Chapter 14

Biochemistry 2/e - Garrett & Grisham
Chapter 14
Enzyme Kinetics
to accompany
Biochemistry, 2/e
by
Reginald Garrett and Charles Grisham
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should be mailed to: Permissions Department, Harcourt Brace & Company,
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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
Enzymes
Enzymes endow cells with the
remarkable capacity to exert kinetic
control over thermodynamic potentiality
Enzymes are the agents of metabolic
function
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 !
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
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
14.2 Enzyme Kinetics
Several terms to know!

rate or velocity
rate constant
rate law
order of a reaction
molecularity of a reaction
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!
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!
The Michaelis-Menten Equation
You should be able to derive this!

Louis Michaelis and Maude Menten's theory
It assumes the formation of an enzymesubstrate 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
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
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
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 0order in S
The Michaelis-Menten equation describes a
rectangular hyperbolic dependence of v on
S!
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
The catalytic efficiency

Name 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
Linear Plots of the MichaelisMenten Equation
Be able to derive these equations!

Lineweaver-Burk
Hanes-Woolf
Hanes-Woolf is best - why?
Smaller and more consistent errors
across the plot
Enzyme Inhibitors
Reversible versus Irreversible
Reversible inhibitors interact with an
enzyme via noncovalent associations
Irreversible inhibitors interact with an
enzyme via covalent associations
Classes of Inhibition
Two 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
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

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Biochemistry 2/e - Garrett & Grisham

Biochemistry 2/e - Garrett & Grisham

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

Biochemistry 2/e - Garrett & Grisham

Other Aspects of Enzymes

Copyright 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham

14.2 Enzyme Kinetics

Several terms to know!

Copyright 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham

Copyright 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham

The Transition State

Biochemistry 2/e - Garrett & Grisham

Copyright 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham

What Enzymes Do....

Copyright 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham

Copyright 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham

The Michaelis-Menten Equation

You should be able to derive this!

Biochemistry 2/e - Garrett & Grisham

Copyright 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham

Biochemistry 2/e - Garrett & Grisham

Biochemistry 2/e - Garrett & Grisham

The dual nature of the

Biochemistry 2/e - Garrett & Grisham

The turnover number

Biochemistry 2/e - Garrett & Grisham

The catalytic efficiency

Biochemistry 2/e - Garrett & Grisham

Linear Plots of the MichaelisMenten Equation

Be able to derive these equations!

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

Biochemistry 2/e - Garrett & Grisham