Enzyme Kinetics and Catalysis II

3/24/2003
Kinetics of Enzymes
Enzymes follow zero order kinetics when substrate
concentrations are high. Zero order means there is no increase
in the rate of the reaction when more substrate is added.
Given the following breakdown of sucrose to glucose and
fructose
Sucrose + H
2
0 Glucose + Fructose
O
H
HO
H
HO
H
OH
OH
H
H
OH
OH
HO
H
H
OH
O
H
H
HO
H
H
H
OH
P E ES S E
2
1
1 -
k
k
k
+ +

E = Enzyme S = Substrate P = Product
ES = Enzyme-Substrate complex
k
1
rate constant for the forward reaction
k
-1
= rate constant for the breakdown of the ES to
substrate
k
2
= rate constant for the formation of the
products
When the substrate concentration becomes large
enough to force the equilibrium to form completely
all ES the second step in the reaction becomes rate
limiting because no more ES can be made and the
enzyme-substrate complex is at its maximum value.
| |
| | ES
P
2
k
dt
d
v = =
[ES] is the difference between the
rates of ES formation minus the
rates of its disappearance.
| |
| || | | | | | ES ES S E
ES
2 1 1
k k k
dt
d
=

1
Assumption of equilibrium
k
-1
>>k
2
the formation of product is so
much slower than the formation of the ES
complex. That we can assume:
| || |
| | ES
S E
1
1
= =

k
k
K
s
K
s
is the dissociation constant for the ES complex.
Assumption of steady state
Transient phase where in the course of a reaction the
concentration of ES does not change
| |
0
ES
=
dt
d
2
| | | | | | ES E E
T
+ =
3
Combining 1 + 2 + 3
| | | | ( )| | ( )| | ES k k S ES - E k
2 1 - T 1
+ =
| | | | ( ) | | | | S E k S k k k ES
T 1 1 2 1 -
= + +
| |
| | | |
| | S K
S E
ES
T
+
=
M
1
2 1 -
k
k k
K
+
=
M
rearranging
Divide by k
1
and solve for [ES] Where
| |
| |
| | | |
| | S K
S E
ES
P
T 2
2
0
+
= =
|
.
|

\
|
=
= M t
o
k
k
dt
d
v
v
o
is the initial velocity when the reaction is just starting out.

And is the maximum velocity
| |
T 2 max
E k V =
| |
| | S K
S V
max
+
=
M
o
v
The Michaelis - Menten
equation
The K
m
is the substrate concentration where v
o
equals
one-half V
max

The K
M
widely varies among different enzymes
The K
M

can be expressed as:
1
2
1
2
1
1
K K
k
k
k
k
k
k
s M
+ = + =

As Ks decreases, the affinity for the substrate
increases. The K
M
can be a measure for substrate
affinity if k
2
<k
-1

There are a wide range of K
M
, Vmax , and efficiency
seen in enzymes

But how do we analyze kinetic data?
The double reciprocal plot
| |
max max
M
V
1
S
1
V
K 1
+
|
|
.
|

\
|
=
o
v
Lineweaver-Burk plot: slope = K
M
/Vmax,
1/v
o
intercept is equal to 1/Vmax
the extrapolated x intercept is equal to -1/K
M

For small errors in at low [S] leads to large errors in 1/v
o

| |
T
max
E
V
=
cat
k
k
cat
is how many reactions an
enzyme can catalyze per second
The turnover number
For Michaelis -Menton kinetics k
2
= k
cat
When [S] << K
M
very little ES is formed and [E] = [E]
T
and
| | | | | || | S E
K
k
S E
K
k
M
cat
T
M
2
~ ~
o
v
K
cat
/K
M
is a measure of catalytic efficiency
What is catalytic perfection?
When k
2
>>k
-1
or the ratio
2 1
2 1
k k
k k
+

is maximum
Then
1
M
K
k
k
cat
=
Or when every substrate that hits
the enzyme causes a reaction to
take place. This is catalytic
perfection.
Diffusion-controlled limit- diffusion rate of a substrate
is in the range of 10
8
to 10
9
M
-1
s
-1
. An enzyme lowers
the transition state so there is no activation energy
and the catalyzed rate is as fast as molecules collide.
Reaction Mechanisms
A: Sequential Reactions
All substrates must combine with enzyme
before reaction can occur
Bisubstrate reactions
Random Bisubstrate Reactions
Ping-Pong Reactions
Group transfer reactions
One or more products released before all
substrates added
Kinetic data cannot unambiguously
establish a reaction mechanism.
Although a phenomenological description can be
obtained the nature of the reaction intermediates
remain indeterminate and other independent
measurements are needed.
Inhibition kinetics
There are three types of inhibition kinetics competitive,
mixed and uncompetitive.
Competitive- Where the inhibitor competes with the
substrate.
Competitive Inhibition
| |
| | S K
S V
M
max
+
=
o
o
v
| |
|
|
.
|

\
|
+ =
I
K
I
1 o
| || |
| | EI
I E
K
I
=
HIV protease inhibitors
Competitive Inhibition: Lineweaver-Burke Plot
Uncompetitive Inhibition
Uncompetitive Inhibition: Lineweaver-Burke Plot
Mixed inhibition
Mixed inhibition is when the inhibitor binds to the
enzyme at a location distinct from the substrate
binding site. The binding of the inhibitor will either
alter the K
M
or V
max
or both.
| || |
| | EI
I E
K
I
=
| || |
| | ESI
I ES
K
I
=
'
| |
| | S K
S V
M
max
o o
'
+
=
o
v
| |
|
|
.
|

\
|
'
+ =
'
I
K
I
1 o
The effect of pH on kinetic parameters