The Behavior of Proteins: Enzymes: © 2018 Cengage Learning. All Rights Reserved

Chapter 6
The Behavior of
Proteins: Enzymes
© 2018 Cengage Learning. All Rights Reserved.

Chapter Outline
(6-1) Enzyme kinetics vs. thermodynamics
(6-2) Rate of enzyme-catalyzed reactions
(6-3) Enzyme–substrate binding
(6-4) The Michaelis–Menten approach to enzyme
kinetics
(6-5) Examples of enzyme-catalyzed reactions
(6-6) Enzyme inhibition

Enzymes
• Biological catalysts, usually globular proteins
• Self-splicing RNA is the only exception
• Increase the rate of a reaction by a factor of up to
1020 over an uncatalyzed reaction
• Highly specific to the extent that they can distinguish
stereoisomers of a given compound
• Fine-tuned by regulatory processes

Thermodynamic Principles - Enzyme Catalysis
• Standard free energy change (ΔG°): Difference
between energies of reactants and products under
standard conditions
• Enzymes can speed up reactions but cannot alter the
equilibrium constant or the free energy change
• Reaction rate depends on its activation energy
(ΔG°‡), which is the energy input required to initiate a
reaction
• ΔG°‡ for an uncatalyzed reaction is higher than that for
a catalyzed reaction

The Transition State
• Intermediate stage in a reaction in which old bonds
break and new bonds are formed
• In the plot of the energies for a spontaneous reaction,
transition state lies at the maximum of curve
connecting the reactants and products
• ΔG°‡ can be the amount of free energy required to
bring the reactants to the transition state

The Transition State (continued)
• ΔG° of a reaction remains unchanged when a
catalyst is added, but ΔG°‡ is lowered
• Presence of an enzyme lowers ΔG°‡ needed for
substrate molecules to reach the transition state
• Concentration of the transition state increases

Effect of Catalysts on Activation Energy
• Consider the following reaction:
2H 2 O 2  2H 2 O + O 2
• ΔG°‡ is lowered if the reaction is allowed to proceed on

platinum surfaces but is lowered even more by
enzyme catalase

Enzyme Catalysis
• For a reaction taking place at constant temperature
and pressure, such as:
A B
• Change in free energy is given by the following
expression:
ΔG° = ΔΗ °  TΔS °
• Difference in energies between initial state and final
state is given by the following expression:
ΔG° =  RT lnK eq
• Change in free energy is related to the equilibrium
constant, Keq
Temperature Dependence of Catalysis
• Increasing the temperature of a reaction mixture will
increase the energy available to the reactants to
reach the transition state
• Occurs only to a limited extent with biochemical
reactions because increasing temperature will
eventually lead to enzyme denaturation

Enzyme Kinetics
• In a reaction of the form A + B → P, rate of the
reaction can be expressed in terms of:
• Rate of disappearance of one of the reactants
• Rate of appearance of the product
Rate of disappearance of A =  Δ  A  /Δt

Rate of disappearance of B =  Δ  B /Δt
Rate of appearance of P = Δ  P  /Δt
• Δ - Change
• [ ] - Concentration in moles per liter
• t - Time
Enzyme Kinetics (continued 1)
  A    B  P
Rate = = =
t t t
• Negative sign indicates that A and B are used up in the

reaction, while P is produced
Rate   A   B
f g
Rate = k  A   B
f g
• k - Proportionality constant called the rate constant

• f and g - Small whole numbers that are experimentally
determined
• Overall order of a reaction is the sum total of all
exponents
• Rate of the reaction A → P is given by the following
equation:
Rate = k  A 
1
• This reaction is first order with respect to A and first

order overall
• First order: Description of a reaction whose rate depends on
the first power of the concentration of a single reactant

• Consider the reaction A + B → C + D whose rate
equation is given by the following expression:
Rate = k  A   B
1 1
• Where k is the rate constant, exponent for [A] is 1, and

exponent for [B] is 1
• Reaction is said to be first order with respect to A, first
order with respect to B, and second order overall
• Second order: Description of a reaction whose rate
depends on the product of the concentrations of two
reactants

Second-Order Reactions - Example
• Consider the reaction of glycogenn with inorganic
phosphate, Pi
Glycogen n + Pi  Glucose-1-phosphate + Glycogen n1
• Reaction rate depends on concentrations of both

reactants
Rate = k  Glycogen   Pi  = k  Glycogen  Pi 

1 1
• Reaction is first order with respect to glycogen, first

order with respect to phosphate, and second order
overall

Zero-Order Reactions
• Reaction that proceeds at a constant rate,
independent of the concentration of reactant
For the reaction A  B
Rate = k  A  = k
0
• Rate depends on the presence of catalysts

• Enzyme-catalyzed reactions exhibit zero-order kinetics
when the reactant concentrations are so high that the
enzyme is completely saturated with reactant
molecules

Enzyme–Substrate Binding
• In an enzyme-catalyzed reaction:
• Substrate (S) is a reactant
• Active site: Portion of the enzyme surface to which
the substrate binds via noncovalent forces and at
which the reaction takes place
• Noncovalent forces include hydrogen bonding,
electrostatic attractions, and van der Waals attractions
• First step is binding of substrate to the enzyme

Enzyme–Substrate Binding Models
• Lock-and-key model
• Substrate binds to that portion of the enzyme with a
complementary shape
• Induced fit model
• Binding of the substrate induces a change in the
conformation of the enzyme that results in a
complementary fit
• Binding site has a different three-dimensional shape
before the substrate is bound

Figure 6.4 - Two Models for the Binding of a
Substrate to an Enzyme

Enzyme–Substrate Binding (continued)
• Enzyme and substrate must bind to form the ES
complex for which attraction must exist between E
and S
E+S ES (enzyme  substrate complex)
• Causes the ES complex to be lower on an energy

diagram than the E + S at the start
• Bound ES must attain the conformation of the
transition state EX‡
• Proximity and orientation of the substrate speed up
the reaction

Figure 6.5 - Free Energy of Activation Profile of an ES
Complex Formation

Figure 6.6 - Formation of Product from
Substrate

Michaelis–Menten Approach to Enzyme Kinetics
• Mechanism for an enzyme-catalyzed reaction can be
summarized in the following form:
ES   E+P
k1 k2
E+S k1
• k1 - Rate constant for the formation of ES from the

enzyme, E, and the substrate, S
• k–1 - Rate constant for the dissociation of the ES
complex
• k2 - Rate constant for the conversion of the ES
complex to product P and release of P from the
enzyme

Rate (Velocity) of an Enzymatic Reaction
• Initial rate, or initial
velocity (Vinit or V0), and
observed kinetics
depend on [S]
• [E] is constant
• At infinite [S], the
reaction would proceed
at maximum velocity
(Vmax)

Michaelis–Menten Model
• KM - Inverse measure of the affinity of the enzyme for
the substrate
• Lower the KM, the higher the affinity
• Rate of formation of the ES is given by the following
equation:
Δ  ES
Rate of formation = = k1  E S
Δt
• Δ[ES]/Δt - Change in concentration of the complex
during a given time
• k1 - Rate constant for the formation of the complex

Michaelis–Menten Model (continued 1)
• Rate of the disappearance of the ES complex is
given by the following equation:
Δ  ES
Rate of breakdown = = k1  ES + k2  ES
Δt
• –Δ[ES]/Δt
• Negative sign denotes that the concentration of the
complex decreases as it breaks down
• k–1 - Rate constant for the dissociation of the ES
complex to regenerate enzyme and substrate
• k2 - Rate constant for the reaction of the complex to
give product and enzyme
• Steady state: Condition in which the [ES] remains
constant in spite of continuous turnover
• Reached when the rate of formation of the ES equals
the rate of its breakdown
Δ  ES Δ  ES
=
Δt Δt
k1  E S = k1  ES + k2  ES
• [S] is greater than [E]

• When the steady state is reached, the concentration
of free enzyme, [E], is given by the following reaction:
 E  =  E  T   ES
• Where [E]T is the total concentration of the enzyme
• Substituting for the concentration of free enzyme, [E],
gives:
k1  E T   ES S = k1  ES + k2  ES

• Collecting all rate constants for the individual
reactions gives:
 E    ES S =
T k1 + k2
= KM
 ES k1
• KM is called the Michaelis constant

• In the initial stages of the reaction, formation of
product depends only on the rate of breakdown of ES
k2  E T S
Vinit = k2  ES = (equation 1)
K M + S

• If substrate concentration is so large that the enzyme
is saturated with substrate
[ES] =  E T
Vinit = Vmax = k2  E T
• Substituting k2[E]T = Vmax into equation 1 gives the

Michaelis–Menten equation
Vmax S
Vinit =
K M  S

Graphical Determination of Vmax and KM
• Constant rate when the enzyme is saturated with
substrate is the Vmax for the enzyme
Vmax S
V =
K M + S
• When [S] = KM:
Vmax S Vmax

V = V =
S + S 2

Enzyme Mechanisms
• Common mechanisms for enzymes that catalyze
reactions containing two or more substrates are:
• Ordered mechanism
• Substrates have to bind to the enzyme in a specific
order
• Random mechanism
• Substrates can bind to the enzyme in any order
• Ping-pong mechanism
• Substrate binds to the enzyme and releases a product
before the second substrate binds to the enzyme

Ordered and Random Mechanisms - Examples
• Ordered mechanism
• Random mechanism

Ping-Pong Mechanism - Example

Linearizing the Michaelis–Menten Equation
• It is difficult to determine Vmax experimentally
• Equation for a hyperbola can be transformed into an
equation for a straight line by taking the reciprocals of
both sides:
1 K M + S 1 KM  S
= = 
V Vmax S V Vmax S Vmax S
1 KM 1 1
=  
V Vmax S Vmax
y  mx + b

Lineweaver–Burk Plot
• Lineweaver–Burk double-reciprocal plot gives the:
• Slope of the line, m, which equals KM/Vmax
• y intercept, b, which equals 1/Vmax

Example 6.1
• The following data describe an enzyme-catalyzed
reaction
• Plot these results using the Lineweaver–Burk method,
and determine values for KM and Vmax
• The symbol mM represents millimoles per liter; 1 mM =
1×10–3 mol L–1 (Concentration of the enzyme is the
same in all experiments)

Example 6.1 - Solution
• Reciprocal of substrate concentration and of velocity
gives the following results:

Figure 6.10 - Lineweaver–Burk Plot of the Data

Example 6.1 - Solution (continued)
• Plotting the results gives a straight line as shown in
Figure 6.10
• Visually from the graph, the y intercept is 12 and the x
intercept is –0.155
• Reciprocal of the y intercept is Vmax, which equals
0.083 mM sec–1
• Reciprocal of the negative of the x intercept = KM =
6.45 mM
• Use the exact equation for the line of best fit to the
experimental points, which is 1/V = 75.46 (1/[S]) + 11.8
• Using the equation generates the following: KM = 6.39
mM and Vmax = 0.0847 mM sec–1

Significance of KM and Vmax
• When V = Vmax/2, KM = [S]
• Consider a case in which the reaction E + S → ES
takes place more frequently than ES → E + P
• k–1 is greater than k2 (k–1 ≫ k2)
• Vmax is related to the turnover number of an enzyme
• Turnover number: Number of moles of substrate that
react to form product per mole of enzyme per second
• Also called kcat or kp
V
= turnover number = kcat
ET 

Table 6.2 - Turnover Numbers and KM for Some Typical
Enzymes

Examples of Enzyme-Catalyzed Reactions
• Chymotrypsin catalyzes the hydrolysis of:
• Peptide bonds where the carboxyl is contributed by
Phe and Tyr
• Ester bonds

(continued 1)
• In a typical reaction in which p-nitrophenyl ester is

hydrolyzed by chymotrypsin, the experimental
reaction rate depends on [S]
• In this case, [S] is the concentration of p-nitrophenyl
ester

(continued 2)
• Aspartate transcarbamoylase (ATCase)

• Allosteric enzyme that catalyzes an early reaction in
pyrimidine biosynthesis
Carbamoyl phosphate + Aspartate  Carbamoyl aspartate + HPO 24
Reaction catalyzed by aspartate transcarbamoylase
• Reaction rate depends on [S]

• In this case, [S] is the concentration of aspartate

Velocity Curves of Chymotrypsin and ATCase

Inhibitors
• Substances that decrease the rate of an enzyme-
catalyzed reaction
• Reversible inhibitors
• Substances that bind to an enzyme and subsequently
are released
• Include competitive, noncompetitive, and
uncompetitive inhibitors
• Irreversible inhibitors
• Substances that react with enzymes to produce
proteins that are not enzymatically active and from
which original enzymes cannot be regenerated

Competitive Inhibition
• Decrease in enzymatic activity caused by binding of a
substrate analogue to the active (catalytic) site
• Inhibitor competes with the substrate for the active
site on the enzyme
• Represented by two mutually exclusive equations
E+S ES or E + I EI
• Equilibrium expression for the breakdown of the

enzyme-inhibitor complex (EI) is given by:
KI =
 E  I 
 EI
Figure 6.15 - Substrate or Inhibitor Binding in the
Case of Competitive Inhibition

Identifying a Competitive Inhibitor
• In the presence of a competitive inhibitor:
• Slope of the Lineweaver–Burk plot changes
• y intercept of the graph does not change
• Vmax is unchanged
• KM increases by the following factor:
1+
 I
KI
• Substitute value of KM in the following equation:
1 KM 1 1
= × +
V Vmax S Vmax
Identifying a Competitive Inhibitor (continued)
1 KM  1  1 1
= 1 + × +
V Vmax  K I  S Vmax
y = m × x + b
• In a Lineweaver–Burk double-reciprocal plot of 1/V

versus 1/[S], the slope and the x intercept change

Figure 6.16 - Lineweaver–Burk Double-Reciprocal Plot
of Enzyme Kinetics for Competitive Inhibition

Noncompetitive Inhibition
• Form of enzyme inactivation in which a substance
binds to a site other than the active site but distorts
the active site to inhibit reaction
• Several equilibria are involved
• Involves two distinct binding sites, one for the

substrate and one for the inhibitor

Figure 6.17 - Nature of Substrate and Inhibitor Binding
in Noncompetitive Inhibition

Identifying a Noncompetitive Inhibitor
• Inhibitor does not interfere with substrate binding
• Value of Vmax decreases, and value of KM remains the
same
• Increasing substrate concentration cannot overcome
noncompetitive inhibition
• Inhibitor and substrate are not competing for the same
site

Identifying a Noncompetitive Inhibitor
(continued)
• In presence of a noncompetitive inhibitor, I, maximum

velocity of the reaction, Vimax, has the following form:
I Vmax
V =
1 +  I  /K I
max
• KI - Dissociation constant for the enzyme–inhibitor

complex, EI

Lineweaver–Burk Plot for Noncompetitive
Inhibition
• Slope and y intercept change
• x intercept remains unchanged
No inhibition
1 KM 1 1
= × +
V Vmax  S  Vmax
y = m × x + b
Noncompetitive inhibition
1 KM   I  1 1   I 
= 1 + × + 1 + 
V Vmax  K I   S  Vmax  KI 
y = m × x + b
Figure 6.18 - Lineweaver–Burk Plot of Enzyme Kinetics
for Noncompetitive Inhibition

Example 6.2
• Sucrose (common table sugar) is hydrolyzed to
glucose and fructose in a classic experiment in
kinetics
• Reaction is catalyzed by the enzyme invertase
• Using the following data, determine, by the
Lineweaver–Burk method, whether the inhibition of this
reaction by 2 M urea is competitive or noncompetitive

Example 6.2 - Solution
• Plot the data with the reciprocal of the sucrose
concentration on the x-axis and the reciprocals of the
two reaction velocities on the y-axis as shown in
Figure 6.19
• Note that the two plots have different slopes and
different y intercepts, typical of noncompetitive
inhibition
• Note the same intercept on the negative x-axis, which
gives –1/KM

Figure 6.19 - Lineweaver–Burk Plot of the Data

Mixed Noncompetitive Inhibition
• Similar to noncompetitive inhibition but the binding of
I does affect the binding of S and vice versa
• Dissociation constants KI and K′I are not identical

Uncompetitive Inhibition
• Inhibitor can bind to the ES complex but not to the
free E
• Lineweaver–Burk plot contains parallel lines

• Vmax decreases
• KM decreases

Figure 6.21 - Lineweaver–Burk Double-Reciprocal Plot
of Enzyme Kinetics for Uncompetitive Inhibition

Irreversible Inhibition
• Covalent binding of an inhibitor to an enzyme,
causing permanent inactivation
• Suicide substrates: Molecules used to bind to an
enzyme irreversibly and inactivate it
• Also called Trojan horse substrates
• Used in medicine
• Example - Antibiotic penicillin

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Chapter 6

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• ΔG°‡ is lowered if the reaction is allowed to proceed on

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Rate of disappearance of A =  Δ  A  /Δt

Rate of appearance of P = Δ  P  /Δt

• Negative sign indicates that A and B are used up in the

• k - Proportionality constant called the rate constant

• This reaction is first order with respect to A and first

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• Where k is the rate constant, exponent for [A] is 1, and

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• Reaction rate depends on concentrations of both

Rate = k  Glycogen   Pi  = k  Glycogen  Pi 

• Reaction is first order with respect to glycogen, first

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• Rate depends on the presence of catalysts

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E+S ES (enzyme  substrate complex)

• Causes the ES complex to be lower on an energy

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• k1 - Rate constant for the formation of ES from the

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• [S] is greater than [E]

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• KM is called the Michaelis constant

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• Substituting k2[E]T = Vmax into equation 1 gives the

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• When [S] = KM:

Vmax S Vmax

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• In a typical reaction in which p-nitrophenyl ester is

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• Aspartate transcarbamoylase (ATCase)

• Reaction rate depends on [S]

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