Last Lecture: Enzymes, Introduction

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Last Lecture Enzymes, Introduction Today
Enzymes, Kinetic Analysis
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First Order Reaction
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Second Order Reaction
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Factors Affecting Rates of Enzyme Catalyzed
Reactions
1. Rate affected by Enzyme Concentration
formation of ES rate limiting
measure rate of appearance of product dP/dt
or disappearance of substrate
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Factors Affecting Rates of Enzyme Catalyzed
Reactions
carry out reaction under saturating
concentrations of S ie SgtgtgtE rate is
independent of S, Rate k1E or Rate k2ES
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Factors Affecting Rates of Enzyme Catalyzed
Reactions
2. Temperature rate of most chemical
reactions doubles with 10C increase in T
for many enzymes, triples
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Factors Affecting Rates of Enzyme Catalyzed
Reactions
3. pH pH rate profile indicates proton
transfer in reaction
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Factors Affecting Rates of Enzyme Catalyzed
Reactions
4. Control of transcription, processing,
translation
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Factors Affecting Rates of Enzyme Catalyzed
Reactions
5. Limited Proteolysis
many enzymes biosynthesized as inactive
precursors (zymogens) that are activated by
limited proteolysis to yield mature active
enzyme ex. Chymotripsinogen, synthesizedin
pancreas, 245 aa
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6. Substrate Concentration Effects
Michaelis-Menten
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6. Substrate Concentration Effects
Assumptions 1. Initial rates, P 0
therefore no k-2 2. Steady State
(Briggs-Haldane) 3.
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The Michaelis Constant, KM
(3)
(2)
(1)
(1) (2) (3)
0 order reaction
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Analysis of Kinetic Data
Double Reciprocal Plot (Lineweaver-Burk)
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7. Regulation of Enzyme Activity by Reversible
Inhibitor Binding Small molecule
inhibitors or activators Three types of
reversible inhibitors (a) Competitive
inhibitors resemble substrates, compete
for active
site (b) Non-competitive inhibitors inhibitors
bind to second
site and effect
conformational
change (c) Uncompetitive
inhibitors inhibitor binds to ES complex
not
free enzyme (E)
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Competitive Inhibition
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Competitive Inhibition
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Competitive Inhibition
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Effects of Competitive Inhibition on Enzyme
Kinetics
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Non-competitive Inhibition
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Kinetic Scheme Non-competitive Inhibition
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Non-Competitive Inhibition
intrinsic affinity of substrate/product
unchanged
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Uncompetitive Inhibition
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Uncompetitive Inhibition
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Uncompetitive Inhibition
uncompetitive inhibition
Compound RoundupTM N-phosphonomethylglycine Targ
et 3-phosphoshikimate 3-carboxyvinyltransferase
Compound Li Target myo-inositol
monophosphatase
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Uncompetitive Inhibition
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Summary
-observed kinetics diagnostic of mechanism -KM
can change, Vmax, or both