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Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Chapter 14
Enzyme Kineticsto accompany
Biochemistry, 2/e
by
Reginald Garrett and Charles Grisham
All rights reserved. Requests for permission to make copies of any part of the work should be mailed to: Permissions Department, Harcourt Brace & Company, 6277 Sea Harbor Drive, Orlando, Florida 32887-6777
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Outline
• 14.1 Catalytic Power, Specificity, Regulation
• 14.2 Introduction to Enzyme Kinetics
• 14.3 Kinetics of Enzyme-Catalyzed Reactions
• 14.4 Enzyme Inhibition
• 14.5 Kinetics of Two-Substrate Reactions
• 14.6 Ribozymes and Abzymes
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Enzymes
• Enzymes endow cells with the remarkable capacity to exert kinetic control over thermodynamic potentiality
• Enzymes are the agents of metabolic function
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Catalytic Power
• Enzymes can accelerate reactions as much as 1016 over uncatalyzed rates!
• Urease is a good example: – Catalyzed rate: 3x104/sec
– Uncatalyzed rate: 3x10 -10/sec
– Ratio is 1x1014 !
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Specificity
• Enzymes selectively recognize proper substrates over other molecules
• Enzymes produce products in very high yields - often much greater than 95%
• Specificity is controlled by structure - the unique fit of substrate with enzyme controls the selectivity for substrate and the product yield
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Other Aspects of Enzymes
• Regulation - to be covered in Chapter 15 • Mechanisms - to be covered in Chapter 16 • Coenzymes - to be covered in Chapter 18
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
14.2 Enzyme Kinetics
Several terms to know!
• rate or velocity
• rate constant
• rate law
• order of a reaction
• molecularity of a reaction
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
The Transition State
Understand the difference between G and G‡
• The overall free energy change for a reaction is related to the equilibrium constant
• The free energy of activation for a reaction is related to the rate constant
• It is extremely important to appreciate this distinction!
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
What Enzymes Do....
• Enzymes accelerate reactions by lowering the free energy of activation
• Enzymes do this by binding the transition state of the reaction better than the substrate
• Much more of this in Chapter 16!
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
The Michaelis-Menten Equation
You should be able to derive this! • Louis Michaelis and Maude Menten's theory
• It assumes the formation of an enzyme-substrate complex
• It assumes that the ES complex is in rapid equilibrium with free enzyme
• Breakdown of ES to form products is assumed to be slower than 1) formation of ES and 2) breakdown of ES to re-form E and S
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Understanding Km
The "kinetic activator constant"
• Km is a constant
• Km is a constant derived from rate constants
• Km is, under true Michaelis-Menten conditions, an estimate of the dissociation constant of E from S
• Small Km means tight binding; high Km means weak binding
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Understanding Vmax
The theoretical maximal velocity
• Vmax is a constant
• Vmax is the theoretical maximal rate of the reaction - but it is NEVER achieved in reality
• To reach Vmax would require that ALL enzyme molecules are tightly bound with substrate
• Vmax is asymptotically approached as substrate is increased
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
The dual nature of the Michaelis-Menten equation
Combination of 0-order and 1st-order kinetics
• When S is low, the equation for rate is 1st order in S
• When S is high, the equation for rate is 0-order in S
• The Michaelis-Menten equation describes a rectangular hyperbolic dependence of v on S!
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
The turnover number
A measure of catalytic activity
• kcat, the turnover number, is the number of substrate molecules converted to product per enzyme molecule per unit of time, when E is saturated with substrate.
• If the M-M model fits, k2 = kcat = Vmax/Et
• Values of kcat range from less than 1/sec to many millions per sec
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
The catalytic efficiencyName for kcat/Km
• An estimate of "how perfect" the enzyme is
• kcat/Km is an apparent second-order rate constant
• It measures how the enzyme performs when S is low
• The upper limit for kcat/Km is the diffusion limit - the rate at which E and S diffuse together
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Linear Plots of the Michaelis-Menten Equation
Be able to derive these equations!
• Lineweaver-Burk
• Hanes-Woolf
• Hanes-Woolf is best - why?
• Smaller and more consistent errors across the plot
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Enzyme Inhibitors
Reversible versus Irreversible
• Reversible inhibitors interact with an enzyme via noncovalent associations
• Irreversible inhibitors interact with an enzyme via covalent associations
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Classes of InhibitionTwo real, one hypothetical
• Competitive inhibition - inhibitor (I) binds only to E, not to ES
• Noncompetitive inhibition - inhibitor (I) binds either to E and/or to ES
• Uncompetitive inhibition - inhibitor (I) binds only to ES, not to E. This is a hypothetical case that has never been documented for a real enzyme, but which makes a useful contrast to competitive inhibition
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
14.6 Ribozymes and Abzymes
Relatively new discoveries
• Ribozymes - segments of RNA that display enzyme activity in the absence of protein – Examples: RNase P and peptidyl transferase
• Abzymes - antibodies raised to bind the transition state of a reaction of interest – For a great recent review, see Science, Vol. 269,
pages 1835-1842 (1995)
– We'll say more about transition states in Ch 16
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
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