Chapter Three

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Chapter Three
Enzymes
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Main contents
Introduction
1. Structure and Function of Enzymes
2. Nomenclature and Classification of Enzymes
3. Properties and Catalytic Mechanisms of Enzymes
4. Kinetics of Enzyme-catalyzed Reactions
5. Regulation of Enzymse Activity
6. Clinical Applications of Enzymes
Disease cases
Questions
Concepts
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Concept of Enzyme

• Enzymes are the reaction catalysts of
  biological systems with extraordinary
  catalytic power and high degree of
  specificity for their substrates, which are
  produced by zoetic cells in organisms.

• Category of Biocatalysts

• Enzymes

• Ribozymes ( Deoxyribozymes )

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The importance of enzymes to human
- Essential to every biochemical process

• Act in organized sequences to catalyze 100s of
  stepwise reactions by which

- Nutrient molecules are degraded
- Chemical energy conserved and transformed
- Macromolecules made from simple precursors
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History of Enzyme Research

• BC 2000, there were records about ferment or made
  wine in our country.
• Before one century or so, Pasteur believed that
  the ferment was the result of yeast activation in
  cells.
• 1877,Kuhne first brought forward the concept of
  enzyme
• 1897,Buchner brothers made the ferment success by
  using the extracts of yeast from cells.
• 1926,Sumner successfully isolated the first
  enzyme----urease
• 1982,Cech first found that some special RNA
  molecules have the activity to catalyze the RNA
  splicing, putting forward ribozyme .
• 1995,Jack W.Szostak laboratory first reported
  that deoxyribozyme

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Section OneThe Molecular Structure and Function
of Enzyme
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1. Structure of Enzymes
1.1 Composition of Enzyme molecules
? Simple enzyme
? Conjugated enzyme
Apoenzyme
Prosthetic group or coenzyme
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Classification of cofactors according to how it
combines to apoenzyme
Combine with apoenzyme loosely, it could be
removed by ultrafiltration or dialysis
Combine with apoenzyme tightly through covalent
bonds , it couldnt be removed by
ultrafiltration or dialysis
9
The actions of different components in enzyme
during a catalyst reaction

• The specificity of a reaction decided by
  apoenzyme
• The sort and character of a reaction decided by
  cofactor

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(No Transcript)
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Some common coenzyme, their vitamin precursors
and deficiency disease
Coenzyme Precursor
Deficiency disease
Coenzyme A Pantothenic acid
Dermatitis FAD, FMN
Riboflavin (vit B2) Growth
retardation NAD, NADP Niacin
Pellagra Thiamine
Thiamine (vit B1)
Beriberi Pyrophosphate Tetrahydrofolate
Folic acid
Anemia Deoxyadenosyl Cobalamin (vit
B12) Pernicious anemia Cobalamin Co-subst
rate in the Vitamin C (ascorbic acid)
Scurvy hydroxylation of proline in
collagen Pyridoxal phosphate Pyridoxine (vit
B6) Dermatitis
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• Metalloenzyme
• The combination of metal ion with enzyme part
  is tight. It isnt ease to lose the metal ion
  during the isolation process.
• Metal-activated enzyme
• The combination of metal ion with enzyme part is
  not tight, but the metal ion is needed for the
  activity of enzyme.

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• The actions of metal ions
• Keep the conformation of enzyme stable
• Participate in the catalytic reaction, transfer
  electrons
• Act as a bridge between enzyme and substrate
• Neutralize the negatively charged group, decrease
  the electrostatic expel force

• The actions of small organic molecules
• Act as carrier to transfer electrons, protons
  or other groups during the reaction process

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1.2 The various sorts of enzymes

• Monomeric enzymeonly possess tertiary structure

Such as bovine ribonuclease

• Oligomeric enzymeconsists of multiple homologous
  or heterozygous subunits

Such as lactate dehydrogenase

• Multienzyme systemmultienzyme complex aggregated
  by various functionally different enzymes

Such as pyruvate dehydrogenase complex

• Multifunctional enzyme or tandem enzymean enzyme
  with several different functions on the same
  polypeptide chain because of the fusion event of
  genes during evolution procedure

Such as fatty acid synthase
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2. Active Site of an Enzymes
Essential groups That means the chemical groups
in the polypeptide chain related to the activity
of enzyme molecule.
The primary and spatial structure of chymotrypsin
? ?
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Active center (or active site ) of enzyme
Definition----The active site of an enzyme is the
region of the enzyme that can binds the
substrate, to form an enzyme-substrate complex,
and transforms it into product.
The active site is a three-dimensional entity,
often a cleft or crevice on the surface of the
protein, in which the substrate is bound by
multiple weak interactions.
Two models the lock-and-key model and the
induced-fit model.
17

• The essential groups inside the active site

• The essential groups outside the active site

Be needed for keeping the active conformation of
enzyme, but presented outside of the active site
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substrate
Essential group outside active site
Catalytic group
Binding group
Active site
? ?
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Active site of lysozyme
Glu35 and Asp52 ----catalytic groups
Trp62, 63, Asp101 and Trp108----binding groups
AF are six N-acetylglucosamine ring
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3. Structure and function of Enzymes
3.1 The Primary Structure of Enzymes and its
Function
The primary structure is the structural basis for
enzyme activity.
For example
Trypsin, chymotrypsin, elastase
Serine protease family, 25 homology, 4 S-S-
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-Trypsin cleaves on the carboxyl side of
positively charged Lys or Arg residues
H2N-Gly-Ile-Val-Glu-Gln-Cys-Cys-Thr-Ser-Lys-Ile-Se
r-Leu-Tyr-Gln-Leu-Glu-Asn-Tyr------
-Chymotrypsin cleaves on the carboxyl side of
bulky aromatic and hydrophobic amino acid residues
H2N-Gly-Ile-Val-Glu-Trp-Cys-Cys-Thr-Ser-Ile-Cys-Se
r-Ser-Leu-Gln-Phe-Glu-Asn-------
-Elastase cleaves on the carboxyl side of
residues with small uncharged side-chains
H2N-Ile-Val-Glu-Ala-Cys-Cys-Thr-Ser-Ile-Cys-Trp-Il
e-Gly-Gln-Phe-Glu-Asn-------
22
Their differing specificities are determined by
the nature of the amino acid groups in their
substrate-binding sites which are complementary
to the substrates that they act upon.
But all these three enzymes have a Ser195, His57,
Asp102, which are the main groups involved in the
catalytic mechanism.
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3.2 The Spatial Structure of Enzymes and its
Function
The catalytic activity of enzyme also depends on
its conformation such as trypsin etc
Trypsin binding site
Gly216----Asp189----Gly226
Chymotrypsin binding site
Gly216----Ser189----Gly226
To see fig 5-4 page 101
Elastase binding site
Val216----Ser189----Val190
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Only intact enzyme possesses catalytic activity
For an oligomeric enzyme, each single subunit
couldnt exhibit activity.
-Trypsin EC 3.4.21.4
- Chymotrypsin EC 3.4.21.1
- Elastase EC 3.4.21.36
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Section Two
The Nomenclature and Classification of Enzyme
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1. Nomenclature of Enzyme
International Union of Biochemistry and Molecular
Biology (IUBMB)
Common naming method Each enzyme is named
conventionally with ase as suffix
Systematic naming method Substrate name
reaction type EC 4 digit number awarded by
Enzyme Commission (EC)
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-Enzymes are classified into six major groups
defined by the reaction that they catalyze.
-Each enzyme has a unique four-digit
classification number according to the
classification criterion of enzyme brought
forward by International Enzyme Commission
(EC). Samples Urease EC 3.5.1.5 Hexokinase
EC 2.7.1.1
28
- Many named by adding suffix ase to the name
of their substrate or a word describing their
activity
Such as lactate dehydrogenase Pyruvate
dehydrogenase hexokinase
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2. Classification of Enzyme
(1) Oxidoreductases (2) Transferases (3)
Hydrolases (4) Lyases (5) Isomerases (6)
Ligases, synthetases
OTHLIL
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International classification of enzymes
Class Name Type of reaction catalyzed
Example
1 Oxidoreductases Transfer of
electrons A-B?AB- alcohol


dehydrogenase 2 Transferases
Transfer of functional groups
Hexokinase
A-B C ?A B-C 3
Hydrolases Hydrolysis reactions
Trypsin
A-B H2O ?A-H B-OH 4
Lyases Cleavage of C-C,
C-O, C-N Pyruvate decar-
and other bonds,
often forming boxylase
a double bond
A-B ?AB X-Y 5
Isomerases Transfer of groups
within a molecule Maleate
A-B ?A-B
isomerase 6
Ligases Bond formation coupled
Pyruvate or synthases
to ATP hydrolysis
carboxylase
A B ? A-B
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Section Three
Properties and Catalytic Mechanisms of Enzymes

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1. Properties of Enzyme Catalyzed Reaction
The common characters of an enzyme and a
general catalyst
Before or after a reaction, enzymes are
not changed themselves Enzymes do not
alter the equilibrium position, but do
accelerate the attainment of the equilibrium
position by speeding up the forward and
reverse reactions. The reaction is
thermodynamically permissive.
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The Characters of enzyme catalysis
(1) High effectively catalytic rate ( The
rate can be increased up to
1081020-fold). (2) Highly specificity (3)
Reaction enzyme-catalyzed can be regulated.
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• 1.1 Highly Catalytic Ability of Enzymes

• (1) High effectively catalytic rate ( The
  rate can be increased up to
  1081020-fold).
• (2) No high temperature is needed
• (3) Can more effectively decrease the activation
  energy of reaction

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High effectively catalytic ability
Mechanisms To decrease the activation
energy of reaction The number of active
molecules ? More molecules easily become
active molecules
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Gibbs free energy of activation (?G )
Non-enzyme -catalyzed reaction
Transition state
free energy
?G
General catalyzed reaction
Enzyme-catalyzed reaction
S
?G (total free energy change)
Ssubstrate Pproduct
P
Progress of reaction
The energy changes taking place during the course
of a biochemical reaction
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Activation energy and transition state
Energy barrier ----In all reactions there is an
energy barrier that need to transform the
substrate molecules into the transition
state. Transition state----an unstable chemical
form part-way between the substrates and the
products
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Free energy change The difference in energy level
between the substrates and products is termed the
change in Gibbs free energy (?G ).
A negative ?G indicates that the reaction is
thermodynamically favorable in the direction
indicated, whereas a positive ?G indicates that
the reaction is not thermodynamically favorable
and requires an input of energy to proceed in the
direction indicated.
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An energetically unfavorable reaction is often
driven by linking it to an energetically
favorable reaction, such as the hydrolysis of ATP.
Gibbs free energy of activation (?G ) is equal
to the difference in free energy between the
transition state and the substrate.
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1.2 Highly Specificity of Enzymes
Definition----It is termed as specificity of
enzyme that an enzyme only can catalyze one kind
of substrate to react and to form one kind of
product.
Why? Because there is active site of enzyme which
related to some amino acid residues in peptide
chain.
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The specificity of enzyme can be classified in
light of their substrates and actions

• Absolute specificityOnly catalyze one special
  kind of substrate and produce one kind of product
• Relative specificityCan catalyze one sort of
  compounds or chemical bonds
• Stereo specificityOnly act on a single
  stereoisomer of the substrate

For example LDH
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1.3 Activities of Enzymes Can Be Regulated

• To regulate the amount of enzyme in organism

For example synthesis or degradation of enzymes

• To regulate the catalytic effectiveness of enzyme
  

For example allosteric regulation, covalent
modification to enzymes
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2. Catalytic Mechanism of Enzymes
2.1 formation of ES Complex and Induced-fit
hypothesis
Enzyme-substrate complex
induced-fit hypothesis
When an enzyme approaches to its substrate
mutually, the structure of enzyme could be
changed and adapted each other, then combines
with the substrate. This process is termed
induced-fit hypothesis.
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Induced fit hypothesis
E itself undergoes a change in conformation upon
binding S
- Permits formation of additional weak binding
interactions in transition state - Brings
specific functional groups into proper position
for catalysis
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The flash of induced-fit hypothesis
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The induced-fit of carboxypeptidase
? ?
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2.2 Abzymes
In 1948 Linus Pauling proposed that compounds
resembling the transition state of a catalyzed
reaction should be very effective inhibitors of
enzymes. These mimics are called transition state
analogs. The transition state analogs use the
hapten to generate antibodies with catalytic
activity. These antibodies are called abzymes
which can be used as new enzymes and drugs.
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2.3 Several Factors Contributing to Enzyme
Catalysis
(1) Proximity and orientation effects
(2) Electrostatic effects
(3) Acid-base catalysis
(4) Covalent catalysis
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• Section Four
• Kinetics of Enzyme-Catalyzed Reactions

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• The concept of kinetics of enzyme-catalyzed
  reactions

It is a research about the factors to influence
the velocity of enzyme-catalyzed reactions.

• The factors including
• Conc. of enzyme, Conc. of substrate, pH,
  temperature, inhibitors, activators, etc.

? When one factor is studied, other factors
should be invariableness.
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The relationship between product formation and
time for an enzyme-catalyzed reaction
The relationship between substrate conc S and
initial reaction velocity (V0)
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1. The Effect of Substrate on the Velocity of
enzyme-catalyzed Reactions
1.1 Research Preconditions

• Only single substrate, single product
• The velocity of enzyme-catalyzed reaction under
  certain conditions
• To measure the initial velocity when the change
  of S is less than 5

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What is velocity of enzyme? Enzyme velocity are
normally reported as values at time zero ( symbol
V0 µmol min-1)
In fact, V0 means the rate of an enzyme-catalyzed
reaction at beginning of reaction.

• Why V0 should be used to represent an enzyme
  activity?
• The substrate conc is highest.
• No feedback inhibition by product.
• Enzyme activity is in best state.

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Enzyme velocity
Enzyme activity is commonly expressed by the
initial rate ( V0) of the reaction being
catalyzed. The units of V0 are µmol min-1
Two standard units of enzymes activity unit (U)
or the katal (Kat) 1 µmol min-1 1U 16.67
nanokat.
Total activity refers to the total units of
enzyme in a sample Specific activity is the
number of units per milligram of protein ( units
mg-1)
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1.2 Rectangular Hyperbola Plot of Initial
Velocity Versus Substrate Concentration
-At low S, V0 will increase with the increase
of S -At higher S, enzyme becomes saturated,
there would no increase of V0 even with the
increase of S. -A graph of V0 against S will
give a hyperbolic curve.
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Rectangular Hyperbola Curve
The relationship between substrate conc S and
initial reaction velocity (V0)
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-At low S, V will increase at directly ratio
with the increase of S. It is the first order
reaction
? ?
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With the increase of S, the velocity continues
increasing, but not at directly ratio. It is a
mixed-order reaction.
? ?
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-At higher S, enzyme becomes saturated, there
would no increase of V even with the increase of
S. Here, the velocity has reached its most
velocity. It is a zero-order reaction.
? ?
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1.3 Formulation of the Michaelis-Menten Equation
The model of enzyme-catalyzed reaction
intermediate product hypothesis

• intermediate product ES

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?1913, Michaelis and Menten
Michaelis-Menten Equation
S concentration of substrate V velocity
of reaction at different S Vmaxmaximum
velocity Km Michaelis constant
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The Michaelis-Menten Equation was deduced in
light of two hypothesises The formation of ES
is a quickly equilibrium reaction, but ES
decomposed to be product is slowly, the speed of
reaction dependents on the slow reaction, namely
Vk3ES. (1) The total S is much higher
than E, therefore, the S could be considered
no change at the initial stage of reaction,
SSt.
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The deductive process

• In a steady state, the velocity of formation of
  ES is equal to that of degradation, so, ES is
  equilibrium.
• K1 (Et-ES) SK2 ES K3 ES

then(2)changed to (Et-ES) S Km ES
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Coordinate that
Take (3) instead of (1), then,
Vk3ES (1)
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At very high S, the active sites would be
saturated completely, so EtES, V would
reach the maximum value,
VmaxK3ESK3Et (5)
Substituting (5) into (4), Michaelis-Menten
Equation is yielded
66
How to calculate Km value
When V is equal to half of Vm, it can be deduced
KmS
?Km value is equal to the S at ½ of Vm ,its
unit is mol/L?
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1.4 The Significance of Km and Vm

• Km value
• ? Km value is equal to the S at ½ of Vm ,its
  unit is mol/L.
• ? Significances
• a) Km is one of characteristic constants of an
  enzyme
• b) Km can be used to represent the affinity of
  enzyme to its substrate the larger, the less
  affinity
• c) There are different Km values for an enzyme
  to different substrates

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• Vmax

DefinitionVm is the velocity of enzyme-catalytic
reaction when enzyme is completely saturated,
directly proportional to E.
SignificanceVmaxK3 E
If total E has been known, the turnover number
of an enzyme or kinetic constant K3 can be
calculated in light of Vm.
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Turnover number of an enzyme
Definition The number of substrate molecules
are transferred into product in each unit of time
when the enzyme molecules are fully saturated by
substrates
Significance ----It can be used to compare the
catalytic ability of each unit of enzyme.
Generally, it is about 1104 /sec for the
turnover number of most enzymes.
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1.5 Measurement of Km and Vm Values

• Double Reciprocal Plot , or Lineweaver- Burk Plot

By taking the reciprocal of both sides of the
Michaelis-Menten equation
71

• Double reciprocal plot(LineweaverBurk plot )

Slope Km/Vmax
Intercept
Intercept
against
Plotting
Resulting in a double reciprocal or
Lineweaver-Burk plot
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2. The Effect of Enzyme on the Velocity of
enzyme-catalyzed Reactions

• When SgtgtE,enzymes could be saturated by
  substrates, the velocity of enzyme-catalytic
  reaction is directly proportional to the E .
• The equation should be
• V K3 E

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3. The Effect of Temperature on the Velocity of
enzyme-catalyzed Reactions
Optimum temperature
Temperature affects the rate of enzyme-catalyzed
reactions in two ways.
A rise in temperature increases the thermal
energy of the substrate molecules (at less than
40 ?).
At higher temperature( gt50?), enzymes are more
easily denatured.
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The overall effect of a rise in temperature on
the reaction rate of the enzyme is a balance
between these two opposing effects.
The optimum Temperature
The temperature of reaction at point which enzyme
possesses maximal efficiency is termed the
optimum temperature for this enzyme.
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4. The Effect of pH on the Velocity of
enzyme-catalyzed Reactions
Acetylcholine esterase
amylase
pepsin
Enzyme activity
Optimum pH The pH at which an enzyme has
maximal activity in solution.
The effect of pH on V
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5. The Effect of Inhibitors on the Velocity of
enzyme-catalyzed Reactions

• Inhibitors of enzymes
• The substances which can make the activity of
  enzyme decreasing but not cause the enzyme
  denaturation can be called the inhibitor of
  enzymes

• Different from the denaturation of enzymes

• Inhibitor has itself selection for enzymes
• The factors causing enzyme denaturation have no
  selection for enzymes

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Enzyme inhibition
The activities of an enzyme are easily affected
by many types of molecule which exist in
organisms. Some of them can decrease the
activities of enzymes, but others improve the
activities of enzymes.
Inhibitor----Any molecule which acts directly on
an enzyme to lower its catalytic rate is called
an inhibitor. Source----normal cellular
metabolites, drugs, toxins (foreign substances )
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• The category of inhibition

Irreversible inhibition
Reversible inhibition
Competitive inhibition Non-competitive
inhibition Uncompetitive inhibition
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5.1 Irreversible Enzyme Inhibition

• Concept
• Irreversible inhibitors usually bind covalently
  to the enzyme, often active groups , and make the
  enzyme lose its activity.
• For example
• Organophosphorus compound? hydroxy enzyme
• Detoxification --- PAM ( pyridine aldoxime
  methyliodide)
• Heavy metal ions or As (arsenic) ?? sulfhydryl
  enzyme
• Detoxification --- BAL (?????)

80
Organophosphorus compound
Hydroxy enzyme
Inactive E
acid
Lewisite
Sulfhydryl enzyme
Inactive E
acid
BAL binding with arsenic
Inactive E
BAL
Sulfhydryl enzyme
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5.2 Reversible Enzyme Inhibition

• Concept
• Reversible----Inhibitors usually bind to enzyme
  uncovalently. Enzyme inhibition can be overcame
  by removing the inhibitors from the enzyme, such
  as by dialysis, ultrafiltration

Category
a. Competitive inhibition b. Noncompetitive
inhibition c. Uncompetitive inhibition
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(1) Competitive Inhibition

• Definition
• If a inhibitor could bind to the active site of
  enzyme competitively instead of substrate due to
  that the structure of inhibitor is similar to
  substrates, the activity of the enzyme would be
  inhibited and decreased. The inhibition is termed
  competitive inhibition.

Reaction model
83
Competitive inhibition
Characteristics 1) Inhibitor structure is
similar to the enzymes 2) Inhibitor and
substrate can competitively bind to the active
site on an enzyme. 3) At high S, the inhibition
can be overcome. 4) Vmax keep unchanged but Km
increase.
84
Competitive inhibition
85
Competitive inhibition
Vmax keep unchanged but Km increase.
Km ?, Vmax-
86
For example 2
For example 1
Malonate and succinate can competitively bind to
succinate dehydrogenase
87
(2) Noncompetitive Inhibition

• Reaction model

88
Noncompetitive inhibition
Characteristics -A noncompetitive inhibitor
binds reversibly at a site other than the active
site and leads to a decrease in catalytic
activity. -An enzyme can bind the inhibitor or
substrate or both. -The effects of a
noncompetitive inhibitor cannot be overcome by
increasing S -Resulting in Vmax ?, Km keep the
same.
89
Noncompetitive inhibition
Km keep unchanged but Vmax decrease.
Km -, Vmax ?
90
(3) Uncompetitive Inhibition

• Reaction model

91
Uncompetitive Inhibition
Characteristics -A uncompetitive inhibitor only
binds reversibly ES complex, and leads to a
decrease in catalytic activity. -The inhibition
depends on the conc. of S and I. -The effects
of an uncompetitive inhibitor cannot be overcome
by increasing S. -Resulting in Vmax ?, Km ?.
92
Uncompetitive Inhibition
Km decrease and Vmax decrease too.
Vmax ?, Km ?
93
Comparison of inhibition by different reversible
inhibitors
94
6. Activators of Enzymes

• Activator
• Any substance which can cause enzyme activity
  increase can be termed as activator of enzyme.
• essential activator
• non-essential activator

Generally, many kinds of metal ions are
activators for some enzymes, such as Mg2, K,
and Mn2
Magnesium, Mg2 Potassium, K, Manganese, Mn2
95
Section Five Regulation of Enzyme Activity

96

• The object to be regulated
• Key enzymes associated with the committed steps

• The manners of regulation

97
1. Allosteric Regulation

• Definition

The regulation of enzyme activity occurs when
some metabolites combine reversibly to an
allosteric site spatially remote from the
catalytic site of an enzyme and change the
conformation of the enzyme ,resulting in the
change of enzyme activity. This regulation is
termed allosteric regulation.
98

• allosteric enzyme

• allosteric site

• allosteric effectors

99
1.1 The Mechanism of Allosteric Regulations
The conformational change of an allosteric enzyme
can cause itself activity change, increasing or
decreasing, depending on the effectors.
An alloseric activator increase the rate of
enzyme activity, while an allosteric inhibitor
decreases the activity of the enzyme.
Example Aspartate transcarbamoylase ( ATCase
) Its structure 6 catalytic subunits 6
regulatory subunits
100
CO2 glutamine ATP
CPS II
Activation
CPS II----carbamoyl phosphate synthetase II
Carbamoyl phosphate
ATCase----Aspartate transcarbamoylase
ATCase
Formation of N-carbamoylaspartate by ATCase is
the committed step in pyrimidine biosynthesis and
a key control point
H2PO4-
N-Carbamoylaspartate
Inhibition
CTP
101
ATP
hyperbolic-shaped curve
V0
No allosteric effectors
- CTP
sigmoidal-shaped curve
30
10
0
20
Aspartate (mM)
Plot of initial reaction velocity (V0) against
substrate concentration for the allosteric enzyme
ATCase
102
1.2 General Properties of Allosteric Enzymes
Key points 1) An allosteric enzyme is regulated
by its effectors (activator or inhibitor). 2)
Allosteric effectors bind noncovalently to the
enzyme they regulate 3) Allosteric enzymes are
often multi-subunit proteins. Allosteric enzymes
have often more than one active site. 4) A plot
of V0 against S for an allosteric enzyme gives
a sigmoidal-shaped curve. 5) The binding of one
of subunits of an allosteric enzyme with an
effector will induce a conformational change
103
2. Covalent Modification

• Definition----
• Reversible covalent modification is the
• making and breaking of a covalent bond
• between a nonprotein group and an enzyme
• molecule.

• Forms of Covalent Modification Phosphorylation /
  dephosphorylation
• adenylylation/deadenylylation
• methylation/demethylation
• -SH / -S-S , etc

104
Covalent Modification
Protein-OH
Pi
ATP
Protein phosphatase
Protein kinase
ADP
H2O
The reversible phosphorylation and
dephosphorylation of an enzyme
105
Covalent Modification
Key points 1) Change of a covalent bond 2) The
most common is the addition and removal of a
phosphate group, phosphorylation or
dephosphorylation 3) Enzymes----protein kinases
or phosphatases 4) The activity of an enzyme
after the modification can increase or
decrease 5) The modification is a rapid,
reversible and effective process
106
3. Zymogen
Concept---- Several enzymes are synthesized as
larger inactive precursor forms called proenzymes
or zymogens. That process involved irreversible
hydrolysis of one or more peptide bonds on a
zymogen is termed the activation of zymogen.
Examples Trypsin, chymotrypsin and elastase in
the pancreas
107
trypsinogen

enteropeptidase

trypsin
proelastase
Chymotrypsinogen


Chymotrypsin
elastase
The central role of trypsin in activating the
pancreatic zymogens
108
Blood clotting cascade Another example of the
occurrence of inactive zymogens is found in the
enzymes involved in the blood clotting
cascade. The whole process of blood clotting is
brought about by series of zymogen activations.
109
trypsin
enteropeptidase
The process of trypsinogen activation
110
4. Isoenzymes
Definition Multiple forms of an enzyme that can
catalyze the same reaction but differ from each
other in their amino acid sequences, substrate
affinity, Vm, and /or regulatory properties are
called isoenzymes (or isozymes )

111
For example
Lactate dehydrogenase, LDH1 LDH5)
112
Significances It can be used in diagnostic
differentiation of the liver diseases and
myocardium.
113
5. Genetic Control
-The amount of enzyme present is a balance
between the rates of its synthesis and
degradation. -The level of induction or
repression of the gene encoding the enzyme, and
the rate of degradation of its mRNA, will alter
the rate of synthesis of the enzyme protein.
-Once the enzyme protein has been synthesized,
the rate of its breakdown (half-life ) can also
be altered as a means of regulating enzyme
activity.
114
Section Six Clinical Applications of Enzymes

115
1. Enzymes and Pathogenesis
- defects basis of genetic disorders (eg.
enzyme activity missing)
- basis of some cancers (eg. growth controlling
enzyme permanently on)
116
Enzymes defects induce molecular diseases
Tyrosinase Glucose-6-phosphate
dehydrogenase Hypoxanthine guanine
phosphoribosyltransferase Cystathionine
synthase Phenylalanine hydroxylase
Albinism Favism Lesch-nyham syndrome
Homocystinuria Phenylketonuria
117
Phenylalanine hydroxylase
Individuals lacking this enzyme suffer from
phenylketonuria, PKU, an inborn error of
metabolism
118
Lesch-Nyhan syndrome (LNS) is a rare inherited
disease that disrupts the metabolism of the raw
material of genes. These raw materials are called
purines, and they are an essential part of DNA
and RNA. The body can either make purines (de
novo synthesis) or recycle them (the resalvage
pathway). Many enzymes are involved in these
pathways. When one of these enzymes is missing, a
wide range of problems can occur.
119
In LNS, there is a mutation in the HPRT1 gene
located on the X chromosome. The product of the
normal gene is the enzyme hypoxanthine-guanine
phosphoribosyltransferase, which speeds up the
recycling of purines from broken down DNA and
RNA. Many different types of mutations affect
this gene, and the result is a very low level of
the enzyme.
120
The molecular basis of glycogen storage disease
type 1a structure and function analysis of
mutations in glucose-6-phosphatase.
1 J Biol Chem 2002 Feb 15277(7)5047-53Related
Articles, Links
121
1 Blood 2003 Jan 1101(1)345-7Related Articles,
Links   HK Utrecht missense mutation in the
active site of human hexokinase associated with
hexokinase deficiency and severe nonspherocytic
hemolytic anemia.van Wijk R, Rijksen G, Huizinga
EG, Nieuwenhuis HK, van Solinge WW.Department of
Clinical Chemistry and the Department of
Hematology, University Medical Center Utrecht,
The Netherlands.
122
PABA, p-amino-benzoic acid
Glu dihydropterin
sulfanilamide inhibiting
1
dihydrofolic acid
2
1-dihydrofolate synthetase
Tetrahydrofolic acid
2-dihydrofolate reductase
Go to 87
123
2. Enzymes and Diagnosis of the Diseases
2.1 Blood Plasma Enzymes and Diagnosis of
Diseases
Some enzymes presenting in high concentration in
blood plasma have important functions such as
blood coagulation ( thrombin ), fibrin
dissolution ( plasmin ) and processing of
chylomicrons (lipoprotein lipase )
Some enzymes presenting in trace amount in blood
plasma generally come from other tissues, which
is called intracellular enzymes. The change of
the amount of them in plasma would give some
information about the tissues normal or nonnormal.
124
The blood plasma enzymes for disease diagnosis
125
2.2 Immobilized Enyzme and diagnosis of Diseases
2.3 Enzyme-linked Immunoassays and Diagnosis of
Diseases
2.4 Isoenzyme and Diagnosis of Diseases
2.5 Insoluble enzymes and Diagnosis of Diseases
126
3. Enzymes and Therapy of Diseases
Some enzymes can be used as therapeutic agents.
Strepotokinase, for the clearing of blood clots
Asparaginase therapy, for the leucocythemia
Intravenous administration of asparaginase could
reduce the host plasma level of asparagine,
which causes tumor regression.
127
Disease cases
1. Hepatitis (??)
2. Adenosine deaminase, ADA (???????)
3. Macrocytic anemia ( ??????)
128
Concepts
1 Biocatalysts 2 Enzymes, apoenzyme, coenzyme 3
Active Site of Enzyme 4 Allosteric regulation 5
chemical modification 6 Isoenzymes 7
zymogen 8 Km, Vm 9 reversible inhibition,
irreversible inhibition 10 competitive inhibition
129
Questions
1 What is the substrate specificity of an
enzyme? 2 If an enzyme has the EC number
4.3.2.1, What kind of enzyme does it belong to? 3
What is the optimum pH for an enzyme? 4 Why
does not the activity of an enzyme increase when
its substrate concentration is too high in
reaction system?
130
5 What is a holoenzyme? What is an apoenzyme?
What are cofactors?
6 What are Km and Vm? How to get Km and Vm of an
enzyme ?
7 How to analyze enzyme inhibition ?
8 What are the differences between competitive
and noncompetitive inhibition?