INTERNATIONAL UNIVERSITY

SCHOOL OF BIOTECHNOLOGY

CHAPTER 1. ENZYMES

BIOCHEMISTRY
1
 Learning objectives
1. Comprehend how enzyme
kinetics relates to the chemical
kinetics presented in general
chemistry courses.
2. Perceive how kinetic parameters
are experimentally determined.
3. Learn the Michaelis–Menten
equation and the meaning of KM
and Vmax.
4. Understand the enzyme kinetics 2
 Brief Contents
1. What is an enzyme?
2. Classification of enzyme
3. Properties of enzyme
4. Enzyme kinetics
5. Catalysis
6. Enzyme regulation

3
 Detailed Contents
1. WHAT IS ENZYME?
2. PROPERTIES OF ENZYME
3. CLASSIFICATION OF ENZYMES
4. ENZYME KINETICS
Michaelis- Menten kinetics
Lineweaver Burk plots
Enzyme inhibition
5. CATALYSIS
Catalytic mechanisms
The role of cofactor in enzyme catalysis
Effects of temperature and pH on enzyme-
catalyzed reactions
Detailed mechanisms of enzyme catalysis
6. ENZYME REGULATION
Covalent modification
Allosteric regulation
4
 1. What is an enzyme?
 The most important functions of
enzyme is their role as catalyst. All
enzymes were considered to be
protein but some examples of RNA
molecules can also be catalyst.
 Living processes consists almost
entirely of biochemical reactions.
Without catalysts these reactions
would not occur fast enough to
sustain life. 5
How a biochemical reaction can happen?
 A reaction happens need the energy to
vibrate the molecules and the reactant
concentration enough. The energy here is
often provided by heat.
 However, in living system, high
temperature may harm the biological
structure
 Truly that the concentration in living
system is very low. So living organisms
solve these problems by using enzyme

6
Which properties help enzyme a biocatalyst

1. The rates of enzymatically catalyzed

reactions are often phenomenally
high
2. The enzyme are highly specific to
the reaction
3. Enzyme has the complex structures
so it can be regulated

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 2. Properties of enzymes

 The active site

 Activation energy
 Cofactor

8
2. The active site of enzyme:
• The active site takes up a relatively small part of the
total volume of an enzyme
• The active site is a three dimentional entily
• Substrates are bound to enzymes by multiple weak
attraction
• Active sites are clefts or crevices
• The specificity of binding depends on the precisely
defined arrangement of atoms in active site
• Functional residues in enzyme binding directly to
substrate to form or break down the bonding in
substrates to produce products 9
2. Active sites of an enzyme:
- Properties of active sites

+ Many different functional residues of amino acids,

water in bonding, metal ions, functional residues of
coenzyme

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2. Amino acids found in active sites

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2. The active site of enzyme:

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2. The active site of enzyme

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 4. Catalytic mechanism of enzyme
2-phosphoglycerate Enolic intermediate

Phosphoenolpyruvate

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2. The active site of enzyme
Lock and key model (Emil Fischer - 1890)

Induced fit model (Daniel Koshland - 1958)

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2. The active site of enzyme

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 2. Cấu tạo hóa học của enzyme
- Enzyme dị lập thể (alosteric):

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2. Active site of enzyme
- Some enzymes contain active sites at the structure not available for
reaction (unactivated) (zymogens): pepsinogen, trypsinogen,
chymotrypsinogen,...

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Zimogen và the activation of zimogen:
- Zimogen is an inactive molecule, it can not catylize substrate
- Activation of zimogen: zimogen  enzyme , by itseft or by
protease

Enzyme xúc tác cho

Zimogen Enzyme
quá trình hoạt hóa
Pepsinogen Pepsin Pepsin
Chymotrypsinog Trypsin,
Chymotrypsin
en chymotrypsin
Trypsin,
Trypsinogen Trypsin
enteropeptidase
Procarboxypepti Carboxypeptid
Trypsin
dase ase
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Proelastase Elastase Trypsin
2. Coenzyme
- What is Coenzyme?

is not protein (nonprotein), serve as

important role for catalyzing a reaction

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2. Coenzyme:
Coenzyme are derivatives of Vitamin:
Chemical group(s)
Coenzyme Vitamin
transferred
NAD+ and
Niacin (B3) Electrons
NADP+
Pantothenic acid Acetyl group and
Coenzyme A
(B5) other acyl groups
Methyl, formyl,
Tetrahydrofolic
Folic acid (B9) methylene and
acid
formimino groups
Carbonyl group
Menaquinone Vitamin K
and electrons
Ascorbic acid Vitamin C Electrons
Coenzyme F420 Riboflavin (B2) Electrons 22
 Coenzymes are NOT derivatives of Vitamin
Coenzyme Chemical group(s) transferred

Adenosine triphosphate Phosphate group

S-Adenosyl methionine Methyl group
3'-Phosphoadenosine-5'-
Sulfate group
phosphosulfate
Coenzyme Q Electrons
Tetrahydrobiopterin Oxygen atom and electrons
Diacylglycerols and lipid head
Cytidine triphosphate
groups
Nucleotide sugars Monosaccharides
Glutathione Electrons
Coenzyme M Methyl group
Coenzyme B Electrons
Methanofuran Formyl group 23

Tetrahydromethanopterin Methyl group

2.2. Coenzyme:
- Types of coenzyme:
+ Coenzyme of an oxido-reduction enzyme : NAD, NADP,
FAD, Ubiquinon,

+ Coenzyme of transport enzymes:

- Coenzyme A, Lipoic acid,...
- Biotin, Thiamin pyrophosphate, pyridoxal

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2.3. Cofactors
Are metal ions which is essential to enzymatic activities

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 3. CLASSIFICATION OF
ENZYMES
 There are the six major enzyme
categories:
1. Oxidoreductases:
Oxidoreductase catalyze oxidation-
reduction reactions. Subclasses of
this group include the
dehydogenases, oxidases,
oxygenases, reductases, peroxidase,
and hydroxylases
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 6. Cách gọi tên và phân loại enzyme

1. Oxidoreductase
- Enzymes have 2 parts, and coenzyme as followings:

+ NAD+ (Nicotinamide Adenine Dinucleotide)

+ NADP+ (Nicotinamide Adenine Dinucleotide Phosphate)

+ FAD (Flavin Adenine Dinucleotide)

+ FMN (Flavin Mononucleotide)

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 6. Cách gọi tên và phân loại enzyme

1. Oxidoreductase

Including sub-classes:

+ Dehydrogenase

+ Reductase

+ Oxygenase

+ Peroxydase

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1. Oxidoreductase
 Dehydrogenase:
- Catalyze the reaction in which H+ from substrate transfering
to NAD+, NADP+, FAD, FMN
Þ These enzymes can be found some first stages of ETC
-The reactions: H from NADH, NADPH, FADH2, FMNH2, can be
transferred to substrate and reduce the substrates
 Catalyze the synthesis reaction

30
 6. Cách gọi tên và phân loại enzyme
1. Oxidoreductase
 Dehydrogenase:
 Alcoholdehydrogenase
CH3CH2OH + NAD+  CH3CHO + NADH + H+
 important role in alcohol fermentation
Glutamatedehydrogenase
L-glutamic + H2O + NAD+  -ketoglutaric + NH3 +
NADH + H+
 N from soil to plant and microorganism by absorbing NH3
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1. Oxidoreductase
 Reductase:
Catalize the process in which electron can be transferred to
Oxygen and then Oxygen can combine with proton
4 Ferocytochrom c + O2 + 4H+ = 4 Fericytochrom c + 2H2O

 Oxygenase
Catalyze oxido-reduction reactions in which Oxygen can
combine with substrate to form functional groups as -OH,
-COOH
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1. Oxidoreductase
 Peroxydase:
- Including peroxydase and Catalase
- Coenzyme is hem
- Catalyze organic substrates in the presence of H2O2
Peroxydase:

Donating substrate + H2O2 = oxidized substrate + H2O

Catalase:

H2O2 + H2O2 = O2 + 2H2O

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2. Transferases
Transferases catalyze reactions that
involve the transfer of groups from
one molecule to another such as
amino, carboxyl, carbonyl, methyl,
phosphoryl, and acyl (RC=O) and the
enzyme names often go with trans
such as transcarboxylase,
transmethylase, and transaminases
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 3. Hydrolases: Hydrolases catalyze
reaction in which the cleavage of
bonds is accomplished by adding
water
Hydrolases include the esterases,
phosphatase, and peptidase

35
 4. Lyases: Lyases catalyze reaction
in which groups H2O, CO2, and NH3
are removed to form double bond or
are added to a double bond.
Examples: Decarboxylases,
hydratases, deaminases, and
syntases

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 5. Isomerases: This is a
heterogeneous group of enzyme.
Izomerases catalyze several types of
intramoleucular rearrangements. The
epimerases catalyze the inversion of
a symmetric carbon atoms. Mutases
catalyze the intramolecular transfer
of functional groups

37
 6. Ligases: Ligases catalyze bond in
formation between two substrate
molecules. The energy for the these
reaction is always supplied by ATP
hydrolysis. The name of many
ligases include the term synthetase
several other ligases are called
carboxylase

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 Enzyme kinetics
Kinetics is the study of the rates of
chemical reactions. For any reacting
system, thermodynamics can be used to
predict whether the reaction will
spontaneously occur. The kinetics of the
reaction indicate how fast the reaction
actually goes. Most of the biological
reactions that occur in the cells of living
organisms are greatly sped up by protein
catalysts called enzymes. Recall that
catalysts dramatically increase the rate of
a reaction without affecting the
equilibrium. 40
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 Enzyme kinetics
ELEMENTARY KINETICS

Our first objective for this exercise is to

show how enzyme kinetics relates to the
elementary kinetics you studied in General
Chemistry.
Let’s begin with a simple reaction, a
unimolecular reaction that can be
described as A being converted into B.
A B
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 Enzyme kinetics
Recall that since there is only one reactant, this is a
first order reaction whose rate, or velocity, can be
described as the disappearance of the reactant over
time or the appearance of the product over time.
The reaction rate is represented by the slopes of
these lines and is not constant. For this first-order
reaction, the rate is directly proportional to the
concentration of reactant. Here K is the first-order
rate constant for the reaction. Although the rate
constant does not change, the velocity of the
reaction begins to decrease as the concentration of
the reactant diminishes and the product
accumulates. The rate of the reaction eventually
slows to zero as the product of the initial forward
reaction becomes a reactant for the reverse
reaction. Equilibrium is the pointwhere the forward
and reverse reaction rates are equal, and the overall 46

rate of the reaction is zero.

47
 Enzyme kinetics

In a simple enzyme-catalyzed reaction, the

reactants are labeled S for substrates and
products are labeled P. This looks like our
first example, a simple first-order reaction in
which substrate is converted to product.
Similarly, the rate of an enzyme-catalyzed
reaction is either the disappearance of the
substrate or the formation of the product
over time. As the reaction proceeds, the
reverse reaction begins to compete with the
forward reaction until the rates are equal,
and equilibrium has been reached. 48
49
 Enzyme kinetics
The rate equation for a enzyme-catalyzed
reaction with a single substrate is the
same as the rate equation for the simple
nonenzymatic reaction, but only when the
concentration of substrate is so low that
the enzyme has very little chance to
convert it to product. Under these unique
conditions, it appears that the reaction is
first order with respect to substrate.
However, this special case is much too
limiting and rarely applies in biological and
experimental systems. Consequently,
biochemists must use a different model to
describe the kinetics of biological reactions
50

catalyzed by enzymes.
51
 Enzyme kinetics
ENZYME KINETICS
The kinetics of an enzyme-catalyzed
reaction can be studied when the
concentration of the enzyme is small
compared to the concentration of the
substrate. As long as the substrate is in
large excess over enzyme, altering its
concentration does not change the rate.
This is quite different from the first-order
reaction in the previous example, where
the rate depended on the substrate
concentration. Here, the rate does not
depend on the substrate concentration,
and the reaction is said to be zero order 52

with respect to substrate.

53
 Enzyme kinetics
Near the turn of the 20th century, Adrian Brown
first observed this conflicting behavior of enzyme-
catalyzed reactions. It became apparent to him
that the substrate must form a complex with the
enzyme. This observation marked the beginning
of the study of enzyme kinetics. Brown correctly
deduced that a zero-order reaction with respect
to substrate occurred when the enzyme was
saturated with substrate. Essentially, the limited
number of active sites put a ceiling on how fast
the reaction could proceed. Because the enzyme
concentration is rate limiting, the rate is
independent of substrate concentration. The zero-
order rate constant is referred to as the maximal
velocity, or Vmax.
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 Enzyme kinetics
To account for this kinetic behavior caused by the
substrate’s interaction with enzyme, the model
for an enzyme-catalyzed reaction must include an
enzyme-substrate complex, or ES complex. The
reaction scheme is shown here. The rate
constants k1 and k–1 are the forward and reverse
rate constants for the formation of the ES
complex, and k2 is the rate at which the ES
complex converts the bound substrate into
product. Notice that in order to simplify this
model, we assume that the product formation
step is irreversible (that is, there is no k–2).

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 Enzyme kinetics
Catalysts are generally not
considered reactants because they
facilitate reactions without being
altered. However, in an enzyme-
catalyzed reaction, the enzyme does
participate as a “pseudo-reactant”
because it must bind and form a
complex with substrate, and the
availability of its active sites affects
the overall rate of the reaction. 60
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62
 Enzyme kinetics
MICHAELIS–MENTEN EQUATION
We have seen that for an enzyme-
catalyzed reaction, we can write a
simple rate equation only when the
concentration of substrate is
saturating or when the concentration
of enzyme is saturating. In between
these two extremes, we need to use
a slightly more complex rate
equation called the Michaelis–Menten
equation. 63
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 Enzyme kinetics
The Michaelis–Menten equation is limited
to the initial rate v0 so that we can ignore
the possibility of the product accumulating
and undergoing the reverse reaction. The
Michaelis–Menten equation and its
hyperbolic graph describe how the
reaction rate varies with substrate
concentration for a one-substrate reaction.
It is important to note that despite the
similarities in shape, this graph is NOT a
graph of the concentration of product
forming over time.
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 Enzyme kinetics

This graph shows the reaction

progress of an enzyme-catalyzed
reaction. The Michaelis–Menten
model makes a steady-state
assumption, meaning that the
concentration of the ES complex
remains constant. This assumption is
valid only when the concentration of
substrate is much greater than the
total enzyme concentration.
72
 Enzyme kinetics
KINETIC PARAMETERS
Vmax is the maximal velocity that can be
achieved by an enzyme under the special
case of saturating substrate concentration.
KM is a “lumped” rate constant
incorporating all the rate constants for ES
and P formation: k1, k–1, and k2. It is equal
to the substrate concentration that gives
1/2-saturation of the enzyme. It is
therefore also equal to the substrate
concentration at one-half the Vmax.
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74
 Enzyme kinetics

For a reaction that follows the Michaelis–Menten

kinetic model, the KM is inversely related to the
proportion of total enzyme (Et) which is in complex
with substrate. In other words, the higher the KM,
the lower the fraction of enzyme with a bound
substrate. Although loosely related, the KM is not
the same as the enzyme’s affinity for substrate, for
it also takes in to account the decomposition of the
ES complex to product. The most useful definition
for KM is that it is a constant that describes the
dependence of v0 on S, and dictates the steepness
of the shape of the Michaelis–Menten curve.
Experiment with the sliders to change Vmax and
KM. Note how the shape of the line changes with
KM. 75
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 Enzyme kinetics
A KINETIC EXPERIMENT

To help you understand the

hyperbolic relationship between the
substrate concentration and the rate
of an enzyme-catalyzed reaction
which is described by the Michaelis–
Menten equation, we will now
demonstrate a kinetic experiment.
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 Enzyme kinetics
To experimentally determine the relationship
between substrate concentration and initial
velocity, a biochemist sets up a series of test
tubes for the reaction. Each tube contains a
constant amount of enzyme. But the amount
of the substrate placed in each tube varies.
For easy analysis, the biochemist often
chooses a substrate that yields a product that
has a different color or different fluorescence.
In order to measure the initial velocity, the
reaction is only run for a short length of time.
This is done so that only a small percentage
of substrate is converted into product, and
therefore there will be no kinetic contribution
from the reverse reaction of product
formation, and k–2 can still be ignored. 80
81
 Enzyme kinetics
The amount of product formed over
time is monitored. The amount of
product formed divided by the brief
time of incubation is the slope of the
early linear phase of the reaction.
This rate of product formation is the
initial velocity of the reaction. Note
that the tubes containing the most
substrate yield the highest initial
velocities. 82
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 Enzyme kinetics
A plot of initial velocity versus substrate
concentration yields a hyperbolic curve
consistent with the Michaelis–Menten
model of enzyme activity. The important
kinetic parameters Vmax and KM can be
estimated from this graph. The Vmax is
the point where the enzyme is fully
saturated and cannot achieve a higher
initial velocity Vmax and is therefore the
upper limit for v0. The KM is the substrate
concentration that corresponds to 1/2 of
the Vmax. More precise values for Vmax
and KM can be obtained by using a curve-
fitting program. Remember that the
Michaelis–Menten equation is a
mathematical expression for a hyperbola. 88
 Enzyme kinetics
VIRTUAL ENZYME KINETICS

Now imagine that you are a biochemist

attempting to characterize the kinetic behavior of
a new mutant of the motor protein kinesin You
will need to set up reaction tubes and collect data
to determine the values for Vmax and KM. You
will be monitoring the ATPase activity of the
kinesin by quantifying the conversion of
radiolabeled ATP to ADP.

Plan your experiment by indicating the proper

order of the steps.

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 Enzyme kinetics
First, prepare six 1 milliliter reaction
samples. Use the micropipettor to
add samples drop-wise to the tubes.
Be careful: sometimes the order of
addition matters. Be sure to leave
room in the tube for substrate, and
do not add substrate to any tube
until all samples are ready. When all
the samples are prepared, add
substrate to each tube and start the
timer. 91
 Enzyme kinetics
Now that you are ready to start the
reactions, add substrate and start the
timer. After the desired incubation time,
quench the reactions with detergent to
inactivate the enzyme.
You now need to determine the amount
of product formed in each reaction tube.
You will use thin layer chromatography to
separate the ADP product from the
leftover ATP substrate and quantify the
radiolabeled ADP by phosphoimaging and
densitometry. The values obtained are
now added to the table.
92
 Enzyme kinetics
From the experimental data, calculate the initial
velocities and enter the values into the last row of
the table.
Now use your data to construct a Michaelis–
Menten plot. First, select the proper axis labels,
then add your x and y data to plot a curve. While
you could roughly estimate Vmax and KM by eye
or achieve more precise values by fitting the data
computationally, it is instructive to do the curve
fitting by hand. Use the sliders for Vmax and KM
to create a curve that is computed from the
Michaelis–Menten equation and that fits your
data. Note how changing each kinetic parameter
affects the shape of the curve. Manipulate the
parameters until you are satisfied with the fit,
and then press "Accept fit."
93
 Enzyme kinetics
From your data you have obtained values
for Vmax and KM. You can now use these
kinetic parameters to compare the ATPase
activities of your kinesin mutant and wild-
type kinesin, in order to determine the
effect of the mutation. This information,
coupled with the location of the mutation
in the three-dimensional structure of
kinesin, could lead to new insights into the
mechanism of this motor protein.

94
 Enzyme kinetics
LINEWEAVER-BURK PLOTS

While computers can easily determine kinetic

parameters from a hyperbolic Michaelis–Menten
plot, these graphs are unwieldy for visually
estimating values for Vmax and KM. For this
purpose, the Michaelis–Menten equation can be
rearranged to a linear equation with the form y =
mx + b. The resulting plot is called a Lineweaver-
Burk or double-reciprocal plot. KM and Vmax can
be readily obtained from the x and y intercepts of
a Lineweaver-Burk plot. The y-intercept is
1/Vmax and the x-intercept is –1/KM.Try varying
the Vmax and KM on the following graphs.

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 Enzyme kinetics
CONCLUSION

Chemical kinetics and enzyme kinetics are

related, but because of the complex of enzyme
with bound substrate, enzyme kinetics only
correlates well with chemical kinetics in the
extreme cases with very limited substrate or vast
excess of substrate.

Michaelis–Menten enzyme kinetics describes the

behavior of enzymes over a wide range of
substrate concentrations. Two important kinetic
parameters are Vmax and KM.

97
 Enzyme kinetics
Vmax is the maximal velocity of an
enzyme-catalyzed reaction at saturating
substrate concentration. KM is loosely
related to substrate affinity, but a more
correct description is that it determines the
shape of the Michaelis–Menten hyperbolic
curve. Kinetic experiments can quantify
how the rate of an enzyme-catalyzed
reaction changes with the concentration of
substrate. Data obtained can be plotted
and fit to the Michaelis–Menten equation or
rearranged into a double-reciprocal plot.
From these graphs, the kinetic parameters
KM andVmax can be obtained. 98
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 Enzyme inhibitor
INTRODUCTION
Almost all therapeutic drugs are enzyme
inhibitors, from old medicine box
standards such as aspirin and penicillin to
the newest compounds used to treat HIV
infection. Understandably, enzyme kinetics
plays an outstanding role in this effort to
produce effective therapeutics, for kinetic
studies can quantify the degree that
inhibitors inactivate or slow down the
targeted enzyme’s catalytic rate and
describe its potential efficacy as a drug. 102
 Enzyme inhibitor
In addition, many naturally produced
toxic substances and chemical warfare
agents are also enzyme inhibitors. For
example, a peptide found in snake venom
became a lead compound in the
development of a class of hypertension
drugs that inhibit angiotensin-converting
enzyme (ACE inhibitors). The nerve gas
sarin is a potent inhibitor of an enzyme
important for synaptic transmission
innerve tissue. 103
 Enzyme inhibitor
Enzyme inhibitors alter the activity
of an enzyme by decreasing the
enzyme’s ability to bind substrate
Lowering its catalytic turnover, or
both.

104
 Enzyme inhibitor
OVERVIEW: REVERSIBLE VS.
IRREVERSIBLE INHIBITORS

There are two main classes of enzyme

inhibitors, reversible and irreversible, that
are differentiated by the magnitude of
their affinity for enzyme. Reversible
enzyme inhibitors bind and dissociate with
their enzyme in a equilibrium process.
Irreversible inhibitors bind tightly to an
enzyme to form an essentially permanent
complex.
105
 Enzyme inhibitor

IRREVERSIBLE INHIBITORS

Because they form such a tight complex

with the enzyme that they permanently
remove its catalytic activity, irreversible
inhibitors are also termed inactivators. For
example, kinetic studies on the male pattern
baldness drug finasteride (Propecia®)
reveal it to be an essentially irreversible
inhibitor of the 5-α-reductase. This enzyme
catalyzes one step in the conversion of
testosterone to dihydrotestosterone, which
is a hormone implicated in hair loss. 106
 Enzyme inhibitor
Some irreversible inhibitors even
become covalently bound to amino
acids in the active site of the enzyme
as they are brought through the
early chemical steps of catalysis.
Aspirin is such an example.

107
 Enzyme inhibitor
REVERSIBLE INHIBITORS
Reversible inhibitors can be classified as
competitive, mixed, or noncompetitive
inhibitors. If the detailed mechanism of
inhibition is known, then the classification
can be made by identifying where on the
enzyme the inhibitor binds, or the order
with which it binds, relative to substrate.
Alternatively, a determination of simple
kinetic parameters can generally be used
to classify the inhibitor.
108
 Enzyme inhibitor
COMPETITIVE INHIBITION

Competitive inhibitors compete with

substrate for an enzyme’s active site,
lowering the enzyme’s likelihood of
binding substrate and slowing the
observed reaction velocity.

109
 Enzyme inhibitor
Competitive kinetics
Kinetic studies can be used to determine
the type and potency of inhibition for an
unknown inhibitor. Typical steady-state
kinetic experiments can be performed
where reaction velocity is measured in the
presence of varying concentrations of
substrate. If inhibitor is then added, and
the data shows an increase in KM, yet the
Vmax is unaffected, this is the signature of
a competitive inhibitor.
110
 Enzyme inhibitor
MIXED INHIBITION
A mixed inhibitor binds to a site on the
enzyme and interferes with both apparent
substrate affinity and catalytic turnover,
thus affecting both the observed KM and
kcat for the enzyme-catalyzed reaction.
Mixed inhibitors do not bind directly in
the active site, and therefore do not block
substrate binding, but instead bind at sites
that can be proximal or distal from the
active site.
111
 Enzyme inhibitor
Mixed inhibitors can therefore bind to
free enzyme prior to substrate,
distorting the active site to a
nonoptimal conformation for
catalysis. The inhibitor-distorted
active site has trouble converting the
substrate to product before it
dissociates, resulting in a lowered
apparent substrate binding affinity.
112
 Enzyme inhibitor
Mixed inhibitors distort the active site,
interfering with the chemistry performed
by the enzyme. Therefore the enzymatic
turnover rate is slowed.
Mixed inhibitors can also bind after
substrate to the enzyme-substrate
complex. Similar to before, the inhibitor
binding to the ES complex distorts the
active site and its bound substrate to a
nonoptimal conformation, so that less ES
complex productively form product before
the substrate dissociates.
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 Enzyme inhibitor
Binding of the mixed inhibitor to the
ES complex also interferes with the
subsequent chemistry step.

114
 Enzyme inhibitor
Mixed kinetics

Steady-state experiments performed

in the presence of a mixed inhibitor
demontrate an increase in KM, and a
decrease in Vmax. Remember, Vmax
is simply kcat multiplied by the total
enzyme concentration. Therefore, a
decrease in kcat is a decrease in
Vmax. 115
 Enzyme inhibitor
NONCOMPETITIVE INHIBITION

Noncompetitive inhibition is a special case

of mixed inhibition where the affinity of
inhibitor for E and ES is the same.
Noncompetitive kinetics Steady-state
experiments performed in the presence of
a noncompetitive inhibitor demonstrate a
decrease in Vmax, yet KM is unaffected.

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 5. CATALYSIS

Catalytic mechanisms

 The role of cofactor in enzyme

catalysis
 Effects of temperature and pH on
enzyme-catalyzed reactions
 Detailed mechanisms of enzyme
catalysis
117
 6. ENZYME REGULATION
Covalent modification
Allosteric regulation

118
 7. Enzyme sources

1. Animal

Pancreatine Pepsin
119
 7. Enzyme sources
2. Plant

120

Papain Bromelain
 7. Enzyme sources
2. Plant

Amylase

-glucosidase 121
 7. Enzyme sources
3. Microorganism

Bacteria Yeast

122
 7. Enzyme sources
3. Microorganism

Molds 123
 7. Enzyme sources

Isolating
enzyme
(42oC/40h) Filling
the Enzyme
HCl 5% precipitati
enzyme
solution on by
NaCl 22%
Drying enzyme

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8. Enzyme synthesis

125
8. Enzyme synthesis

126
8. Enzyme synthesis

127
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8. Enzyme synthesis

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8. Enzyme synthesis

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8. Enzyme synthesis

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 8. Enzyme synthesis

Including 5 stages:

1. Amino acid activation

2. Inition

3. Elongation

4. Termination

5. Fold and mature enzyme

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8. Enzyme synthesis

134
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Initial complex formation

136
Initial complex formation

137
Initial complex formation

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Thank you for your kindly
listening

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