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Lecture - 17-Enzyme Kinetics and Catalysis II

The document discusses enzyme kinetics and catalysis, including how enzymes can follow zero-order or Michaelis-Menten kinetics depending on substrate concentration. It also covers reaction mechanisms, inhibition kinetics like competitive and uncompetitive inhibition, and how pH can affect kinetic parameters.

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Nagarjuna Vuchuru
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© © All Rights Reserved
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22 views

Lecture - 17-Enzyme Kinetics and Catalysis II

The document discusses enzyme kinetics and catalysis, including how enzymes can follow zero-order or Michaelis-Menten kinetics depending on substrate concentration. It also covers reaction mechanisms, inhibition kinetics like competitive and uncompetitive inhibition, and how pH can affect kinetic parameters.

Uploaded by

Nagarjuna Vuchuru
Copyright
© © All Rights Reserved
Available Formats
Download as PPT, PDF, TXT or read online on Scribd
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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 + H20 Glucose + Fructose
H
H H
H OH O
OH
HO H OH
H O H
HO OH H
HO H H
HO
H OH

H OH
k1
E  S  ES  E  P k2

k -1

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
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.

d P  [ES] is the difference between the


v  k 2 ES rates of ES formation minus the
dt rates of its disappearance. 1

d ES
 k1 E S  k 1 ES  k 2 ES
dt
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:

k 1 E S
Ks  
k1 ES
Ks 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

d ES
0
dt
ET  E  ES 3

Combining 1 + 2 + 3

k1 E T - ESS  k -1  k 2 ES


rearranging

ESk -1  k 2  k1 S  k1 ET S


Divide by k1 and solve for [ES] Where
k -1  k 2
ES  E T S KM 
k1
K M  S
 d P   k 2 E T S
vo     k 2 ES 
 dt t 0 K M  S

vo is the initial velocity when the reaction is just starting out.

And Vmax  k 2 E T is the maximum velocity

Vmax S The Michaelis - Menten


vo 
K M  S
equation
The Km is the substrate concentration where vo equals
one-half Vmax
The KM widely varies among different enzymes

The KM
k 1 k 2 k2
can be expressed as: KM    Ks 
k1 k1 k1

As Ks decreases, the affinity for the substrate


increases. The KM can be a measure for substrate
affinity if k2<k-1
There are a wide range of KM, Vmax , and efficiency
seen in enzymes

But how do we analyze kinetic data?


The double reciprocal plot

1  KM  1 1
   
vo  Vmax  S Vmax
Lineweaver-Burk plot: slope = KM/Vmax,
1/vo 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

kcat is how many reactions an


Vmax enzyme can catalyze per second
kcat 
E T The turnover number
For Michaelis -Menton kinetics k2= kcat
When [S] << KM very little ES is formed and [E] = [E]T

k2 k cat
ET S  ES
and
vo 
KM KM
Kcat/KM is a measure of catalytic efficiency
What is catalytic perfection?
k1k 2
When k2>>k-1 or the ratio is maximum
k 1  k 2

kcat Or when every substrate that hits


Then
 k1 the enzyme causes a reaction to
KM 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.
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
KI 
E I
EI
Vmax S
vo 
K M  S
 I 
  1  
 KI 
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 KM or Vmax or both.

E I KI 
ESI
KI 
EI ESI
Vmax S  I 
   1  
vo 
K M   S  K 
I 
The effect of pH on kinetic parameters

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