This document discusses enzyme kinetics and inhibition. It notes that enzymes follow zero-order kinetics at high substrate concentrations, meaning the reaction rate does not increase with more substrate. It then describes Michaelis-Menten kinetics using the rate equation and defines terms like KM and Vmax. KM is the substrate concentration at half the maximum rate and is a measure of substrate affinity. Different types of inhibition are also summarized, including competitive, uncompetitive, and mixed inhibition and how they affect the Michaelis-Menten equation and Lineweaver-Burk plots.

1. M.Prasad Naidu
MSc Medical Biochemistry,
Ph.D.Research Scholar

2. 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 + H20 Glucose + Fructose
O
H
HO
H
HO
H
OH
OHH
H
OH
OH
HO
H
H OH
O
H
H
HO
H
H
H
OH

3. PEESSE 2
1
1-
k
k
k
E = Enzyme S = Substrate P = Product
ES = Enzyme-Substrate complex
k1 rate constant for the forward reaction
k-1 = rate constant for the breakdown of the ES to
substrate
k2 = rate constant for the formation of the
products

4. 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
2k
dt
d
v
[ES] is the difference between the
rates of ES formation minus the
rates of its disappearance.
ESESSE
ES
211 kkk
dt
d
1

5. Assumption of equilibrium
k-1>>k2 the formation of product is so
much slower than the formation of the ES
complex. That we can assume:
ES
SE
1
1
k
k
Ks
Ks is the dissociation constant for the ES complex.

6. 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

7. ESEE T 3
Combining 1 + 2 + 3
ESkkSES-Ek 21-T1
SEkSkkkES T1121-
SK
SE
ES T
M
1
21-
k
kk
KM
rearranging
Divide by k1 and solve for [ES] Where

8. SK
SE
ES
P T2
2
0 Mt
o
k
k
dt
d
v
vo is the initial velocity when the reaction is just starting out.
And is the maximum velocityT2max EkV
SK
SVmax
M
ov
The Michaelis - Menten
equation

9. The Km is the substrate concentration where vo equals
one-half Vmax

10. The KM
can be expressed as:
1
2
1
2
1
1
KK
k
k
k
k
k
k
sM
As Ks decreases, the affinity for the substrate
increases. The KM can be a measure for substrate
affinity if k2<k-1

11. There are a wide range of KM, Vmax , and efficiency
seen in enzymes
But how do we analyze kinetic data?

12. maxmax
M
V
1
S
1
V
K1
ov

14. Lineweaver-Burk plot: slope = KM/Vmax,
1/vo intercept is equal to 1/Vmax
the extrapolated x intercept is equal to -1/KM
For small errors in at low [S] leads to large errors in 1/vo
T
max
E
V
catk
kcat is how many reactions an
enzyme can catalyze per second
The turnover number

15. For Michaelis -Menton kinetics k2= kcat
When [S] << KM very little ES is formed and [E] = [E]T
and
SE
K
k
SE
K
k
M
cat
T
M
2
ov
Kcat/KM is a measure of catalytic efficiency

16. When k2>>k-1 or the ratio
21
21
kk
kk
is maximum
Then
1
MK
k
kcat
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 108 to 109 M-1s-1. An enzyme lowers
the transition state so there is no activation energy
and the catalyzed rate is as fast as molecules collide.

17.  All substrates must combine with enzyme
before reaction can occur

18. Bisubstrate reactions

20.  Group transfer reactions
 One or more products released before all
substrates added

21. Although a phenomenological description can be
obtained the nature of the reaction intermediates
remain indeterminate and other independent
measurements are needed.

22. There are three types of inhibition kinetics competitive,
mixed and uncompetitive.
•Competitive- Where the inhibitor competes with the
substrate.

23. Competitive Inhibition

24. SK
SV
M
max
ov
IK
I
1
EI
IE
KI

27. Competitive Inhibition: Lineweaver-Burke Plot

28. Uncompetitive Inhibition

29. Uncompetitive Inhibition: Lineweaver-Burke Plot

32. 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 KM or Vmax or both.
EI
IE
KI
ESI
IES
KI
SK
SV
M
max
ov
IK
I
1