Enzyme Kinetics

Enzymes Are Uniquely Powerful Catalysts

Enzymes are proteins that can accelerate
biochemical reactions by factors of 10
5
to 10
17
!
This is much higher than chemical catalysts.
Enzymes can be extremely specific in terms of
reaction substrates and products.
Enzymes catalyze reactions under mild
conditions (e.g., pH 7.4, 37C).
The catalytic activities of many enzymes can be
regulated by allosteric effectors.

Chemical Kinetics
Irreversible First-Order Reactions
A B

v = d[B]/dt = -d[A]/dt = k[A]

(k = first-order rate constant (s
-1
))

Change in [A] with time (t):
[A]= [A]
o
e
kt
or
[A]/[A]
o
= e
kt

ln([A]/[A]
o)
= kt

([A]
o
= initial concentration)

k
Reversible First-Order Reactions
A B


v = -d[A]/dt = k
1
[A] - k
-1
[B]

At equilibrium: k
1
[A]
eq
- k
-1
[B]
eq
= 0


[B]
eq
/[A]
eq
= k
1
/k
-1
= K
eq

k
1
k
-1


Second-Order Reactions
2A P

v = -d[A]/dt = k[A]
2

Change in [A] with time:
1/[A] = 1/[A]
o
+ kt

A + B P

v = -d[A]/dt = -d[B]/dt = k[A][B]

(k = second-order rate constant (M
-1
s
-1
))
k
k
Note: third-order reactions rare, fourth- and higher-order reactions unknown.
Free Energy Diagrams
K
eq
= e
G/RT

For A A


[A]

/[A]
o
= e
G/RT

[A]

= [A]
o
e
G/RT
K
eq
= equilibrium constant
[A]

= concentration of molecules having the activation energy
[A]
o
= total concentration of A
G

= standard free energy change of activation (activation energy)

Relationship of Reaction Rate Constant to
Activation Energy and Temperature: The
Arrhenius Equation
k = A e
-Ea/RT


Reaction rate constant (k) determined by activation
energy (E
a
or

G

, applying transition state theory)

and temperature (T) and proportional to frequency of
forming product
(A or Q = k
B
T/h, where k
B
= Boltzmanns constant, h =
Plancks constant):

k = (k
B
T/h) e
-G/RT

k =

Q e
-G/RT


G = H - T S, so:
k = Q e
S/R
e
-H/RT

k = Q e
-H/RT
(where Q = Q e
S/R
)
So: ln k = ln Q - H

/RT
L-malate
fumarate + H
2
0
ln k
Relation of Equilibrium Constant to
Activation Energy
K
eq
= k
1
/k
-1

K
eq
= (Q e
-G
1
/RT
)/(Q e
-G
-1
/RT
)

K
eq
= e
-(G
1
-

G
-1
)/RT


G = G
1

- G
-1

K
eq
= e
G/RT
Equilibrium constant K
eq
says nothing about rate of
reaction, only free energy difference between final and
initial states. The activation energy barrier opposes
reaction in both directions
Effect of a Catalyst on Activation Energy
Catalysts do not affect G
A
(initial) or G
B
(final) and so do not affect overall free
energy change (G = G
B
- G
A
) or equilibrium constant K
eq
.
Equilibrium concentrations of A and B still determined solely by overall free
energy change.
Catalysts only affect G

, lowering the activation energy.

They accelerate both the forward and reverse reaction (increase kinetic rate
constants k
1
and k
-1
).
Intermediate States in Multistep
Reactions
Enzyme Kinetics
The Effect of Substrate Concentration on
Reaction Velocity
Michaelis-Menten Kinetics (1)
v = k
2
[ES]
(Note: k
2
also referred to as k
cat
)

[Enzyme]
total
= [E]
t
= [E] + [ES]

How to solve for [ES]?

1. Assume equilibrium, if k
-1
>> k
2
:
K
S
= k
-1
/k
1
= [E][S]/[ES]
or
2. Assume steady state:
d[ES]/dt = 0
(Michaelis and
Menten, 1913)
(Briggs and
Haldane, 1925)
E = enzyme, S = substrate,
ES = enzyme-substrate complex,
P = product
The Steady State in Enzyme Kinetics
Michaelis-Menten Kinetics Continued (2)
Rate of formation of ES complex = k
1
[E][S]
Rate of breakdown of ES complex = k
-1
[ES] + k
2
[ES]

Because of steady state assumption:
k
1
[E][S] = k
-1
[ES] + k
2
[ES]

Rearranging: [ES] = (k
1
/(k
-1
+ k
2
))[E][S]

Substituting Michaelis constant = K
M
= (k
-1
+ k
2
)/k
1
) = K
S
+ k
2
/k
1
:
[ES] = ([E][S])/K
M

So:

K
M
[ES] = [E][S]
Michaelis-Menten Kinetics Continued (3)
Substituting [E] = [E]
t
- [ES]:
K
M
[ES] = [E]
t
[S] - [ES][S]

Rearranging: [ES](K
M
+ [S]) = [E]
t
[S]

So: [ES] = [E]
t
[S]/(K
M
+ [S])



Michaelis-Menten Kinetics Continued (4)
Now we can substitute for [ES] in the rate equation
v
o
= k
2
[ES].

But first note that the velocity in v = k
2
[ES] we use is the initial
velocity, v
o
, the velocity of the reaction after the pre-steady state
and in the early part of the steady state, i.e., before ~10% of
substrate is converted to product. This is because at this stage of
the reaction, the steady-state assumption is reasonable ([ES] is still
approximately constant). Also, since not much P has yet
accumulated, we can approximate the kinetics for even reversible
reactions with this equation if we limit ourselves to v
o
.



The Michaelis-Menten Equation
v
o
= k
2
[E]
t
[S]/(K
M
+ [S])

or

v
o
= V
max
[S]/(K
M
+ [S])

(since V
max
= k
2
[E]
t
when [S] >> K
M
)
A Lineweaver-Burk (Double Reciprocal)
Plot
An Eadie-Hofstee Plot
Multistep Reactions
E + S ES ES E + P


v
o
= k
cat
[E]
t
[S]/(K
M
+ [S])
k
2
k
3
k
1
k
-1


k
cat
= empirical rate constant that reflects rate-
determining component. Mathematically, for the
reaction above,
k
cat
= k
2
k
3
/(k
2
+ k
3
).
However, k
2
and k
3
often very hard to establish
with precision as individual rate constants.



Catalytic Efficiency (k
cat
/K
M
)
Perfect enzyme

Diffusion-controlled limit:
10
8
-10
9
M
-1
s
-1
Substrate
preferences of
chymotrypsin
pH-Dependence of Enzyme Activity

Enzyme-Catalyzed Bisubstrate Reactions:
Two Examples
Bisubstrate Reactions
S1 + S2 P1 + P2

A-X + B A + B-X (in transferase reactions)

Sequential binding of S1 and S2 before catalysis:
Random substrate binding - Either S1 or S2 can
bind first, then the other binds.
Ordered substrate binding - S1 must bind before
S2.
Ping Pong reaction - first S1 P1, P1 released
before S2 binds, then S2 P2.


E


E



Ping Pong reaction
Sequential binding
Ternary
complex
Indicative of ternary complex formation and a sequential
mechanism
Indicative of a Ping Pong mechanism
Enzyme Inhibition
Types of Enzyme Inhibition
Reversible inhibition
(Inhibitors that can reversibly bind and dissociate from
enzyme; activity of enzyme recovers when inhibitor diluted
out; usually non-covalent interaction.)
Competitive
Mixed (noncompetitive)
Uncompetitive
Irreversible inhibition
(Inactivators that irreversibly associate with enzyme; activity
of enzyme does not recover with dilution; usually covalent
interaction.)
Competitive Inhibition
Effects of Competitive Inhibitor on
Enzyme Kinetics
K
app
M
= K
M
(1 + [I]/K
I
)

> K
M
V
app
max
= V
max
K
I
(inhibitor dissociation
constant)

=

k
off
/k
on


a = 1 + [I]/K
I

A Substrate and Its Competitive Inhibitor
HIV Protease Inhibitors
Relationship of K
I
to Half-Maximal
Inhibitory Concentration (IC
50
)

For a competitive inhibitor of an enzyme that follows
Michaelis-Menton kinetics:

v
I
/v
0
= (V
max
[S]/(K
M
a + [S]))/(V
max
[S]/(K
M
+ [S])) = (K
M
+
[S])/(K
M
a + [S])
v
I
= initial velocity with inhibitor
v
0
= initial velocity without inhibitor
a = 1 + [I]/K
I


When v
I
/v
0
= 0.5, [I] = IC
50
= K
I
(1 + [S]/K
M
)

If measurement made when [S] << K
M
, IC
50
= K
I
Uncompetitive Inhibition

Effects of Uncompetitive Inhibitor on
Enzyme Kinetics
K
app
M
= K
M
/(1 + [I]/K
I
)

< K
M
V
app
max
= V
max
/(1 + [I]/K
I
)

< V
max

a = 1 + [I]/K
I

Mixed (Noncompetitive) Inhibition
Effects of Mixed (Noncompetitive)
Inhibitor on Enzyme Kinetics
K
app
M
= (1 + [I]/K
I
)K
M
/(1 + [I]/K
I
)

(= K
M
, when K
I
= K
I
, which is often the case.)


V
app
max
= V
max
/(1 + [I]/K
I
) < V
max

k
1


k
-1
Not the same as uncompetitive inhibition.
In mixed inhibition, inhibitor can bind E or
ES.

a = 1 + [I]/K
I

a = 1 + [I]/K
I


a = 1 + [I]/K
I

a = 1 + [I]/K
I

(For mixed inhibitor,
generally, ~ K
M)
Irreversible Inhibition
k
1


k
-1
k
2
E + I EI E-I

Plot:
ln(residual enzyme activity) vs. time

If [I]>>[E], conditions are pseudo-first
order and slope is -k
obs
(pseudo-first
order inactivation rate constant)

k
inact
(second-order inactivation constant)
= k
1
k
2
/k
-1
= k
obs
/[I]
Slope = -k
obs

Irreversible Inhibition by Adduct
Formation
(diisopropylfluorophosphate)
Irreversible Inhibition of Chymotrypsin by
TPCK
(N-tosyl-L-phenylalanine
chloromethylketone)