) or electronic energy transfer (EET). The technique is based on the
observation that a molecule excited by the absorption of light can transfer energy to a nearby molecule if the emission spectrum of
the first molecule overlaps with the excitation spectrum of the second (Figure 8.9.1) This transfer of energy can only take place if
the two molecules are sufficiently close together (no more than a few nanometers apart).

Figure 8.9.1 : Excitation and emission spectra for donor and acceptor fluorophores in FRET. Wikipedia
In the technique, a donor fluorophore or an acceptor fluorophore is covalently attached to two molecules of interest. The acceptor
fluorophore is designed to accept energy from the donor molecule (orange dotted line in Figure 8.9.2) and fluoresce at a unique
wavelength (red arrow) when it receives that energy from the donor.

Figure 8.9.2 : Fluorescence resonance energy transfer between donor and acceptor chromophores. Image by Pehr Jacobson
Further, the wavelength of light that the donor absorbs is uniquely tailored for the donor fluorophore and has no effect on the
acceptor fluorophore. The only way the acceptor can fluoresce is if it is close enough to receive energy transferred from the donor
(red arrow). This fluorescence will have a unique wavelength, as well. If the donor and acceptor are not close enough together, the
donor fluoresces and emits light corresponding to the green or black arrow. These are different wavelengths than that of the red
arrow.
The experiment begins in the cell with one protein with a donor fluorophore and the other protein with an acceptor fluorophore.
Light of a wavelength that excites the donor fluorophore is shined on the cell. If a protein with a donor interacts with the protein
carrying an acceptor, then energy transfer occurs from the donor fluorophore to the acceptor and the unique fluorescence (red line)
of the acceptor is detected. If the two proteins do not interact, then little or no fluorescence from the acceptor is detected.

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8.10: Genome Editing (CRISPR)
The development of tools that would allow scientists to make specific, targeted changes in the genome has been the Holy Grail of
molecular biology. An ingenious new tool that is both simple and effective in making precise changes is poised to revolutionize the
field, much as PCR did in the 1980s. Known as the CRISPR/Cas9 system, and often abbreviated simply as CRISPR, it is based on
a sort of bacterial immune system that allows bacteria to recognize and inactivate viral invaders.

CRSPR
CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats, short repeated sequences found in prokaryotic
DNA, separated by spacer sequences derived from past encounters with, for example, a bacteriophage. Like the glass slipper left
behind by Cinderella that was later used to identify her, the pieces of the invader's sequences are a way for the bacteria to identify
the virus if it attacks again. Inserted into the bacterial genome, these sequences can later be transcribed into a guide RNA that
matches, and base-pairs with, sections of the viral genome if it was encountered again. A nuclease associated with the guide RNA
then cleaves the sequence base-paired with the guide RNA. (The nucleases are named Cas for CRISPR-associated.)
The essential elements of this system are a guide RNA that homes in on the target sequence and a nuclease that can make a cut in
the sequence that is bound by the guide RNA. By engineering guide RNAs complementary to a target gene, it is possible to target
the nuclease to cleave within that gene. In the CRISPR/Cas9 system, the Cas9 endonuclease cuts both strands of the gene sequence
targeted by the guide RNA (Figure 8.10.1). This generates a double-strand break that the cell attempts to repair.

Figure 8.10.1 : A guide RNA directs the Cas9 nuclease to its target gene. Image by Pehr Jacobsen
As you may remember, double-strand breaks in DNA can be repaired by simple, nonhomologous end joining (NHEJ) or by
homologous recombination. When a break is fixed by NHEJ, there is good chance that there will be deletions or insertions that will
inactivate the gene they are in. Thus, targeted cleavage of a site by CRISPR/Cas9 can easily and specifically inactivate a gene,
making it easy to characterize the gene's function.

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But, what if you wished to simply mutate the gene at a specific site to study the effect of the mutation? This, too, can be achieved.
If a homologous sequence bearing the specific mutation is provided, homologous recombination can repair the break, and at the
same time insert the exact mutation desired. It is obvious that if you can insert a mutation as just described, it should be possible to
correct a mutation in the genome by cleaving at the appropriate spot and providing the correct sequence as a template for repair by
homologous recombination. The simplicity of the system holds great promise for curing genetic diseases.
Scientists have also come up with some creative variations on the CRISPR/Cas9 system. For instance, one variant inactivates the
nuclease activity of Cas9. The guide RNA in this system pairs with the target sequence, but the Cas9 does not cleave it. Instead, the
Cas9 blocks the transcription of the downstream gene (Figure 8.10.2) This method allows specific genes to be turned off without
actually altering the DNA sequence.

Figure 8.10.2 : Inactive Cas9 can block transcription of a target gene. Image by Pehr Jacobsen
Another variation also uses a disabled Cas9, but this time, the Cas9 is fused to a transcriptional activation domain. In this situation,
the guide RNA positions the Cas9-activator domain in a place where it can enhance transcription from a specific promoter (Figure
8.10.3). Other variations on this theme attach histone-modifying enzymes or DNA methylases to the inactive Cas9. Again, the

guide RNA positions the Cas9 in the desired spot, and the enzyme attached to Cas9 can methylate the DNA or modify the histones
in that region.

Figure 8.10.3 : A Cas9-activator domain fusion can activate transcription of a target gene. Image by Pehr Jacobsen
CRISPR has already been used to edit genomes in a wide variety of species (and in human cell cultures). It may not be long before
the technique is approved for clinical use. In the meanwhile, CRISPR is transforming molecular biology.

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8.11: Protein Cleavage
Because of their large size, intact proteins can be difficult to study using analytical techniques, such as mass spectrometry.
Consequently, it is often desirable to break a large polypeptide down into smaller pieces. Proteases are enzymes that typically break
peptide bonds by binding to specific amino acid sequences in a protein and catalyzing their hydrolysis.
Chemical reagents, such as cyanogen bromide, which cleaves peptide bonds on the C-terminal side of a methionine residue can
also be used to cut larger proteins into smaller peptides. Common proteins performing this activity are found in the digestive
system and are shown below.
Subtilisin - C-terminal side of large uncharged side chains
Chymotrypsin - C terminal side of aromatics (Phe, Tyr, Trp)
Trypsin - C-terminal side of lysine and arginines (not next to proline)
Carboxypeptidase - N-terminal side of C-terminal amino acid
Elastase - Hydrolyzes C-side of small AAs (Gly, Ala)
Cyanogen Bromide (chemical) - Hydrolyzes C-side of Met

Figure 8.45 - Protease cleavage sites on a polypeptide

Determining mass and protein sequence

Mass spectrometry, as its name suggests, is a method that can be used to determine the masses of molecules. Once limited to
analyzing small molecules, it has since been adapted and improved to allow the analysis of biologically important molecules like
proteins and nucleic acids. Mass spectrometers use an electrical field to accelerate an ionized molecule toward a detector. The time
taken by an ionized molecule to move from its point of ionization to the detector will depend on both its mass and its charge and is
termed its time of flight (TOF).
MALDI-TOF

Figure 8.46 - A desktop MALDI-TOF system

MALDI-TOF (Matrix-assisted Laser Desorption Ionization - Time of Flight) is an analytical technique allowing one to determine
the molecular masses of biologically relevant molecules with great precision. It is commonly used in proteomics and determination
of masses of large biomolecules, including nucleic acids. The development of MALDI, which permits the production of ionic
forms of relatively large molecules, was crucial to the successful use of mass spectrometry of biomolecules. Figure 8.46 shows a
compact MALDI-TOF system.
The MALDI-TOF process involves three basic steps. First, the material to be analyzed is embedded in solid support material
(matrix) that can be volatilized in a vacuum chamber by a laser beam. In the second part of the process, a laser focused on the
matrix volatilizes the sample, causing the molecules within it to vaporize and, in the process, to form ions by either gaining or
losing protons. Third, the ions thus created in the sample are accelerated by an electric field towards a detector. Their rate of
movement towards the detector is a function of the ratio of their mass to charge (m/z). An ion with a mass of 100 and a charge of
+1 will move twice as fast as an ion with a mass of 200 and a charge of +1 and at the same rate as an ion with a mass of 200 and a
charge of +2. Thus, by precisely determining the time it takes for an ion to go from ionization (time zero of the laser treatment) to
being detected, the mass to charge ratio for all of the molecules in a sample can be readily determined.

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Ionization may result in destabilization of larger molecules, which fragment into smaller ones in the MALDI-TOF detection
chamber. The size of each of the sub-fragments of a larger molecule allows one to determine its identity if this is not previously
known. This fragmentation can be intentionally enhanced by having the accelerated ions collide with an inert gas, like argon.
Fragmentation of a molecule may also be carried out prior to analysis, as for example, by cleaving a protein into smaller peptides
by the use of enzymes or chemical agents. The amino acid sequence of a protein may be determined by using MALDI-TOF by
analyzing the precise molecular masses of the many short peptide fragments obtained from a protein. When one amino acid, for
example, fragments from a larger peptide, this can be detected as the difference in mass between the fragment with and without the
amino acid, since each amino acid will have a characteristic molecular mass. By peptide mass fingerprinting and analysis of
smaller fragments of individual peptides, the entire sequence of a polypeptide can, thus, be determined.

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8.12: Membrane Dynamics (FRAP)
Understanding the dynamics of movement in the membranes of cells is the province of the Fluorescence Recovery After
Photobleaching (FRAP) technique (Figure 8.47). This optical technique is used to measure the two dimensional lateral diffusion of
molecules in thin films, like membranes, using fluorescently labeled probes. It also has applications in protein binding.
In the method, a lipid bilayer is uniformly labeled with a fluorescent tag (Figure 8.47, Step A) and then a subset of the tag is
bleached using a laser (Step B). The spread of the bleached molecules is followed using a microscope (Step C). Information
obtained in this manner provides data about the rate of lateral diffusion occurring in a lipid bilayer (Step D).

Figure 8.47 - FRAP analysis. Wikipedia

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9: Chapter 10
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9.5: Section 5-

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10: Chapter 11
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 CHAPTER OVERVIEW

11: Point by Point

Topic hierarchy
11.1: In the Beginning
11.2: Structure and Function
11.3: Membranes
11.4: Catalysis
11.5: Energy
11.6: Metabolism
11.7: Information Processing
11.8: Techniques

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11.2: Structure and Function

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11.3: Membranes

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11.4: Catalysis

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11.5: Energy

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11.6: Metabolism

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11.7: Information Processing
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11.8: Techniques
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Index
A H R
actin Hind III replication
2.5: Structure and Function- Protein Function II 8.6: Isolating Genes 7.3: DNA Replication
restriction enzymes
C I 8.6: Isolating Genes
Cardiolipins Isoelectric focusing
2.8: Structure and Function - Lipids and Membranes 8.3: Electrophoresis S
Chemiosmotic model Isoprenoids secondary structure
5.2: Electron Transport and Oxidative 6.4: Other Lipids 2.4: Structure and Function- Proteins II
Phosphorylation serine proteases
Chromatography K 4.3: Mechanisms of Catalysis
8.2: Fractionation and Chromatography Techniques signaling
keratins
Chymotrypsin 2.4: Structure and Function- Proteins II 7.9: Signaling
4.3: Mechanisms of Catalysis Size Exclusion Chromatography
Kinesins
citric acid cycle 2.5: Structure and Function- Protein Function II 8.2: Fractionation and Chromatography Techniques
6.2: Citric Acid Cycle & Related Pathways SNARE proteins
Clotting L 3.3: Other Considerations in Membranes
4.4: Blood Clotting Southern blot
lysis
Collagen 8.4: Detection, identification and quantitation of
8.1: Cell Lysis
2.4: Structure and Function- Proteins II specific nucleic acids and proteins
CRISPR sphingolipids
8.10: Genome Editing (CRISPR)
M 6.4: Other Lipids
Macropinocytosis Squalene
D 3.3: Other Considerations in Membranes
6.4: Other Lipids
Microarrays statins
DNA (Z Form)
8.4: Detection, identification and quantitation of
2.6: Structure and Function - Nucleic Acids 6.4: Other Lipids
specific nucleic acids and proteins
dyneins microfilaments subtilisin
2.5: Structure and Function- Protein Function II 4.3: Mechanisms of Catalysis
2.5: Structure and Function- Protein Function II
microtubules
E 2.5: Structure and Function- Protein Function II
T
electrophoresis Morpheein model tertiary structure
8.3: Electrophoresis 4.2: Control of Enzymatic Activity 2.4: Structure and Function- Proteins II
Endocytosis transcription
3.3: Other Considerations in Membranes N 7.5: Transcription
Expression Cloning northern blot transcriptome
8.6: Isolating Genes 8.5: Transcriptomics
8.4: Detection, identification and quantitation of
Expression vectors specific nucleic acids and proteins transglutaminase
8.6: Isolating Genes Nucleic acids 4.4: Blood Clotting
2.6: Structure and Function - Nucleic Acids translation
F 7.7: Translation
Fluorescence Recovery After P tubulin
2.5: Structure and Function- Protein Function II
Photobleaching (FRAP) Palindromes
8.12: Membrane Dynamics (FRAP) 8.6: Isolating Genes
Fractionation Perilipin U
8.2: Fractionation and Chromatography Techniques 6.3: Fats and Fatty Acids urea cycle
phagocytosis 6.5: Amino Acids and the Urea Cycle
FRET
8.9: FRET 3.3: Other Considerations in Membranes
Photophosphorylation V
G 5.3: Energy - Photophosphorylation von Willebrand factor
gluconeogenesis Polyacrylamide gel electrophoresis 4.4: Blood Clotting
6.1: Metabolism - Sugars 8.3: Electrophoresis
glycolytic pathway polymerase chain reaction (PCR) W
6.1: Metabolism - Sugars 8.7: Polymerase Chain Reaction (PCR) warfarin
Porphyria 4.4: Blood Clotting
6.4: Other Lipids Western blot
promoter 8.4: Detection, identification and quantitation of
7.5: Transcription specific nucleic acids and proteins

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Glossary
Sample Word 1 | Sample Definition 1

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Detailed Licensing
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specific nucleic acids and proteins - CC BY-NC-SA 10.2: Section 2- - CC BY-NC-SA 4.0
4.0 10.3: Section 3- - CC BY-NC-SA 4.0
8.5: Transcriptomics - CC BY-NC-SA 4.0 10.4: Section 4- - CC BY-NC-SA 4.0
8.6: Isolating Genes - CC BY-NC-SA 4.0 10.5: Section 5- - CC BY-NC-SA 4.0
8.7: Polymerase Chain Reaction (PCR) - CC BY-NC- 10.6: Section 6- - CC BY-NC-SA 4.0
SA 4.0 11: Point by Point - CC BY-NC-SA 4.0
8.8: Reverse Transcription - CC BY-NC-SA 4.0 11.1: In the Beginning - CC BY-NC-SA 4.0
8.9: FRET - CC BY-NC-SA 4.0 11.2: Structure and Function - CC BY-NC-SA 4.0
8.10: Genome Editing (CRISPR) - CC BY-NC-SA 4.0 11.3: Membranes - CC BY-NC-SA 4.0
8.11: Protein Cleavage - CC BY-NC-SA 4.0 11.4: Catalysis - CC BY-NC-SA 4.0
8.12: Membrane Dynamics (FRAP) - CC BY-NC-SA 11.5: Energy - CC BY-NC-SA 4.0
4.0 11.6: Metabolism - CC BY-NC-SA 4.0
9: Chapter 10 - CC BY-NC-SA 4.0 11.7: Information Processing - CC BY-NC-SA 4.0
9.1: Section 1- - CC BY-NC-SA 4.0 11.8: Techniques - CC BY-NC-SA 4.0
9.2: Section 2- - CC BY-NC-SA 4.0 Back Matter - Undeclared
9.3: Section 3- - CC BY-NC-SA 4.0
Index - Undeclared
9.4: Section 4- - CC BY-NC-SA 4.0
Glossary - Undeclared
9.5: Section 5- - CC BY-NC-SA 4.0
Detailed Licensing - Undeclared
9.6: Section 6- - CC BY-NC-SA 4.0
10: Chapter 11 - CC BY-NC-SA 4.0

2 https://bio.libretexts.org/@go/page/97845