Enzyme

Kinetics
Course Outcomes
Write enzyme kinetic
models and solve related
problems.
Demonstrate how to set up
and solve enzyme kinetic
expressions.

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 ?
Enzyme Kinetics
Enzymes endow cells with the remarkable capacity to
exert kinetic control over thermodynamic potentiality
Enzymes are the agents of metabolic function
What we want to be able to determine:
Maximum velocity
Substrate affinity
Inhibitor affinity
What it can tell us:
Flow through metabolic pathways
Utilization of substrates
What can we do with the information:
Control and manipulate metabolic events

Kinetics is the study of the rates of reactions
Virtually All Reactions in Cells Are
Mediated by Enzymes
Living systems use enzymes to accelerate and
control the rates of vitally important biochemical
reactions.
Enzymes provide cells with the ability to exert
kinetic control over thermodynamic potentiality.
(glucose pyruvate)
Enzymes are the agents of metabolic function.
Most enzymes are proteins.
Some enzymes require cofactors or coenzymes.
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
G

, thereby
accelerating
rate.
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.
Coenzymes and cofactors are nonprotein
components essential to enzyme activity.
What Characteristic Features Define
Enzymes ?
Enzymes can accelerate reactions as much as
10
16
over uncatalyzed rates.
Urease is a good example:
Catalyzed rate Uncatalyzed rate Ratio
3x10
4
/sec 3x10
-10
/sec 1x10
14

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 yield.
Enzymes are the Agents of Metabolic
Function
Figure 13.2
The breakdown of glucose by
glycolysis provides a prime
example of a metabolic pathway
with many sequential steps.

All pathways are regulated but
not all enzymes are regulated .
Regulation of glycolysis occurs at
several points, the major control
point is through the enzyme
phosphofructokinase I.
90% yield in each step = 35% over 10 steps
Figure 13.3
Yields in biological
reactions must be
substantially
greater than 90%.
Enzymes Nomenclature
As in organic, there is a system of nomenclature
devised for enzymes as well as a lot of commonly
used names.
There are six main classes used for naming
enzymes. Know these six classes.
There is also a numerical assignment for each
enzyme developed by the Enzyme Commission.
E.g.: EC 1.1.1.1 denotes alcohol dehydrogenase
Its systematic name is:
alcohol:NAD
+
oxidoreductase
donor:acceptor main class
The Six Classes of Enzymes
Class# Class and reaction catalyzed
1. Oxidoreductases (dehydrogenases)
oxidation-reduction reactions
2. Transferases group transfer reactions
3. Hydrolases hydrolysis reactions
4. Lyases lysis, forming a double bond
5. Isomerases isomerization reactions
6. Ligases (synthetases) joining of two substrates,
uses ATP
Enzyme Nomenclature Provides a Systematic
Way of Naming Metabolic Reactions
Know the six main classes.
Coenzymes and Cofactors Are Nonprotein
Components Essential to Enzyme Activity
Coenzymes are organic cofactors.
A Mathematical statement of the Rate of an
Enzyme-Catalyzed Reaction
Enzyme kinetics seeks to determine the
maximum reaction velocity that enzymes can
attain and 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 of reaction
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:








The rate is proportional to the concentration of A
[ ] [ ]
[ ]
[ ]
A P
d P d A
v
dt dt
A
v k A
dt

= =

= =
Chemical Kinetics Provides a Foundation
for Exploring Enzyme Kinetics
The simple elementary reaction of AP is a first-
order reaction, v = k[A] and exponent on [A] is 1.
This is a unimolecular reaction.
For a second order reaction, the rate law is:
v = k[A][B]. This reaction may be bimolecular or
unimolecular depending on the mechanism.
Kinetics cannot prove a reaction mechanism.
Kinetics can only rule out various alternative
hypotheses because they dont fit the data.
Kinetic vs Chemical Mechanism
An enzyme kinetic mechanism is the
order of substrate addition and product
release in an enzyme catalyzed reaction
A chemical mechanism is the chemical
pathway of conversion of S P,
including the structures of any
intermediates

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, G

.
Decreasing G

increases the reaction rate.

The activation energy is related to the rate
constant by:
/ G RT
k Ae
A
=
Catalysts Lower the Free Energy of
Activation for a Reaction
Figure 13.5 Energy diagram for a chemical reaction (AP)
and the effects of (a) raising the temperature from T
1
to T
2
, or
(b) adding a catalyst.
The Transition State
Understand the difference between AG and AG

The overall free energy change for a reaction is
related to the equilibrium constant.
The free energy of activation for a reaction is
related to the rate constant.
It is extremely important to appreciate this
distinction.
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.
What Equations Define the Kinetics of
Enzyme-Catalyzed Reactions ?
Figure 13.6
A plot of v versus
[A] for the
unimolecular
chemical
reaction, AP,
yields a straight
line having a
slope equal to k.
As [S] increases, kinetic behavior changes
from 1
st
order to zero-order kinetics
Figure 13.7 Substrate saturation curve for
an enzyme-catalyzed reaction.
Enzyme Kinetics
Michaelis-Menten kinetics or saturation kinetics
which was first developed by V.C.R. Henri in 1902 and
developed by L. Michaelis and M.L. Menten in 1913.

This model is based on data from batch reactors
with constant liquid volume.

- Initial substrate, [S0] and enzyme [E0]
concentrations are known.
- An enzyme solution has a fixed number of
active sites to which substrate can bind.

- At high substrate concentrations, all these sites
may be occupied by substrates or the enzyme is
saturated.
Saturation Enzyme Kinetics
Substrate Saturation of an Enzyme
A. Low [S] B. 50% [S] or K
m
C. High, saturating [S]

The Michaelis-Menten Equation is the
Fundamental Equation of Enzyme Kinetics
Louis Michaelis and Maud Menten's theory.
It assumes the formation of an enzyme-substrate
complex.
It assumes that the ES complex is in rapid
equilibrium with free enzyme.
Breakdown of ES to form products is assumed to
be slower than 1) formation of ES and 2)
breakdown of ES to re-form E and S.
Briggs and Haldane later introduced the steady
state assumtion.
Steady State Assumption
The M-M equation was derived in part by making
several assumptions. An important one was: the
concentration of substrate must be much greater
than the enzyme concentration. In the situation
where [S] >> [E] and at initial velocity rates, it is
assumed that the changes in the concentration of the
intermediate ES complex are very small over time (v
o
).
This condition is termed a steady-state rate, and is
referred to as steady-state kinetics. Therefore, it
follows that the rate of ES formation will be equal
to the rate ES breakdown.

At this point, an assumption is required to
achieve an analytical solution.

- The rapid equilibrium assumption
Michaelis - Menten Approach.

- The quasi-steady-state assumption.
Briggs and Haldane Approach.
Enzyme Kinetics
The Michaelis-Menten Equation is the
Fundamental Equation of Enzyme Kinetics
E = enzyme concentration.
S = Substrate concentration.
ES = Enzyme-substrate complex concentration
(noncovalent).
P = product concentration.
k
1
= rate constant for formation of ES from E + S.
k
-1
= rate constant for decomposition of ES to E + S.
k
2
= rate constant for decomposition of ES to E + P.
E + S ES E + P
k
1

k
2

k
-1

Development of the Michaelis-Menten
Equation
1. The overall rate of product formation: v = k
2
[ES]
2. Rate of formation of [ES]: v
f
= k
1
[E][S]
3. Rate of decomposition of [ES]:
v
d
= k
-1
[ES] + k
2
[ES]
4. The steady state assumption requires that:
Rate of ES formation = Rate of ES decomposition
5. So: k
1
[E][S] = k
-1
[ES] + k
2
[ES]
E + S ES E + P
k
1

k
2

k
-1

6. In solving for [ES], use the enzyme balance to
eliminate [E]. E
T
= [E] + [ES]
7. k
1
(E
T
- [ES])[S] = k
-1
[ES] + k
2
[ES]
k
1
E
T
[S] - k
1
[ES][S] = k
-1
[ES] + k
2
[ES]
8. Rearrange and combine [ES] terms:
k
1
E
T
[S] = (k
-1
+ k
2
+ k
1
[S])[ES]
k
1
E
T
[S]
9. Solve for [ES] = -----------------------
(k
-1
+ k
2
+ k
1
[S])
Michaelis-Menten Derivation
E
T
[S]
10. Divide through by k
1
:

[ES] = -----------------------

(k
-1
+ k
2
)/k
1
+ [S]

11. Defined Michaelis constant: K
M
= (k
-1
+ k
2
) / k
1

12. Substitute K
M
into the equation in step 10.
13. Then substitute [ES] into v = k
2
[ES] from step1
and replace V
max
with k
2
E
T
to give:
V
max
[S]
v
o
= -----------
K
M
+ [S]
Michaelis-Menten Derivation
[ES] Remains Constant Through Much of
the Enzyme Reaction Time Course
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.
Comparison of the Two Approaches
1
1 '
k
k
m
K

=
] [
] [
S
m
K
S
m
V
v
+
=
]
0
[
2
E k
m
V =
1
2 1
k
k k
m
K
+

=
Michaelis-Menten
when k2 << k-1,
1
2 1
'
k
k k
m
K
m
K
+

= =
] [
'
] [
S
m
K
S
m
V
v
+
=
]
0
[
2
E k
m
V =
Briggs-Haldane
d[ES]/dt 0
] [
1
] ][ [
1
ES k S E k

=
Assumption:
Equation:
Maximum forward
reaction rate:
Constant:
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.
The relation of the rectangular hyperbola to the
enzyme kinetics profile is described in references
at the end of the chapter.
Understanding V
max
The theoretical maximal velocity

V
max
is a constant.
V
max
is the theoretical maximal rate of the
reaction - but it is NEVER achieved in reality.
To reach V
max
would require that ALL enzyme
molecules are tightly bound with substrate.
V
max
is asymptotically approached as substrate
is increased.
Understanding K
m
The "kinetic activator constant"

K
m
is a constant.
K
m
is a constant derived from rate constants.
K
m
is, under true Michaelis-Menten conditions, an
estimate of the dissociation constant of E from S.
A measure of ES binding.
Small K
m
means tight binding; large K
m
means
weak binding. Where k
2
is small then K
m
K
d
.

Significance of K
m
Useful to compare Km for different substrates for one
enzyme
Hexokinase : D-fructose 1.5 mM
D-glucose 0.15 mM
Useful to compare Km for a common substrate used
by several enzymes
Hexokinase: D-glucose 0.15 mM
Glucokinase: D-glucose 20 mM
Table 13.3 gives the K
m
values for some
enzymes and their substrates
Table 13.3 gives the K
m
values for some
enzymes and their substrates
The Turnover Number Defines the Activity
of One Enzyme Molecule
A measure of catalytic activity

k
cat
, 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. A measure of rate of
enzyme activity.
If the M-M model fits, k
2
= k
cat
= V
max
/E
t
.
Values of k
cat
range from less than 1/sec to
many millions per sec.
The Turnover Number Defines the Activity
of One Enzyme Molecule
The Ratio k
cat
/K
m
Defines the Catalytic
Efficiency of an Enzyme
The catalytic efficiency: k
cat
/K
m
An estimate of "how perfect" the enzyme is

k
cat
/K
m
is an apparent second-order rate constant.
It measures how the enzyme performs when S is
low.
The upper limit for k
cat
/K
m
is the diffusion limit -
the rate at which E and S diffuse together.
The maximum rate of diffusion for small
molecules is 10
9
M
-1
-sec
-1
.
The Ratio k
cat
/K
m
Defines the Catalytic
Efficiency of an Enzyme
Superoxide dismutase O
2

(radical) 1 x 10
6
5 x 10
-4
2 x 10
9

-Chymotrypsin Acetyl-Phe-amide 1.4 x 10
-1
1.5 x 10
-2
9.3
Enzyme Activity
Specific Activity is the number of units of activity per
amount of total protein.


Ex. A crude cell lysate might have a specific activity of
0.2 units/mg or ml protein upon which purification may
increase to 10 units/mg or ml protein.
One unit would be formation of one mol product per
minute at a specific pH and temperature with a
substrate concentration much greater than the value
of Km.
Units of Enzyme Activity
Terms in discussing enzyme activity

Units of enzyme activity:
mol S/min mol S/sec

Specific activity: (to follow purification)
mol S/min/mg E mol S/sec/kg E

Molecular activity: (turn-over number, TON = k
cat
)
mol S/min/ mol E mol S/sec/ mol E
Turnover Number
Example calculation

An enzyme (1.84 gm, MW 36800), in presence of excess
substrate catalyzes at a rate of 4.2 mol substrate/min.
Calculate the TON. (mol S/min/mol E)

1.84 gm
mol E: = --------------------- = 5 x 10
-5
mol E
36800 gm/mol

V
max
4.2 mol S/min
TON = ------ = ----------------------- = 84000 min
-1

E
t
5 x 10
-5
mol E
Michaleis-Mention Equation
Example calculation
The rate of an enzyme catalyzed reaction is 35 mol/min
at [S] = 10
-4
M. K
M
for this substrate is = 2 x 10
-5
M.
Calculate the rate where [S] = 2 x 10
-6
mol/min.

V
M
[S] V
M
(10
-4
)
v = ------------- so 35 = ---------------------
K
M
+ [S] (2 x 10
-5
) + (10
-4
)
And V
M
= 1.2(35) = 42 mol/min

(42)(2 x 10
-6
) (84 x 10
-6
)
v = -------------------------- = ------------- = 3.8 mol/min
-1

(2 x 10
-5
) + (2 x 10
-6
) (22 x 10
-6
)
Specific Activity
Assume that an assay of 0.8 ml of the crude extract
gives 0.518 activity units.
(3800/0.8)(0.518) = 2460 units in the 3.8 l.
2460 units/22800 mg protein = 0.108 units/mg
As purification proceeds, the specific activity
increases due to loss of extraneous protein.
By purifying to a constant specific activity, one has
reached a limit in purification.
Graphical Determination of K
M
and V
M

The Michaleis-Menten plot only permits an estimate of
V
max
, so K
M
is also an estimate at V
M
/2.
There are several graphical methods which provide a
better determination of V
M
and K
M
.
We will focus on the Lineweaver-Burk equation which
is obtained by taking the reciprocal of the Michaleis-
Menten equation (See the next slide).
A Lineweaver-Burke plot is frequently referred to as a
double reciprocal plot since one plots 1/v vs 1/[S].
The plot gives a straight line which has a slope of
K
M
/V
M
, a y-intercept of 1/V
M
and an x-intercept of
-1/K
M
.
Linear Plots Can Be Derived from the
Michaelis-Menten Equation
Be able to develop this equation

Lineweaver-Burk:
Begin with v = V
max
[S]/(K
m
+ [S]) and take the
reciprocal of both sides.
Rearrange to obtain the Lineweaver-Burk equation:




A plot of 1/v versus 1/[S] should yield a straight line.
max max
1 1 1
[ ]
m
K
v V S V
| |
| |
= +
|
|
\ .
\ .
Linear Plots Can Be Derived from the
Michaelis-Menten Equation
The Lineweaver-
Burk double-
reciprocal plot.
The Lineweaver-
Burk equation.
Eadie-Hofstee Plot
] [S
v
m
K
m
V v =
- the slope is Km
- y-axis intercept is Vm.
-Can be subject to large error since both coordinates contain
dependent variable v,
but there is less bias on points at low [s].
Hanes-Woolf (Langmuir) Plot
- the slope is 1/Vm
- y-axis intercept is Km/Vm
- better fit: even weighting of the data
] [
1 ] [
S
m
V
m
V
m
K
v
S
+ =
Influence of enzyme concentration
v = k
3
[E], as
[S]>>[E]
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.
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 K
m
or
V
max
or both.
Enzymatic Activity is Strongly Influenced by
pH
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
10C 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.
Enzyme Inhibition
Enzyme inhibitors are important for a variety of reasons

1) they can be used to gain information about the shape on
the enzyme active site and the amino acid residues in the
active site.
2) they can be used to gain information about the chemical
mechanism.
3) they can be used to gain information about the regulation
or control of a metabolic pathway.
4) they can be very important in drug design.

Enzyme Inhibition
Reversible inhibitor: a substance that binds to an
enzyme to inhibit it, but can be released
usually involves formation of non-covalent bonds
Generally two types
Dead end
Product
Irreversible inhibitor: a substance that causes
inhibition that cannot be reversed
usually involves formation or breaking of covalent
bonds to or on the enzyme

Inhibitors

Irreversible inhibition
Reversible inhibition
competitive inhibition
non-competitive inhibition
uncompetitive inhibition
Irreversible inhibition
Irreversible inhibition:
The inhibitor combine with essential group of
enzyme active center by covalent bond, resulting in
enzymatic activity loss.
Inhibition Patterns
An inhibitor may bind at the same site as
one of the substrates
these inhibitors structurally resemble the
substrate
An inhibitor may bind at an alternate site
affecting catalytic activity without
affecting substrate binding
Many inhibitors do both
I nhibitors act in a variety of mechanisms
Inhibition of Enzyme Activity
Enzymes may be inhibited
reversibly or irreversibly.
Reversible inhibitors may bind
at the active site or at some
other site.
Reversible inhibitors typically
change V
M
, K
M
or both.
Inhibition of Enzyme Activity
Enzymes may also be inhibited in an
irreversible manner.
Iodoacetate is an irreversible inhibitor.
Penicillin is an irreversible inhibitor.
Irreversible inhibitors are also called suicide
inhibitors.
Competitive Inhibition
Competitive inhibitor competes with a substrate for
the enzyme - substrate binding site

Malonate is a
competitive
inhibitor of
succinate for
succinate
dehydrogenase

A competitive inhibitor reduces the amount of
free enzyme available for substrate binding
thus increasing the Km for the substrate




The effect of a competitive inhibitor can be
overcome with high concentrations of the
substrate

Competitive Inhibition
Competitive Inhibition
Unimolecular
Reaction






Bimolecular
Reaction

Competitive Inhibition
Uncompetitive Inhibition
An uncompetitive
inhibitor binds to
the enzyme substrate
complex but not to
free enzyme
The result is a
decrease in Vmax
and Km
The effect of an
uncompetitive
inhibitor can not be
overcome by high
concentrations of the
substrate
Uncompetitive Inhibition
Uncompetitive
Mixed or Non-Competitive Inhibition
The inhibitor can bind to both free enzyme and the ES
complex
The affinity of the inhibitor to the two complexes might be
different
If binding of inhibitor changes the affinity for the substrate, Km will be
changed and called mixed inhibition
If only Vmax affected called Non-competitive inhibitor
Mixed Inhibition
The result will be decrease in
Vmax and either an increase or
decrease in Km
The effect of an non-competitive
inhibitor can only be partially
overcome by high concentrations
of the substrate

Mixed Inhibition
Non-Competitive
Enzyme Inhibition
Competitive
I binds at the active site
V
M
does not change
K
M
changes
Uncompetitive
I binds with ES only
V
M
changes
K
M
changes
Slope does not
change
Noncompetitive (Pure & Mixed)
I does not effect
binding of S
V
M
changes
K
M
does not change
Uninhibited: Competitive:
V
max
[S] V
max
[S]
v = ----------- v = ------------------------
K
M
+ [S] K
M
(1 + [I]/K
i
) + [S]

Noncompetitive: Uncompetitive:
(V
max
/(1 + [I]/K
i
)) [S] (V
max
/(1 + [I]/K
i
))[S]
v = ------------------------- v = -------------------------
K
M
+ [S] K
M
(1 + [I]/K
i
) + [S]
Michaelis-Menten Equations
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].
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 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 K
m
but that it decreases
V
max
.
Mixed Noncompetitive Inhibition: binding of
I by E influences binding of S by E
Figure 13.16 Lineweaver-Burk plot of mixed noncompetitive
inhibition. Note that both intercepts and the slope change in
the presence of I.
Uncompetitive Inhibition, where I combines
only with E, but not with ES
Figure 13.17
Lineweaver-Burk plot of
uncompetitive inhibition.
Note that both
intercepts change but
the slope (K
m
/V
max
)
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,
the enzyme which
catalyzes an essential
step in bacterial cell wall
synthesis.
It covalently blocks the
active site serine.
Kinetic Behavior of Enzymes Catalyzing
Bimolecular Reactions
Enzymes often catalyze reactions involving two
(or more) substrates.
Reactions may be sequential (single-
displacement) reactions.
These can be of two distinct classes:
Random, where either substrate may bind
first, followed by the other substrate.
Ordered, where a leading substrate binds
first, followed by the other substrate.
Reactions may be ping-pong (double
displacement) reactions.
Conversion of AEB to PEQ is the Rate-Limiting
Step in Random, Single-Displacement Reactions
In this type of sequential reaction, all possible binary
enzyme-substrate and enzyme-product complexes are
formed rapidly and reversibly when enzyme is added to a
reaction mixture containing A, B, P, and Q. This is a random
sequential reaction.
This symbolism is known as Cleland notation.
Creatine Kinase Acts by a Random,
Single-Displacement Mechanism
The overall direction of the reaction will be determined by
the relative concentrations of ATP, ADP, Cr, and CrP and the
equilibrium constant for the reaction.
An example of a random sequential reaction.
Creatine Kinase Acts by a Random,
Single-Displacement Mechanism
Figure 13.21
The structures of creatine
and creatine phosphate,
guanidinium compounds that
are important in muscle
energy metabolism.
In an Ordered, Single-Displacement Reaction,
the Leading Substrate Must Bind First
The leading substrate (A) binds first, followed by B.
Reaction between A and B occurs in the ternary complex
and is usually followed by an ordered release of the
products, P and Q. This is an ordered sequential reaction.
An Alternative way of Portraying the
Ordered, Single-Displacement Reaction
This is another view of ordered sequential.
Figure 13.19 Single-displacement bisubstrate mechanism.
13.5 - Kinetic Behavior of Enzymes
Catalyzing Bimolecular Reactions
The Double Displacement (Ping-Pong)
Reaction
Double-Displacement (Ping-Pong) reactions proceed via
formation of a covalently modified enzyme intermediate.
Reactions conforming to this kinetic pattern are characterized
by the fact that the product of the enzymes reaction with A
(called P in the above scheme) is released prior to reaction of
the enzyme with the second substrate, B.
An Alternative Presentation of the Double-
Displacement (Ping-Pong) Reaction
Other views of the ping-pong mechanism.
The Double Displacement (Ping-Pong)
Reaction
Figure 13.22
Double-
displacement
(ping-pong)
bisubstrate
mechanisms are
characterized by
parallel lines.
Glutamate:aspartate Aminotransferase
Figure 13.23
An enzyme
conforming to
a double-
displacement
bisubstrate
mechanism
(ping-pong).
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.
How Can Enzymes Be So Specific ?
A drawing, roughly to scale, of H
2
O, 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.
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.
Assignment: (2 pager report on abzymes)
For a good review of abzymes, see Science,
Vol. 269, pages 1835-1842 (1995).
Transition states
Is It Possible to Design An Enzyme to
Catalyze Any Desired Reaction ?
A known enzyme can be engineered by in vitro
site-directed 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.
And the cellular environment may provide
complications not apparent in the computer
modeling.