# BIOC12H3 Lecture Notes - Lecture 8: Enzyme Kinetics, Enzyme Inhibitor, Enzyme

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Lecture 8: Mechanisms of Enzyme Action
Measurement of Km and Vmax: Double-reciprocal (lineweaver-Burk) plot to determine Vmax
and Km
The MichaelisMenten equation can be rewritten in order to obtain values for Vmax and
Km from straight lines on graphs. The most commonly used transformation is the double-
reciprocal, or LineweaverBurk, plot in which the values of 1/v0 are plotted against 1/[S]
(Figure 5.6 Slide 2).
The absolute value of 1/Km is obtained from the intercept of the line at the x axis, and the
value of 1/Vmax is obtained from the y intercept. Although double-reciprocal plots are
not the most accurate methods for determining kinetic constants, they are easily
understood and provide recognizable patterns for the study of enzyme inhibition.
Values of kcat can be obtained from measurements of Vmax only when the absolute
concentration of the enzyme is known. Values of Km can be determined even when
enzymes have not been purified provided that only one enzyme in the impure preparation
can catalyze the observed reaction.
Kinetics of Multisubstrate reactions
Let’s consider a reaction in which two substrates, A and B, are converted to products P
and Q.
E + A + B ↔ (EAB) → E + P + Q
Kinetic measurements for such multisubstrate reactions are a little more complicated than
simple one-substrate enzyme kinetics.
Can determine Km for each reaction in the presence of saturating amount of each
substrate.
Multisubstrate reactions can occur by several different kinetic schemes. These schemes
are called kinetic mechanisms because they are derived entirely from kinetic experiments.
Sequential reactions require all the substrates to be present before any product is
released. Sequential reactions can be either ordered, with an obligatory order for the
addition of substrates and release of products, or random with no specific order of
binding or release required for the substrate and product.
In ping-pong reactions, a product is released before all the substrates are bound. In a
bisubstrate ping-pong reaction, the first substrate is bound, the enzyme is altered by
substitution, and the first product is released. Then the second substrate is bound, the
altered enzyme is restored to its original form, and the second product is released. A
ping-pong mechanism is sometimes called a substituted-enzyme mechanism because of
the covalent binding of a portion of a substrate to the enzyme. The binding and release of
ligands in a ping-pong mechanism are usually indicated by slanted lines. The two forms
of the enzyme are represented by E (unsubstituted) and F (substituted).
In ping-pong reactions, one substrate is bound and a product is released, leaving a
substituted enzyme. A second substrate is then bound and a second product released,
restoring the enzyme to its original form.
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Specific Inhibition of enzyme action
An enzyme inhibitor (I) is a compound that binds to an enzyme and interferes with its
activity. Inhibitors can act by preventing the formation of the ES complex or by blocking
the chemical reaction that leads to the formation of product.
As a general rule, inhibitors are small molecules that bind reversibly to the enzyme they
inhibit.
Some inhibitors bind covalently to enzymes causing irreversible inhibition but most
biologically relevant inhibition is reversible.
Usually inhibitors are small molecules that bind reversibly to the enzyme, although some
bind irreversibly (can be determined if inhibitor can be removed by dialysis or gel
filtration chromatography it is reversible).
Most biologically relevant inhibitors bind reversibly via noncovalent forces similar to
substrate enzyme interactions.
There are natural inhibitors in cells and artificial inhibitors used in experiments.
Reversible inhibitors are bound to enzymes by the same weak, noncovalent forces that
bind substrates and products.
The equilibrium between free enzyme (E) plus inhibitor (I) and the EI complex is
characterized by a dissociation constant. In this case, the constant is called the inhibition
constant, Ki.
E + I ↔ EI
Kd = Ki = [E][I]/[EI]
The basic types of reversible inhibition are competitive, uncompetitive, noncompetitive
and mixed.
Competitive inhibition
Competitive inhibitors are the most commonly encountered inhibitors in biochemistry. In
competitive inhibition, the inhibitor can bind only to free enzyme molecules that have not
bound any substrate.
In this scheme only ES can lead to the formation of product. The formation of an EI
complex removes enzyme from the normal pathway.
Once a competitive inhibitor is bound to an enzyme molecule, a substrate molecule
cannot bind to that enzyme molecule. Conversely, the binding of substrate to an enzyme
molecule prevents the binding of an inhibitor. In other words, S and I compete for
binding to the enzyme molecule. Most commonly, S and I bind at the same site on the
enzyme, the active site. This type of inhibition is termed classical competitive
inhibition.
This is not the only kind of competitive inhibition. In some cases, such as allosteric
enzymes, the inhibitor binds at a different site and this alters the substrate binding site
preventing substrate binding. This type of inhibition is called nonclassical competitive
inhibition. When both I and S are present in a solution, the proportion of the enzyme that
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## Document Summary

Measurement of km and vmax: double-reciprocal (lineweaver-burk) plot to determine vmax and km. The michaelis menten equation can be rewritten in order to obtain values for vmax and. The most commonly used transformation is the double- reciprocal, or lineweaver burk, plot in which the values of 1/v0 are plotted against 1/[s] (figure 5. 6 slide 2). The absolute value of 1/km is obtained from the intercept of the line at the x axis, and the value of 1/vmax is obtained from the y intercept. Although double-reciprocal plots are not the most accurate methods for determining kinetic constants, they are easily understood and provide recognizable patterns for the study of enzyme inhibition. Values of kcat can be obtained from measurements of vmax only when the absolute concentration of the enzyme is known. Values of km can be determined even when enzymes have not been purified provided that only one enzyme in the impure preparation can catalyze the observed reaction.