Reginald H.

Garrett
Charles M. Grisham
www.cengage.com/chemistry/garrett

Chapter 13
Enzyme Kinetics

Reginald Garrett & Charles Grisham • University of Virginia

Essential Questions

• What are enzymes, and what do they do?

 Outline
• What characteristic features define enzymes?
• Can the rate of an enzyme-catalyzed reaction be defined
in a mathematical way?
• What equations define the kinetics of enzyme-catalyzed
reactions?
• What can be learned from the inhibition of enzyme
activity?
• What is the kinetic behavior of enzymes catalyzing
bimolecular reactions?
• How can enzymes be so specific?
• Are all enzymes proteins?
• Is it possible to design an enzyme to catalyze any desired
reaction?
Virtually All Reactions in Cells Are
Mediated by Enzymes
• Enzymes catalyze thermodynamically favorable
reactions, causing them to proceed at extraordinarily
rapid rates (see Figure 13.1)
• Enzymes provide cells with the ability to exert kinetic
control over thermodynamic potentiality
• Living systems use enzymes to accelerate and
control the rates of vitally important biochemical
reactions
• Enzymes are the agents of metabolic function
Virtually All Reactions in Cells Are
Mediated by Enzymes

Figure 13.1 Reaction profile showing the

large free energy of activation for glucose
oxidation. Enzymes lower ΔG‡, thereby
accelerating rate.
13.1 What Characteristic Features Define
Enzymes?
• Catalytic power is defined as the ratio of the enzyme-
catalyzed rate of a reaction to the uncatalyzed rate
• Specificity is the term used to define the selectivity of
enzymes for their substrates
• Regulation of enzyme activity ensures that the rate
of metabolic reactions is appropriate to cellular
requirements
• Enzyme nomenclature provides a systematic way of
naming metabolic reactions
• Coenzymes and cofactors are nonprotein
components essential to enzyme activity.
13.1 What Characteristic Features Define
Enzymes?

• Enzymes can accelerate reactions as much

as 1021 over uncatalyzed rates

• Urease is a good example:

• Catalyzed rate: 3x104/sec
• Uncatalyzed rate: 3x10 -10/sec
• Ratio (catalytic power) is 1x1014
 Specificity
• Enzymes selectively recognize proper substrates
over other molecules
• Enzymes produce products in very high yields -
often much greater than 95%
• Specificity is controlled by structure - the unique
fit of substrate with enzyme controls the
selectivity for substrate and the product that’s
formed.
Enzymes are the Agents of Metabolic
Function

Figure 13.2 The breakdown of

glucose by glycolysis provides a
prime example of a metabolic
pathway.
90% yield in each step; 35% over 10 steps

Figure 13.3 Yields in biological

reactions must be substantially
greater than 90%.
Enzyme Nomenclature Provides a Systematic
Way of Naming Metabolic Reactions
Coenzymes and Cofactors Are Nonprotein
Components Essential to Enzyme Activity
13.2 Can the Rate of an Enzyme-Catalyzed
Reaction Be Defined in a Mathematical Way?
• Kinetics is the branch of science concerned with the
rates of reactions
• Enzyme kinetics seeks to determine the maximum
reaction velocity that enzymes can attain and the
binding affinities for substrates and inhibitors
• Analysis of enzyme rates yields insights into enzyme
mechanisms and metabolic pathways
• This information can be exploited to control and
manipulate the course of metabolic events
Several Kinetics Terms to Understand

• rate or velocity
• rate constant
• rate law
• order of a reaction
• molecularity of a reaction
Chemical Kinetics Provides a Foundation for
Exploring Enzyme Kinetics
• Consider a reaction of overall stoichiometry as
shown:
A® P
d[P] - d[ A]
v= =
dt dt
- [ A]
v= =k[ A]
dt
• The rate is proportional to the concentration of A
Chemical Kinetics Provides a Foundation for
Exploring Enzyme Kinetics
• The simple elementary reaction of A→P is a first-
order reaction
• Figure 13.4 shows the course of a first-order reaction
as a function of time
• This is a unimolecular reaction
• For a bimolecular reaction, the rate law is:
v = k[A][B]
• Kinetics cannot prove a reaction mechanism
• Kinetics can only rule out various alternative
hypotheses because they don’t fit the data
The Time-Course of a First-Order Reaction

Figure 13.4 Plot of the course of a first-order reaction. The

half-time, t1/2 is the time for one-half of the starting amount of
A to disappear.
Catalysts Lower the Free Energy of Activation for
a Reaction
• A typical enzyme-catalyzed reaction must pass
through a transition state
• The transition state sits at the apex of the energy
profile in the energy diagram
• The reaction rate is proportional to the concentration
of reactant molecules with the transition-state energy
• This energy barrier is known as the free energy of
activation
• Decreasing ΔG‡ increases the reaction rate
• The activation energy is related to the rate constant
by: - DG/ RT
k =Ae
Catalysts Lower the Free Energy of Activation for a
Reaction

Figure 13.5 Energy diagram for a chemical reaction (A→P)

and the effects of (a) raising the temperature from T1 to T2, or
(b) adding a catalyst.
 The Transition State

Understand the difference between G and G‡

• The overall free energy change for a reaction,
G, is related to the equilibrium constant
• The free energy of activation for a reaction, G‡,
is related to the rate constant
• It is extremely important to appreciate this
distinction
13.3 What Equations Define the Kinetics of
Enzyme-Catalyzed Reactions?
• Simple first-order reactions display a plot of the
reaction rate as a function of reactant
concentration that is a straight line (Figure 13.6)
• Enzyme-catalyzed reactions are more
complicated
• At low concentrations of the enzyme substrate,
the rate is proportional to S, as in a first-order
reaction
• At higher concentrations of substrate, the enzyme
reaction approaches zero-order kinetics
• This behavior is a saturation effect
13.3 What Equations Define the Kinetics of
Enzyme-Catalyzed Reactions?
Figure 13.6 A plot of υ versus [A] for the
unimolecular chemical reaction, A→P, yields
a straight line having a slope equal to k. This
reaction is a first-order reaction.
As [S] increases, kinetic behavior changes from
1st order to zero-order kinetics

Figure 13.7 Substrate saturation

curve for an enzyme-catalyzed
reaction.
The Michaelis-Menten Equation is the
Fundamental Equation of Enzyme Kinetics
Louis Michaelis and Maud Menten's theory
•assumes the formation of an enzyme-substrate
complex
•assumes that the ES complex is in rapid equilibrium
with free enzyme
•assumes that the breakdown of ES to form products
is slower than
1) formation of ES and
2) breakdown of ES to re-form E and S

k1 k2
E + S ES P
k-1
[ES] Remains Constant Through Much of the Enzyme
Reaction Time Course in Michaelis-Menten Kinetics

Figure 13.8 Time course for a

typical enzyme-catalyzed reaction
obeying the Michaelis-Menten,
Briggs-Haldane models for
enzyme kinetics. The early state
of the time course is shown in
greater magnification in the
bottom graph.
The Michaelis-Menten equation

Vmax [S]
v=
K m +[S]
where Vmax =k2 [E T ]

and Km =
( k- 1 + k 2 )
k1
 Understanding Km
The "kinetic activator constant"
• Km is a constant
• Km is a constant derived from rate constants
• Km is, under true Michaelis-Menten conditions,
an estimate of the dissociation constant of E
from S
• Small Km means tight binding; high Km means
weak binding
 Understanding Vmax
The theoretical maximal velocity
• Vmax is a constant
• Vmax is the theoretical maximal rate of the reaction
- but it is NEVER achieved in reality
• To reach Vmax would require that ALL enzyme
molecules are tightly bound with substrate
• Vmax is asymptotically approached as
substrate is increased
The dual nature of the Michaelis-Menten
equation
Combination of 0-order and 1st-order kinetics
• When S is low, the equation for rate is 1st order in S
• When S is high, the equation for rate is 0-order in S
• The Michaelis-Menten equation describes a
rectangular hyperbolic dependence of v on S
• See Figure 13.7
Table 13.3 gives the Km values for some enzymes
and their substrates
The Turnover Number Defines the Activity of
One Enzyme Molecule
A measure of catalytic activity
• kcat, the turnover number, is the number of substrate
molecules converted to product per enzyme
molecule per unit of time, when E is saturated with
substrate.
• If the M-M model fits, k2 = kcat = Vmax/Et
• Values of kcat range from less than 1/sec to many
millions per sec
The Turnover Number Defines the Activity of One
Enzyme Molecule
The Ratio kcat/Km Defines the Catalytic Efficiency
of an Enzyme
The catalytic efficiency: kcat/Km
An estimate of "how perfect" the enzyme is
• kcat/Km is an apparent second-order rate constant
• It measures how well the enzyme performs when
S is low
• The upper limit for kcat/Km is the diffusion limit - the
rate at which E and S diffuse together
The Ratio kcat/Km Defines the Catalytic Efficiency
of an Enzyme
Linear Plots Can Be Derived from the Michaelis-
Menten Equation
Be able to derive these equations
• Lineweaver-Burk:
• Begin with v = Vmax[S]/(Km + [S]) and take the
reciprocal of both sides
• Rearrange to obtain the Lineweaver-Burk equation:

1 æ Km ö æ 1 ö 1
=ç ÷ ç ÷+
v è Vmax ø è[S] ø Vmax
• A plot of 1/v versus 1/[S] should yield a straight line
Linear Plots Can Be Derived from the Michaelis-
Menten Equation

Figure 13.9 The Lineweaver-

Burk double-reciprocal plot.
Linear Plots Can Be Derived from the Michaelis-
Menten Equation
• Hanes-Woolf:
• Begin with Lineweaver-Burk and multiply both sides
by [S] to obtain:
[S] æ 1 ö Km
=ç ÷ [S] +
v è Vmax ø Vmax
• Hanes-Woolf is best - why?
• Because Hanes-Woolf has smaller and more
consistent errors across the plot
Linear Plots Can Be Derived from the Michaelis-
Menten Equation
Figure 13.11 A Hanes-Woolf
plot of [S]/v versus [S]
Enzymatic Activity is Strongly Influenced by pH

• Enzyme-substrate recognition and catalysis are

greatly dependent on pH
• Enzymes have a variety of ionizable side chains that
determine its secondary and tertiary structure and
also affect events in the active site
• The substrate may also have ionizable groups
• Enzymes are usually active only over a limited range
of pH
• The effects of pH may be due to effects on Km or Vmax
or both
Enzymatic Activity is Strongly Influenced by pH

Figure 13.11 The pH activity profiles of four different enzymes.

The Response of Enzymatic Activity to
Temperature is Complex
• Rates of enzyme-catalyzed reactions generally
increase with increasing temperature
• However, at temperatures above 50°to 60°C,
enzymes typically show a decline in activity
• Two effects here:
• Enzyme rate typically doubles in rate for ever 10º
C as long as the enzyme is stable and active
• At higher temperatures, the protein becomes
unstable and denaturation occurs
The Response of Enzymatic Activity to
Temperature is Complex

Figure 13.12 The effect

of temperature on
enzyme activity.
13.4 What Can Be Learned from the Inhibition of
Enzyme Activity?
• Enzymes may be inhibited reversibly or
irreversibly
• Reversible inhibitors may bind at the active site or
at some other site
• Enzymes may also be inhibited in an irreversible
manner
• Penicillin is an irreversible suicide inhibitor
Reversible Inhibitors May Bind at the Active Site
or at Some Other Site
Competitive Inhibitors Compete With Substrate for
the Same Site on the Enzyme

Figure 13.13 Lineweaver-Burk plot of competitive inhibition,

showing lines for no I, [I], and 2[I]. Note that when [S] is
infinitely large (1/[S] = ~0), Vmax is the same whether I is
presence or not.
It means high [S] can overcome the effect of I.
Succinate Dehydrogenase – a Classic Example
of Competitive Inhibition

Figure 13.14 Structures of succinate, the substrate of succinate

dehydrogenase (SDH), and malonate, the competitive inhibitor.
Fumarate (the product of SDH) is also shown.
Pure Noncompetitive Inhibition – where S and I
bind to different sites on the enzyme

Figure 13.15 Lineweaver-Burk plot of pure noncompetitive

inhibition. Note that I does not alter Km but that it decreases
Vmax. I interacts with both E and ES.
Uncompetitive Inhibition, where I combines only
with ES, but not with E

Figure 13.17 Lineweaver-Burk plot of

uncompetitive inhibition. Note that
both intercepts change but the slope
(Km/Vmax) remains constant in the
presence of I.
Enzymes Can Be Inhibited Irreversibly

Figure 13.18 Penicillin is

an irreversible inhibitor of
the enzyme glycoprotein
peptidease, which
catalyzes an essential step
in bacterial cell all
synthesis.
13.7 – How Can Enzymes Be So Specific?
• The “Lock and key” hypothesis was the first
explanation for specificity
• The “Induced fit” hypothesis provides a more
accurate description of specificity
• Induced fit favors formation of the transition state
• Specificity and reactivity are often linked. In the
hexokinase reaction, binding of glucose in the
active site induces a conformational change in
the enzyme that causes the two domains of
hexokinase to close around the substrate,
creating the catalytic site
13.7 – How Can Enzymes Be So Specific?

Figure 13.24 A drawing, roughly to scale, of H2O, glycerol,

glucose, and an idealized hexokinase molecule. Binding of
glucose in the active site induces a conformational change
in the enzyme that causes the two domains of hexokinase
to close around the substrate, creating the catalytic site.
13.7 – Are All Enzymes Proteins?
• Ribozymes - segments of RNA that display
enzyme activity in the absence of protein
• Examples: RNase P and peptidyl transferase
• Abzymes - antibodies raised to bind the transition
state of a reaction of interest
• For a good review of abzymes, see Science,
Vol. 269, pages 1835-1842 (1995)
• Transition states are covered in more depth in
in Chapter 14
RNA Molecules That Are Catalytic Have Been
Termed “Ribozymes”

Figure 13.25 RNA splicing in

Tetrahymena rRNA maturation.
RNA Molecules That Are Catalytic Have
Been Termed “Ribozymes”

Figure 13.26 (a) The 50S subunit from H. marismortui. (b) The
aminoacyl-tRNA (yellow) and the peptidyl-tRNA (orange) in the
peptidyl transferase active site.
RNA Molecules That Are Catalytic Have Been
Termed “Ribozymes”

Figure 13.27 The peptidyl transferase reaction.

Antibody Molecules Can Have Catalytic Activity

Figure 13.28 (a) Intramolecular hydrolysis of a hydroxy ester

yields a δ-lactone.

(b) The cyclic

phosphonate ester
analog of the cyclic
transition state.
13.8 Is It Possible to Design An Enzyme to
Catalyze Any Desired Reaction?
• A known enzyme can be “engineered” by in vitro
mutagenesis, replacing active site residues with new
ones that might catalyze a desired reaction
• Another approach attempts to design a totally new
protein with the desired structure and activity
• This latter approach often begins with studies “in
silico” – i.e., computer modeling
• Protein folding and stability issues make this
approach more difficult
• Further, the cellular environment may provide
complications not apparent in the computer
modeling
13.8 Is It Possible to Design An Enzyme to
Catalyze Any Desired Reaction?

Figure 13.29 cis-1,2-Dichloroethylene (DCE) is an

industrial solvent that poses hazards to human health.

Site-directed mutations have enabled the conversion of a

bacterial epoxide hydrolase to catalyze the chlorinated
epoxide hydrolase reaction.