Lecture 1.

Enzyme Kinetics 2
CH148 Biochemical Engineering
Contents
• Allostery
• Enzyme Inhibition
• Effects of pH and Temperature

2
Allostery

3
Kinetics of Allosteric Binding
Some enzymes have multiple binding sites, and the
binding of one substrate can facilitate the binding of the
subsequent substrates. This is known as cooperative
binding or allostery.
The activity of allosteric enzymes can be altered by
regulatory molecules binding on allosteric sites; their
properties can thus be adjusted to meet the immediate
needs of a cell.
Michaelis-Menten kinetics fails to describe the kinetics of
cooperative binding; allosteric enzymes often display
sigmoidal plots.

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Kinetics of Allosteric Binding

5
Kinetics of Allosteric Binding
The rate expression in the case of allosteric enzymes becomes a three-parameter rate law:

where n is the Hill coefficient, and n > 1 indicates cooperativity. For n = 1, the expression reduces
to the Michaelis-Menten expression.
The Hill coefficient can be determined by linear regression:

𝑣
A plot of ln versus ln 𝑆 gives a straight line whose slope is n.
𝑣𝑚𝑎𝑥 −𝑣

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Enzyme Inhibition

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Enzyme Inhibition
Enzyme activity can be inhibited by the binding of specific small molecules and ions.
Like allostery, inhibition also serves as a major control mechanism in biological systems. In
addition, many drugs and toxic agents act by inhibiting enzymes.
Enzyme inhibition can be irreversible or reversible.
Irreversible inhibitors dissociate very slowly from its target enzyme; they are either covalently
or non-covalently bound to it. Examples are heavy metals (Pb, Cd, Hg, etc.)
Reversible inhibitors rapidly dissociate from the enzyme-inhibitor complex, and can be
competitive, uncompetitive, or noncompetitive. In some cases, the substrate can also be
inhibitory if accumulated at high concentrations.

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Enzyme Inhibition: Competitive
In competitive inhibition, the enzyme binds either
substrate or inhibitor. Competitive inhibitors are
usually substrate analogs and compete with substrate
for the active site of the enzyme.
The inhibition scheme can be described as:

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Enzyme Inhibition: Competitive
(derivation)

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Enzyme Inhibition: Competitive
(derivation)

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Enzyme Inhibition: Competitive
The rate law for competitive inhibition is:

where

The net effect of competitive inhibition is an apparently higher value of Km. Since 𝑣𝑚𝑎𝑥 is unaffected,
high concentrations of substrate can overcome the inhibition to reach 𝑣𝑚𝑎𝑥 .
A competitive inhibitor reduces the rate of catalysis by reducing the proportion of enzyme
molecules bound to a substrate.

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Enzyme Inhibition: Noncompetitive
In noncompetitive inhibition, the inhibitors are not
substrate analogs. Inhibitors bind sites other than the
active site and reduce enzyme affinity to the substrate.
Noncompetitive inhibitors can bind free enzyme or the
ES complex.
The inhibition scheme can be described as:

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Enzyme Inhibition: Noncompetitive
The rate law for noncompetitive inhibition is:

where

The net effect of noncompetitive inhibition is an apparently lower value of 𝑣𝑚𝑎𝑥 . Therefore,
substrate concentrations cannot overcome noncompetitive inhibition; the initial 𝑣𝑚𝑎𝑥 cannot be
restored.
A noncompetitive inhibitor lowers the concentration of functional enzyme. The resulting
solution behaves as a more dilute solution of the enzyme does.

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Enzyme Inhibition: Uncompetitive
In uncompetitive inhibition, the inhibitors bind
to the ES complex only; this implies that substrate
must bind first before uncompetitive inhibitors can
take effect.
The inhibition scheme can be described as:

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Enzyme Inhibition: Uncompetitive
The rate law for uncompetitive inhibition is:

The net effect of uncompetitive inhibition is a decrease in both 𝐾𝑚 and 𝑣𝑚𝑎𝑥 . The rate is more
sensitive to changes in 𝑣𝑚𝑎𝑥 , so the net result is a decrease in reaction rate. As in uncompetitive
inhibition, high substrate concentrations cannot overcome uncompetitive inhibition.
Because some unproductive ESI complex will always be present, 𝑬 𝟎 will be lower and so
will 𝒗𝒎𝒂𝒙 . Also, because ES is consumed to form ESI, the equilibrium shifts to more binding of
S, lowering the apparent value of 𝑲𝒎 .

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Enzyme Inhibition
Measurements of the rates of catalysis at different concentrations of substrate and inhibitor can
serve to distinguish the three types of reversible inhibition.

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Effects of pH and Temperature

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Effects of pH
Recall that some amino acid residues have side chains that are ionizable; certain enzymes have
these groups on their active sites, and their extents of protonation depend on the prevailing pH of
the solution.
Changes in solution pH result in changes in enzyme activity due to different ionizations, which may
also result in changes in the three-dimensional shape of the enzyme. In some cases, the pH of the
medium can also affect the ionization state of the substrate, and hence its affinity to the enzyme.
For these reasons, enzymes are only active over a certain pH range.

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Effects of pH
The following scheme may be used to describe the pH dependence of enzymatic reaction rates for
ionizing enzymes:
The resulting rate law would be:

or

where

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Effects of pH
As a result of this behavior, the pH optimum of the enzyme is between pK1 and pK2.
Theoretical prediction of the pH optimum of enzymes requires a knowledge of the active site
characteristics of enzymes, which are very difficult to obtain. It is usually determined
experimentally.

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Effects of Temperature
The rate of enzyme-catalyzed reactions increases with temperature up to a certain limit only,
above which, activity decreases with temperature due to denaturation.
The initial increase in the activity with temperature is
called temperature activation, and this follows the
Arrhenius equation:

The descending part is called the temperature

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Effects of Temperature
The rate of enzyme-catalyzed reactions increases with temperature up to a certain limit only,
above which, activity decreases with temperature due to denaturation.
The denaturation constant also follows the Arrhenius
equation:

Since 𝑣𝑚𝑎𝑥 = 𝑘2 𝐸 (not 𝐸 0 if it is changing),

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Effects of Temperature
The activation energies of enzyme-catalyzed reactions are within the 4-20 kcal/mol range, whereas
deactivation energies vary between 40-130 kcal/mol range.
As a result, enzyme denaturation is more sensitive to temperature changes; a rise in temperature
from 30o to 40oC results in a 1.8-fold increase in enzyme activity, but a 41-fold increase in enzyme
denaturation.

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 Lecture 1.5

Enzyme Kinetics 2
CH148 Biochemical Engineering
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