Fundamentals of

Biochemistry
Fourth Edition
Donald Voet Judith G. Voet
Charlotte W. Pratt

Chapter 12
Enzyme Kinetics, Inhibition, and Control

Copyright 2013 by John Wiley & Sons, Inc. All rights reserved.

Atorvastatin (Lipitor)

Atorvastatin
PDBid 1HWK

Chapter 12
Reaction Kinetics
Key Concepts 12.1
Simple rate equations describe the progress of first-order
and second-order reactions.
The MichaelisMenten equation relates the initial velocity
of a reaction to the maximal reaction velocity and the
Michaelis constant for a particular enzyme and substrate.
An enzymes overall catalytic efficiency is expressed as
kcat/KM.
A LineweaverBurk plot can be used to present kinetic
data and to calculate values for KM and Vmax.
Bisubstrate reactions can occur by an Ordered or Random
sequential mechanism or by a Ping Pong mechanism.

Kinetics
1.

Kinetics: the study of the rates at which chemical reactions occur; the rate of
a reaction and how this rate changes in response to different conditions is
intimately related to the path followed by the reaction and is therefore indicative
of its reaction mechanism
2. Enzyme Kinetics
a. Through kinetic studies the binding affinities of substrates and inhibitors to
an enzyme can be determined and the maximum catalytic rate of an
enzyme can be established.
b. By observing how the rate of an enzymatic reaction varies with the
reactions conditions and combining this information with that obtained from
chemical and structural studies of the enzyme, the enzymes catalytic
mechanism may be elucidated.
c. Most enzymes function as members of metabolic pathways; the study of
the kinetics of an enzymatic reaction leads to an understanding of that
enzymes role in an overall metabolic process.
d. Under proper conditions, the rate of an enzymatically catalyzed reaction is
proportional to the amount of the enzyme present and therefore most
enzyme assays are based on kinetic studies of the enzyme; Measurements
of enzymatically catalyzed reaction rates are therefore among the most
4
commonly employed procedures in biochemical and clinical analyses

Rates of Reaction
1. Simpler molecular processes by which a reaction may occur
A I1 I2 P
where I1 and I2 symbolize intermediates and thus its mechanistic description

2. Rates of Reactions
a. The order of a reaction can be experimentally determined by
measuring [A] or [P] as a function of time:
b.

v = - d[A] = d[P]
dt
dt
where v is the instantaneous rate (velocity) of the reaction

c.

The order of a specific reaction can be determined by

measuring the reactant or product concentrations as a function
of time and comparing the fit of these data to equations
5
describing this behavior for reactions of various orders.

Rate Equation
1. Rate of a process is proportional to the frequency with which the
reacting molecules simultaneously come together to the products of
the concentrations of the reactants
2.

Rate = k [A]a [B]b . . . [Z]z

where k is a proportionality constant
rate constant order of a reaction is defined as (a + b + + z)

3. Rate order corresponds to the molecularity of the reactionthe # of

molecules that must simultaneously collide in the elementary reaction

First Order Reaction

1. A plot of ln [A] versus t will
yield a straight line whose
slope is -k and whose intercept
on the ln [A] axis is ln [A]0
2. The time for half of the reactant
initially present to
decomposeits half-lifeis a
constant and hence
independent of the initial
concentration of the reactant
3. Unstable substances such as
radioactive nuclei decompose
through first-order reactions.
7

Second Order Reaction

1. Second-order progress curve descends more steeply than the firstorder curve before the first half-time, after which the first-order curve
is the more rapidly decreasing of the two
2. The half-time for a second-order reaction is expressed t1/2 = 1/k [A]0;
therefore it is dependent on the initial reactant concentration

9
Box 12-1a

Enzyme Kinetics

Nomenclature
Kinetic Mechanism --- A detailed
description of a series of elementary
reactions that describe an enzyme-catalyzed
reaction.
Chemical Mechanism --- A detailed
description of the chemistry of each step
including structures of transition states,
resonance etc.
11

Nomenclature

Enzyme --- E
Reactants --- A, B, C..
Products --- P, Q, R..
Inhibitors --- I, J, K.
Non-covalent complex --- ES
Commonly abbreviate [E] by omitting the brackets (eg.
E assumes molar concentration)
Rate constants --- k1, k-1, k2, etc.
12

Rate Equation for an Enzyme-Catalyzed

Unimolecular reaction (The MichaelisMenten Model)

13

MichaelisMenten Model
1. Enzyme-substrate complex: when the substrate concentration
becomes high enough to entirely convert the enzyme to the ES
form (Michaelis complex), the second step of the reaction
becomes rate limiting and the overall reaction rate becomes
insensitive to further increases in substrate concentration
2. Assumption of equilibrium: k-1 >> k2 so that the first step of the
reaction achieves equilibrium
3. Assumption of Steady State: [ES] remains approximately
constant until the substrate is nearly exhausted

14

Progress Curve: Simple EnzymeCatalyzed Reaction

Michaelis-Menten Equation
4.
5.

6.

The Michaelis-Menten Equation for enzyme kinetics

v0 = Vmax [S]/(Km + [S])
KM is the substrate concentration at which the reaction velocity is
half-maximal; KM is also a measure of the affinity of the enzyme for
its substrate providing k2/k1 is small compared with Ks, that is, k2 <<
k-1
Ks is the dissociation constant; as Ks decreases, the enzymes affinity
for substrate increases

16

Michaelis-Menten Kinetics

Enzyme Kinetic Parameters

7.

Catalytic constant: An enzymes kinetic parameter that describes its

catalytic efficiency
kcat = (Vmax/[E]T)

Quantity is also known as turnover number because it is the number of reaction processes
that each active site catalyzes per unit time

8.

Diffusion-controlled limit is in the range of 108 to 109M-1s-1 where k1 can be

no greater than the frequency with which enzyme and substrate molecules
collide with each other in solution; enzymes within this range must catalyze a
reaction almost every time they encounter a substrate molecule

9. At very high values of [S], v0 asymptotically approaches Vmax

19

Lineweaver-Burke Equation
10. Lineweaver-Burk equation for determining the values of Vmax

1/v0 = (Km/Vmax) x (1/[S]) + (1/Vmax)

20
Page 373

Double-Reciprocal
(Lineweaver-Burk) Plot

Enzyme Reactions May Pass Through a

Variety of Intermediates

Steady State Kinetics Incapable of

Revealing Enzyme Intermediates

Bisubstrate Reactions
1.

Enzymatic reactions involving two substrates and yielding two

products account for ~60% of known biochemical reactions.
2. Two types:
a. Transferase reactions in which enzyme catalyzes the transfer
of a specific functional group from one of the substrates to the
other
b. Oxidation-reduction reactions in which reducing equivalents
are transferred between the two substrates

24
Figure 12-5

Bisubstrate Reactions
Terminology by W. W. Cleland for representing enzymatic
reactions:
1.Substrates are designated by letters A, B, C, and D in the order
that they add to the enzyme
2.Stable enzyme forms are designated E, F, and G with E being the
free enzyme. A stable enzyme form is defined as one that by itself is
incapable of converting to another stable enzyme form

25
Page 375

Bisubstrate Reactions
3. Products are designated P, Q, R, and S in the order that
they leave the enzyme
4. The number of reactants and products in a given reaction
are specified, in order, by the terms Uni (one), Bi (two), Ter
(three), and Quad (four)

26

Bisubstrate Reaction: Group Transfer

Bisubstrate Reactions

Bisubstrate Reactions

Sequential reactions
1. Reactions in which all substrates must combine with the
enzyme before a reaction can occur and products be released
2. Ordered mechanism: a compulsory order of substrate
addition to the enzyme
3. Random mechanism: no preference for the order of substrate
addition
4. Characteristic feature of sequential Bi Bi reactions is that the
lines intersect to the left of the 1/v0 axis
29

Page 376

Ordered Bisubstrate Reaction

Random Bisubstrate Reaction

Bisubstrate Reactions

Rate Equations
1.Steady state kinetic measurements can be used to distinguish
among the foregoing bisubstrate mechanisms
2.Vmax is the maximal velocity of the enzyme obtained when both
A and B are present at saturating concentrations; KAM and KBM
are the respective concentrations of A and B necessary to
achieve Vmax in the presence of a saturating concentration of
the other; KAS and KBS are the respective dissociation constants
of A and B from the enzyme E
Page 376

32

Bisubstrate Reactions
Ping Pong Reactions
1.Mechanisms in which one or more products are released before all
substrates have been added
2.Also known as double-displacement reactions where substrates A and B
do not encounter one another on the surface of the enzyme
3.Parallel lines are diagnostic for Ping Pong mechanisms
Isotope Exchange
1.Sequential and Ping Pong bisubstrate mechanisms may be
differentiated using isotope exchange studies
2.Enzymes sucrose phosphorylase and maltose phosphorylase provide
two clearcut examples of enzymatically catalyzed isotopic reactions

33

Double-Displacement (Ping Pong)

Bisubstrate Reaction

Chapter 12
Reaction Kinetics
Checkpoint 12.1
Write the rate equations for a first-order and a secondorder reaction.
If you know a reactions half-life, can you determine its rate
constant? What other information do you need?
What are the differences between instantaneous velocity,
initial velocity, and maximal velocity for an enzymatic
reaction?
Derive the MichaelisMenten equation.

Chapter 12
Reaction Kinetics
Checkpoint 12.1
What do the values of KM and kcat/KM reveal about an
enzyme?
Why cant an enzyme have a kcat/KM value greater than 109
M1 s1?
Write the LineweaverBurk (double reciprocal) equation
and describe the features of a LineweaverBurk plot.
Why cant enzyme kinetics prove that a particular enzyme
mechanism is correct?
Use Cleland notation to describe Ordered and Random
sequential reactions and a Ping Pong reaction.

Chapter 12
Enzyme Inhibition
Key Concepts 12.2
Enzyme inhibitors interact reversibly or irreversibly with an
enzyme to alter its KM and/or Vmax values.
A competitive inhibitor binds to the enzymes active site
and increases the apparent KM for the reaction.
An uncompetitive enzyme inhibitor affects catalytic activity
such that both the apparent KM and the apparent Vmax
decrease.
A mixed enzyme inhibitor alters both catalytic activity and
substrate binding such that the apparent Vmax decreases and
the apparent KM may increase or decrease.

Inhibitors
1.
2.
3.

Inhibitors: Substances that reduce an enzymes activity

Structurally resemble their enzymes substrate but either
do not react or react very slowly compared to substrate
Used to probe the chemical and conformational nature of
a substrate-binding site as part of an effort to elucidate the
enzymes catalytic mechanism

38
Page 378

Oseltamivir (Tamiflu)

Oseltamivir (Tamiflu)-Avian Flu

Neuraminidase Complex

Oseltamivir (Tamiflu)
PDBid 2HU4

Adenosine Deaminase:
Transition State Analog Inhibitor

Competitive Inhibitors
1. Competitive inhibitor: A
substance that
competes directly with a
normal substrate for an
enzyme-binding site
2. Inhibitor resembles the
substrate to the extent
that it specifically binds
to the active site but
differs from it so as to
be unreactive

42

Competitive Inhibitors

43
Page 377

Competitive Inhibitors
3. I, the inhibitor, binds reversibly to the enzyme and
is in rapid equilibrium with it so that KI+ and EI
enzyme-inhibitor complexis catalytically inactive.
4. Acts by reducing the concentration of free enzyme
available for substrate binding.

44
Page 378

Competitive Inhibitors
5. The inhibitor does not affect the turnover number of the enzyme

45
Figure 12-6

6. Inactivator: if the inhibitor binds irreversibly to the enzyme and

somehow inactivates the enzyme

46
Page 380

Competitive Enzyme Inhibition

Competitive Inhibition: Ethanol

Treatment of Methanol Poisoning

Competitive Enzyme Inhibition

Competitive Enzyme Inhibition

Uncompetitive Inhibition
1.
2.
3.

Inhibitor binds directly to the enzyme-substrate complex

but not to the free enzyme
UI need not resemble the substrate
At high values of [S], v0 asymptotically approaches Vmax/
so that the effects of uncompetitive inhibition on Vmax are
not reversed by increasing the substrate concentration

51
Page 381

Uncompetitive Enzyme Inhibition

Uncompetitive Inhibition
4.

A series of Lineweaver-Burk plots at various uncompetitive

inhibitor concentrations consists of a family of parallel lines
indicative of a uncompetitive inhibition

53

Uncompetitive Enzyme Inhibition

Mixed Inhibition (Non-competitive)

1. Both the enzyme and the enzyme-substrate complex bind
inhibitor where both of the inhibitor-binding steps are assumed
to be at equilibrium with different dissociation constants
2. A mixed inhibitor binds to enzyme sites that participate in both
substrate binding and catalysis
3. Michaelis-Menten equation for mixed inhibition

v0 = (Vmax [S]) / (Km + [S])

55
Page 382

Mixed (Noncompetitive)
Enzyme Inhibition

Mixed (Noncompetitive)
Enzyme Inhibition

Mixed Inhibition
4. Mixed inhibitors are therefore effective at both high and low substrate
concentrations

Enzyme Inhibitor Effects

1.

Applied Enzyme
Inhibition

2.

3.

Acquired immunodeficiency
syndrome (AIDS) is caused by
human immunodeficiency virus
type 1
HIV -1 is targeted to and
specifically replicates within
helper T cells, essential
components of the immune
system
Reverse transcriptase inhibitors
are only partially effective
a. 3-azido-3-deoxythymidine
(AZT) was the first drug to be
approved by the FDA to fight
AIDS, but only slowed the
progression of an HIV
infection
b. Under the selective pressure
of an anti-HIV drug such as
AZT, the drugs target
receptor rapidly evolves to a
60
drug-resistant form

Enzyme Inhibition
4. HIV-1 polyproteins are cleaved by HIV-1 protease
5. Aspartic proteases and their catalytic mechanism
a. Proteolytic enzymes have three essential
catalytic components
i.
A nucleophile to attack the carbonyl C
atom of the scissile peptide to form a
tetrahedral intermediate
ii. An electrophile to stabilize the negative
charge that develops on the carbonyl O
atom of the tetrahedral intermediate
iii. A proton donor so as to make the amide N
atom of the scissile peptide a good leaving
group
61

Enzyme
Inhibition
6. HIV-1 protease
inhibitors are effective
anti-AIDS agents
a. HIV-1 protease
differs from
eukaryotic aspartic
proteases in that it
is a homodimer of
99-residue
subunits, even
though its x-ray
structure
resembles those of
eukaryotic aspartic
proteases

62

Chapter 12
Enzyme Inhibition
Checkpoint 12.2
What distinguishes an inhibitor from an inactivator?
Why might an enzymes substrate, transition state, and
product all serve as starting points for the design of a
competitive inhibitor?
Describe the effects of competitive, uncompetitive, and
mixed inhibitors on KM and Vmax.
How can inhibitor binding to an enzyme be quantified?
How does pure noncompetitive inhibition differ from other
forms of inhibition?

Chapter 12
Control of Enzyme Activity
Key Concepts 12.3
Allosteric effectors bind to multisubunit enzymes such as
aspartate transcarbamoylase, thereby inducing cooperative
conformational changes that alter the enzymes catalytic
activity.
Phosphorylation and dephosphorylation of an enzyme such
as glycogen phosphorylase can control its activity by shifting
the equilibrium between more active and less active
conformations.

Regulation of
Enzymatic
Activity

1.

Control of enzyme availability: The

amount of a given enzyme in a cell
depends on both its rate of synthesis
and its rate of degradation
2. Control of enzyme activity: An
enzymes catalytic activity may be
directly regulated through
conformational or structural alterations
a. Rate of enzymatically catalyzed
reaction is directly proportional to the
concentration of its enzymesubstrate complex
b. Catalytic activity of an enzyme can
be controlled through the variation of
its substrate-binding affinity
c. An enzymes substrate-binding
affinity may likewise vary with the
binding of small molecule effectors,
thereby changing the enzymes
65
catalytic activity
Page 386

Aspartate Transcarbamoylase Reaction

Regulation of Enzymatic Activity

The feedback inhibition of ATCase regulates Pyrimidine
Biosynthesis
1. Aspartate transcarbamoylase catalyzes the formation of Ncarbamoylaspartate from carbamoyl phosphate and aspartate
2. ATCase is heterotropically inhibited by cytidine triphosphate (CTP)
and is heterotropically activated by adenosine triphosphate (ATP);
CTP therefore decreases the enzymes catalytic rate, whereas
ATP increases it

67

Allosteric Effectors: ATCase Reaction

Regulation of Enzymatic Activity

3. Feedback inhibition: A
common mode of
metabolic regulation in
which the concentration
of a biosynthetic
pathway product
controls the activity of
an enzyme near the
beginning of that
pathway

69

Regulation of
Enzymatic
Activity

Figure 12-12

Structural Basis of Allosterism in

ATCase
1. Region of protein that
undergoes the most
profound conformational
rearrangement upon the
TR transition is a
flexible loop composed of
residues 230 to 250 in the
catalytic subunit
2. Both the inhibitor CTP and
activator ATP bind to the
same site on the outer
edge of the regulatory
subunit about 60 away
from the nearest catalytic
site
70

Regulation of
Enzymatic
Activity

Page 388

Allosteric changes alter ATCases substrate-binding sites

1. The regulatory subunits allosterically reduce the activity
of the catalytic subunits in the intact enzyme
2. ATP preferentially binds to ATCases R state whereas
CTP bind to the T state
3. TR transition maintains the proteins D3 symmetry
4. ATCases substrates each bind to a separate domain of
the catalytic subunit
5. ATCases tertiary and quaternary shifts are so tightly
coupled through extensive intersubunit contacts that they
cannot occur independently

71

ATCase: T-State vs. R-State

ATCase from E. coli

PDBids 5AT1 and 8ATC

ATCase: Conformational Changes

Control by Covalent Modification:

Phosphorylation

Product of Glycogen Phosphorylase

Rabbit Muscle Glycogen Phosphorylase

Conformational Changes:
Glycogen Phosphorylase

Glycogen phosphorylase
PDBids 8GPB and 7GPB

Glycogen Phosphorylase:
Control by Phosphorylation

Protein Phosphorylation
2. When there is high demand for ATP (low [ATP], low [G6P], and
high [AMP]), glycogen phosphorylase is stimulated and glycogen
synthase is inhibited, so flux favors glycogen breakdown

79
Figure 12-14b

Protein Phosphorylation
3. When [ATP] and [G6P] are high, the reverse is true
and glycogen synthesis is favored
4. AMP promotes phosphorylases T(inactive)R(active)
conformational shift by binding to the R state of the
enzyme at its allosteric effector site

80
Figure 12-15

Chapter 12
Control of Enzyme Activity
Checkpoint 12.3

Compare and contrast the actions of an allosteric effector, a

competitive enzyme inhibitor, and a noncompetitive
inhibitor.
Explain the structural basis for cooperative substrate
binding and allosteric control in ATCase.
Why are such allosteric enzymes composed of more than
one catalytic subunit?
Describe how phosphorylation and dephosphorylation
control the activity of glycogen phosphorylase.
List some advantages of phosphorylation/
dephosphorylation cascade systems over simple allosteric
regulation.

Copyright 2013 John Wiley & Sons, Inc.

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