Enzyme%20Kinetics%20and%20Catalysis%20II

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Enzyme Kinetics and Catalysis II

• 3/24/2003

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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
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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.
ES is the difference between the rates of ES
formation minus the rates of its disappearance.
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Assumption of equilibrium k-1gtgtk2 the formation
of product is so much slower than the formation
of the ES complex. That we can assume
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
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Combining 1 2 3
rearranging
Divide by k1 and solve for ES
Where
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vo is the initial velocity when the reaction is
just starting out. And
is the maximum velocity
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
As Ks decreases, the affinity for the substrate
increases. The KM can be a measure for substrate
affinity if k2ltk-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
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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
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
ltlt KM very little ES is formed and E ET and
Kcat/KM is a measure of catalytic efficiency
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What is catalytic perfection?
When k2gtgtk-1 or the ratio
is maximum
Or when every substrate that hits the enzyme
causes a reaction to take place. This is
catalytic perfection.
Then
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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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.
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The effect of pH on kinetic parameters
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