Biochemical Reaction Rate: Enzyme Kinetics

1
Biochemical Reaction Rate Enzyme Kinetics
Lipitor inhibits HMG-CoA reductase, a critical
step in cholesterol biosynthesis
What affect do enzymes and enzyme inhibitors have
on enzyme catalysis on a quantitative level?
2
Triose Phosphate Isomerase Reaction Progression
3
Product Formation Versus Time
Determining the reaction velocity dependents on
what?
4
Reaction Velocity versus Substrate
A ? P V -dA/dt dP/dt V kA A B ?
P V kAB 2 A ? P V kA2
5
Michaelis-Menten Enzyme Kinetics
k1 k2 k-1

• E S ? ES ? E P
• Assumptions
• Single subunit substrate
• Product low (V0)
• ES constant (steady state)
• Definitions
• Vmax k2ET (ET E ES)
• KM (k-1 k2)/k1 (Michaelis constant)

V0 Vmax S/(S KM)
When does V0 ½Vmax? What is Km? What is V0
when S is much smaller than Km? What is V0 when S
is much larger than Km?
6
Lineweaver-Burk Double-Reciprocal Plot
To calculate Km and Vmax
1/ V0 KM/ Vmax 1/S 1/ Vmax
Y m X b
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Michaelis Constant Value Km
A higher Km value means what?
8
E S ? ES ? E P
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Substrate Preference with Chymotrypsin

• Kcat/KM is a measure of Catalytic Efficiency

10
Varying the Enzyme
For a one-substrate, enzyme-catalyzed reaction,
which of the family of curves would you expect to
be obtained?
Hint What are the equations for Vmax and KM?
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Not a Michaelis-Menten Enzyme-Kinetics Reaction
Why not?
12
Not a Michaelis-Menten Enzyme-Kinetics Reaction
Why not?
13
Not a Michaelis-Menten Enzyme-Kinetics Reaction
An irreversible inhibitor is present
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Reversible Inhibitors Competitive Inhibition
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Competitive Enzyme Inhibition
Reaction Pathway
Substrate can out compete inhibitor ? Vmax
unchanged since Vmax k2ET Inhibitor binds in
the active site ? KM increases since KM (k-1
k2)/k1 How is Lineweaver-Burk plot altered?
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Competitive Inhibition Lineweaver-Burk Plot

• Vmax Unaltered
• KM Increased

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Noncompetitive Enzyme Inhibition
Is Vmax affected? How is KM influenced?
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Noncompetitive Enzyme Inhibition
Reaction Pathway
Taking enzyme out of circulation ? Vmax lowered
since Vmax k2ET Inhibitor binds both E and ES
? KM unchanged since KM (k-1 k2)/k1 How is
Lineweaver-Burk plot altered?
19
Noncompetitive Inhibition Lineweaver-Burk Plot

• Vmax Lowered
• KM Unchanged

20
Uncompetitive Enzyme Inhibition
Reaction Pathway
Is Vmax affected? How is KM influenced?
21
Uncompetitive Enzyme Inhibition
Reaction Pathway
Taking enzyme out of circulation ? Vmax lowered
since Vmax k2ET Inhibitor binds to E after
the substrate binds ? KM decreased since KM
(k-1 k2)/k1 How is Lineweaver-Burk plot
altered?
22
Uncompetitive Inhibition Lineweaver-Burk Plot

• Vmax Lowered
• KM Lowered

23
Virus Inhibition via Reverse Transcriptase
Activity
HIV that causes AIDS
24
Early Developed HIV Drugs
Propose how these molecules function at
inhibitors of HIV.
25
Reverse Transcriptase Inhibition by
Non-Nucleoside Analog
Noncompetitive inhibitor binds to a hydrophobic
patch on surface of reverse transcriptase
26
HIV Protease Cartoon Structure
Substrate with side chains binding in
hydrophobic pockets Catalytic Asp residues
27
HIV Protease Inhibitors
Rational drug design based on a detailed
knowledge of the enzyme structure
Why is it preferable to select a target inhibitor
that is unique to the virus and not the host?
28
Transition State Analogue Inhibitor
Why is the transition state analogue an
inhibitor?
29
Transition State Analogue Inhibitor
Why is the transition state analogue an
inhibitor?
30
Feedback Inhibition of Phosphofructokinase
31
Allosteric Enzyme Regulation of
Phosphofructokinase
What is the difference on these two curves?
32
Mechanism of Allosteric Inhibition for
Phosphofructokinase
Green structure w/o PEP Red configuration w/ PEP
33
Kinetic Relationships
Draw the curve for the appropriate connection
between variables.
34
Lineweaver-Burk Plot
Which reaction has the highest KM and Vmax value?
35
Chapter 7 Problems 5, 7, 18, 19, 21, 25, 27, 29
and 31