ASSIGNMENT

Read and make brief notes on the following:

i. Genes and Genetics

ii. Mutation and Repair.

ENZYMES
Enzymes are proteins that catalyze (i.e., increase the rates of) chemical reactions so that they are compatible
with the processes of life. The fact that they aren't changed by participating in a reaction distinguishes
enzymes (catalysts) from substrates, which are the reactants on which catalysts work. Enzymes catalyze
biochemical reactions. They are similar to other chemical catalysts in many ways:
1. Enzymes and chemical catalysts both affect the rate but not the equilibrium constant of a chemical
reaction. Reactions proceed downhill energetically, in accordance with the Second Law of
Thermodynamics. Catalysts merely reduce the time that a thermodynamically favored reaction requires
to reach equilibrium.
2. Enzymes and chemical catalysts increase the rate of a chemical reaction in both directions, forward and
reverse. This principle of catalysis follows from the fact that catalysts can't change the equilibrium of
a reaction. Because a reaction at equilibrium occurs at the same rate for both directions, a catalyst that
speeds up the forward but not the reverse reaction necessarily alters the equilibrium of the reaction.
3. Enzymes and chemical catalysts bind their substrates, not permanently, but transiently—for a brief
time. There is no action at a distance involved. The portion of an enzyme that binds substrate and carries
out the actual catalysis is termed the active site.

Active site of an enzyme

This is a small region or pocket on the enzyme molecule where substrates specifically bind. The active site
occupies only a small region on the enzyme molecule where bonds are broken and new ones formed. It is
lined with many amino acid side chains that participate in the catalytic process. Each enzyme binds to a
single substrate because the active site and the substrate have complementary structures. The substrate’s
overall shape and charge distribution allow it to enter and interact with the enzyme’s active site. Most
enzymes are much larger than the substrates they act on, and only a small portion of the enzyme (around
3–4 amino acids)-active site, is directly involved in catalysis.

Enzymes differ from ordinary chemical catalysts in several important respects:

1. Enzymes are specific. Chemical catalysts can react with a variety of substrates. For example,
hydroxide ions can catalyze the formation of double bonds and also the hydrolysis of esters. Usually
enzymes catalyze only a single type of reaction, and often they work only on one or a few substrate
compounds.
2. Enzymes work under mild conditions. Chemical catalysts often require high temperature and/or
pressure to function. For example, nitrogen can be reduced to ammonia industrially by the Haber
 process, catalyzed by iron at 500° C and at 300 atmospheres pressure of N 2 . In contrast, the same
reaction is carried out enzymatically at 25°C and less than 1 atmosphere pressure of N 2 . These
gentle conditions of temperature, pressure, and pH characterize enzymatic catalysis, especially
within cells.
3. Enzymes are stereospecific. Chemical catalysis of a reaction usually leads to a mixture of
stereoisomers. For example, the addition of acid-catalyzed water to a double bond leads to an
equimolar (50:50) mixture of D and L isomers where the water is added. In contrast, catalysis of
water addition by enzymes results in complete formation of either the D or L isomer, but not both.
4. Enzymes are macromolecules. The macromolecules are composed of protein, or in a few cases,
RNA. Most chemical catalysts are either surfaces, for example, metals like platinum, or else small
ions, such as hydroxide ions.
5. Enzymes are often regulated. The regulation occurs either by the concentration of substrates, by
binding small molecules or other proteins, or by covalent modification of the enzymes' amino acid
side chains. Thus, an enzyme's effectiveness can be altered without changing the concentration of
the enzyme; on the other hand, the effectiveness of a chemical catalyst is generally determined by
its overall concentration.
Many enzymes require the presence of other compounds - cofactors - before their catalytic activity can be
exerted. This entire active complex is referred to as the holoenzyme; i.e., apoenzyme (protein portion) plus
the cofactor (coenzyme, prosthetic group or metal-ion activator) is called the holoenzyme.

Apoenzyme + Cofactor = Holoenzyme

According to Holum, the cofactor may be:

1. A coenzyme - a non-protein organic substance which is loosely attached (non-covalently) to the protein
part.
2. A prosthetic group - an organic substance which is firmly attached (covalently) to the protein or
apoenzyme portion.
3. A metal-ion-activator - these include K+, Fe2+, Fe3+, Cu2+, Co2+, Zn2+, Mn2+, Mg2+, Ca2+, and Mo3+.

Specificity of Enzymes
One of the properties of enzymes that makes them so important as diagnostic and research tools is the
specificity they exhibit relative to the reactions they catalyze. Specificity is defined as the ability to
discriminate between a substrate and competing molecules. The process involves multiple weak interactions
between the enzyme and the specific substrate molecule. Enzymes selectively recognize proper substrates
over other molecules. Specificity is controlled by structure - the unique fit of substrate with enzyme controls
the selectivity for substrate and the product yield.
In general, there are four distinct types of specificity:

1. Absolute specificity - the enzyme will catalyze only one reaction.

2. Group specificity - the enzyme will act only on molecules that have specific functional groups, such as
amino, phosphate and methyl groups.
3. Linkage specificity - the enzyme will act on a particular type of chemical bond regardless of
the rest of the molecular structure.
4. Stereochemical specificity - the enzyme will act on a particular steric or optical isomer

Classification of enzymes

Enzymes can be classified by the kind of chemical reaction catalyzed. Enzymes are named for the
substrates and type of reaction.

1. Oxidoreductases = catalyze oxidation-reduction reactions e.g dehydrogenases, oxidases,

oxygenases, reductases, peroxidases and hydroxylases.

In this reaction, ethanol is converted to acetaldehyde, and the cofactor, NAD, is converted to
NADH. In other words, ethanol is oxidized, and NAD is reduced.

2. Transferases = catalyze transfer of functional groups (amino, carboxyl, carbonyl, methyl,

phosphoryl and acyl groups) from one molecule to another e.g transcarboxylases, transmethylases
and transaminases.

3. Hydrolases = catalyze reactions in which cleavage of a bond is accompanied by the addition of

water (hydrolytic cleavage). Hydrolases include esterases, hydratases, phosphatases and
peptidases.

4. Lyases = catalyze removal of a group from or addition of a group to a double bond, or other
cleavages involving electron rearrangement. They catalyse reactions in which groups such as H 2 O,
CO2 , and NH3 are removed e.g decarboxylases, hydratases, dehydratases, deaminases and
synthases.
 5. Isomerases = catalyze several types of intramolecular rearrangements e.g epimerases catalyse the
inversion of asymmetric carbon atoms; mutases catalyse the intramolecular transfer of functional
groups.

6. Ligases = catalyze reactions in which two molecules are joined. They catalyse bond formation
between two molecules, the energy of which is always supplied by ATP hydrolysis. Names of many
ligases include the term synthetase. Other ligases are called carboxylases.

Factors affecting rates of enzyme catalyzed reactions

a) Temperature
Like most chemical reactions, the rate of an enzyme-catalyzed reaction increases as the temperature is
raised. A 10o C rise in temperature will increase the activity of most enzymes by 50 to 100%. As shown in
figure below, the reaction rate increases with temperature to a maximum level (optimum temperature), then
abruptly declines with further increase of temperature.

Over a period of time, enzymes will be

deactivated at even moderate
temperatures. Storage of enzymes at 5°C
or below is generally the most suitable.
Some enzymes lose their activity when
frozen.

b) pH
Enzymes are affected by changes in pH. The most favorable pH value - the point where the enzyme is most
active - is known as the optimum pH. Extremely high or low pH values generally result in complete loss
of activity for most enzymes. This is because pH causes a change in ionization state of amino acids in the
active site causing the protein to unfold. pH is also a factor in the stability of enzymes. As with activity, for
each enzyme there is also a region of pH optimal stability. The optimum pH value will vary greatly from
one enzyme to another.
 Effect of pH on reaction
rate

c) Substrate concentration
Enzyme activity increases with increase in substrate concentration if the amount of the enzyme is kept
constant until it reaches a maximum. After this point, increases in substrate concentration will not increase
the reaction velocity. When this maximum velocity had been reached, the entire available enzyme has been
converted to ES, the enzyme substrate complex. This point on the graph is designated Vmax. The Michaelis
constant Km is defined as the substrate concentration at 1/2 the maximum velocity.

Implications of Km
1. A small Km indicates that the enzyme requires
only a small amount of substrate to become
saturated. Hence, the maximu m velocity is reached
at relatively low substrate concentrations.
2. A large Km indicates the need for high substrate
concentrations to achieve maximu m reaction
velocity.
3. The substrate with the lowest Km upon which the
enzyme acts as a catalyst is frequently assumed to be
enzyme's natural substrate, though this is not true for
all enzymes.

d) Enzyme Concentration
In order to study the effect of increasing the enzyme concentration upon the reaction rate, the substrate must
be present in an excess amount; i.e., the reaction must be independent of the substrate concentration. Any
change in the amount of product formed over a specified period of time will be dependent upon the level
of enzyme present.
These reactions are said to be "zero order" because the rates are independent of substrate concentration, and
are equal to some constant k. The formation of product proceeds at a rate which is linear with time. The
addition of more substrate does not serve to increase the rate. In zero order kinetics, allowing the assay to
run for double time results in double the amount of product.
e) Inhibitors
The presence of inhibitors negatively affects enzyme function and activity. However, certain substances
will enhance or promote enzyme activities and these are known as promoters or activators.

f) Cofactors
The presence of cofactors e.g coenzymes enhances enzyme function and activity.

VITAMINS AND COENZYMES

Vitamins are organic constituents of food, which are required for the proper functioning, and maintenance
of body systems. They are required in small quantities and yet play important roles in metabolism. The
most prominent function of vitamins is as coenzymes for enzymatic reactions.

Vitamins are divided into two groups: water-soluble (B-complex and C) and fat-soluble (A, D, E and K).
Unlike water-soluble vitamins that need regular replacement in the body, fat-soluble vitamins are stored in
the liver and fatty tissues, and are eliminated much more slowly than water-soluble vitamins.

Co-enzymes are small organic molecules required by enzymes to help them in catalyzing reactions.
Coenzymes exist free in solution and bind to enzymes in temporary fashion. Many of them are derived from
the B-complex group of vitamins.
Examples of coenzymes

Vitamin Coenzyme form Role

Thiamine (Vit B1) Thiamin pyrophosphate (TPP) Oxidative decarboxylation reaction
(removal of CO2 )
Riboflavin (Vit B2) FAD and FMN Oxidation-reduction reactions
Niacin (Vit B3) NAD and NADP Oxidation-reduction reactions
Pantothenic acid (Vit B5) Coenzyme A (CoA) Carrier of acyl group in fatty acid
metabolism (synthesis)
Pyridoxine (Vit B6) Pyridoxal phosphate (PLP) Group-transfer reactions

Some coenzymes participate in group-transfer reactions that are difficult to carry out with amino acid side
chain chemistries alone. For example, none of the side chains of the normal 20 amino acids can accept an
amino group easily. On the other hand, the coenzyme pyridoxal phosphate has a carbonyl group that is well
adapted to accepting or donating amino groups.