enzyme kinetics and catalysis ii 3/24/2003. kinetics of enzymes enzymes follow zero order kinetics...
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![Page 1: Enzyme Kinetics and Catalysis II 3/24/2003. Kinetics of Enzymes Enzymes follow zero order kinetics when substrate concentrations are high. Zero order](https://reader035.vdocuments.site/reader035/viewer/2022062216/56649da55503460f94a90875/html5/thumbnails/1.jpg)
Enzyme Kinetics and Catalysis II
3/24/2003
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Kinetics of EnzymesEnzymes 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
OHHH
OH
OH
HOH
H OH
O
HH
HO
H
H
H
OH
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P EES S E 2
1
1-
kk
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
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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.
ESP
2kdt
dv
[ES] is the difference between the rates of ES formation minus the rates of its disappearance.
ESESSEES
211 kkkdt
d
1
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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
kK s
Ks is the dissociation constant for the ES complex.
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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
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ES E E T 3
Combining 1 + 2 + 3
ESk k SES-Ek 21-T1
SEk Sk k kES T1121-
S K
SE ES T
M1
21-
k
k k K
M
rearranging
Divide by k1 and solve for [ES] Where
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SK
SEES
P T22
0
Mto
kk
dt
dv
vo is the initial velocity when the reaction is just starting out.
And is the maximum velocity T2max Ek V
SK
SVmax
Mov
The Michaelis - Menten equation
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The Km is the substrate concentration where vo equals one-half Vmax
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The KM widely varies among different enzymes
The KM
can be expressed as:1
2
1
2
1
1 KKk
k
k
k
k
ksM
As Ks decreases, the affinity for the substrate increases. The KM can be a measure for substrate affinity if k2<k-1
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There are a wide range of KM, Vmax , and efficiency seen in enzymes
But how do we analyze kinetic data?
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The double reciprocal plot
maxmax
M
V
1
S
1
V
K1
ov
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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
Tmax
E
Vcatk
kcat is how many reactions an enzyme can catalyze per second
The turnover number
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For Michaelis -Menton kinetics k2= kcat
When [S] << KM very little ES is formed and [E] = [E]T
and SEK
kSE
K
k
M
catT
M
2 ov
Kcat/KM is a measure of catalytic efficiency
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What is catalytic perfection?
When k2>>k-1 or the ratio 21
21
kk
kk
is maximum
Then1
MKk
kcat Or when every substrate that hits the enzyme causes a reaction totake 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.
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Reaction MechanismsA: Sequential Reactions
• All substrates must combine with enzyme before reaction can occur
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Bisubstrate reactions
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Random Bisubstrate Reactions
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Ping-Pong Reactions
• Group transfer reactions
• One or more products released before all substrates added
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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.
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Inhibition kinetics
There are three types of inhibition kinetics competitive, mixed and uncompetitive.•Competitive- Where the inhibitor competes with the substrate.
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Competitive Inhibition
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SK
SV
M
max
ov
IK
I1
EI
IEK I
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HIV protease inhibitors
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Competitive Inhibition: Lineweaver-Burke Plot
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Uncompetitive Inhibition
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Uncompetitive Inhibition: Lineweaver-Burke Plot
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Mixed inhibition
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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
IEK I
ESI
IESK I
SK
SV
M
max
ov
IK
I1
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The effect of pH on kinetic parameters
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