Tymoczko • Berg • Stryer

Biochemistry:
A Short Course
Third Edition

CHAPTER 7
Kinetics and
Regulation

© 2015 W. H. Freeman and Company

Chapter 7 Outline
 Consider a simple reaction:

The velocity or rate of the reaction is determined by measureing

how much A disappears as a function of time or how much B
appears as a function of time.

Suppose that we can readily measure the disappearance of A. The

velocity of the reaction is given by the formula below, where k is a
proportionality constant.

When the velocity of a reaction is directly proportional to reactant

concentration, the reaction is called a first-order reaction and the
proportionality constant has the units s-1.
Many important biochemical reactions are biomolecular or second-order reactions.

or

The rate equations for these reactions are:

and

The proportionality constant for second-order reactions has the units M-1s-1.
A common means of investigating enzyme kinetics to measure velocity as a
function of substrate concentration with a fixed amount of enzyme.

Consider a simple reaction in which the enzyme E catalyzes the conversion of S→P.

with k1, k2 being the rate constant for the indicated reaction steps. So as to ignore
the reverse reaction of P→S, we measure activity when [P] ≈ 0.

Under these conditions, the velocity is called the initial velocity or Vo.
The initial velocity is determined by measuring product formation as a
function of time, and then determining the velocity soon after the reaction
has started.
Leonor Michaelis and Maud Menten derived an equation to describe the
initial reaction velocity as a function of substrate concentration.

where

and

When Vo = ½ Vmax, KM =[S]. Thus, KM is the substrate concentration that yields

½ Vmax.
Two enzymes play a key role in the metabolism of alcohol.
Some people respond to alcohol consumption with facial flushing and rapid
heart beat, symptoms caused by excessive amounts of acetaldehyde in the
blood. There are two different acetaldehyde dehydrogenases in most people,
one with a low KM and one with a high KM. The low KM enzyme is inactivated in
susceptible individuals. The enzyme with the high KM cannot process all of the
acetaldehyde, and so some acetaldehyde appears in the blood.
The Michaelis-Menten equation can be manipulated into one that yields a
straight-line plot.

This double-reciprocal equation is called the Lineweaver-Burke equation.

KM values for enzymes vary widely and evidence suggests that the KM value is
approximately the substrate concentration of the enzyme in vivo.
 If the enzyme concentration, [E]T, is known, then

and

K2, also called kcat, is the turnover number of the enzyme, which is the
number of substrate molecules converted into product per second.
If [S]<<KM, we can assume that free enzyme [E] ≈ [E]T. The Michaelis-Menten
equation can be manipulated to yield:

Under these conditions, is a measure of catalytic efficiency

because it takes into account both the rate of catalysis (kcat) and nature of
the enzyme substrate interaction (KM).
Examination of the kcat/KM ratio reveals that some enzymes approach catalytic perfection.
Expanding the equation for kcat/KM shows that k1, the rate of formation of the enzyme-
substrate complex, is the rate limiting step. Diffusion (≈108-109 s-1 M-1) limits the value of
k1.

Yet, the kcat/KM of some enzymes approaches the rate of diffusion.

Multiple substrate reactions can be divided into two groups.

Sequential reactions are characterized by formation of a ternary complex

consisting of the two substrates and the enzyme.

Double-displacement reactions are characterized by the formation of a

substituted enzyme intermediate. Double-displacement reactions are also called
ping-pong reactions.
Allosteric enzymes control the flux of biochemical reactions in metabolic
pathways. Because of their regulatory properties, allosteric enzymes allow
for the generation of complex metabolic pathways.
The conversion of A to B is the committed step, because once this occurs, B
is committed to being converted into F.

Allosteric enzymes catalyze the committed step of metabolic pathways.

Michaelis-Menten enzymes facilitate the remaining steps.
The amount of F synthesized can be regulated by feedback inhibition.

The pathway product F inhibits enzyme e1 by binding to a regulatory site on

the enzyme that is distinct from the active site.
The regulation of metabolic pathways can be quite complex.

Allosteric enzymes may be inhibited or stimulated by several regulatory molecules.

The reaction velocity of allosteric enzymes displays a sigmoidal relationship to
substrate concentration.
All allosteric enzymes display quaternary structure with multiple
active sites and regulatory sites.

One model that explains the behavior of allosteric enzymes is the concerted
model.
Features of the concerted model:
1. The enzyme exists into two different quaternary structures, designated
T(tense) and R (relaxed).
2. T and R are in equilibrium, with T being the more stable state.
3. The R state is enzymatically more active than the T state.
4. All active sites must be in the same state.
The binding of substrate to one active site traps the other active sites in the R
state and removes the substrate-bound enzyme from the T<->R equilibrium.

This disruption of the T<->R equilibrium by the binding of substrate favors the
conversion of more enzymes to the R state.
Allosteric enzymes are more sensitive to changes in substrate concentration
near their KM values than are Michaelis-Menten enzymes.

This sensitivity is called the threshold effect.

 Allosteric regulators disrupt the R<->T equilibrium when they bind the enzyme.
Inhibitors stabilize the T state while activators stabilize the R state.

The disruption of the T<->R equilibrium by substrates is called the homotropic effect.
The disruption of the T<->R equilibrium by regulators is called the heterotrophic
effect.
The sequential model for allosteric enzymes proposes that subunits
undergo sequential changes in structure.
Phosphoribosylpyrophosphate synthetase (PRS) is an allosteric enzyme
in the purine nucleotide synthesis pathway.

A mutation leading to the loss of regulatory control without an effect

on catalytic activity leads to the overproduction of purine nucleotides.

The overproduction results in the painful disease gout.

Studies of individual enzyme molecules suggest that some enzymes
may exist in multiple conformations that are in equilibrium.

These different conformations may have different catalytic or

regulatory properties.