section 6.6
Mechanisms of Enzyme Action
105
Indinavir
Nelfinavir
F IG U R E 6 -1 4
Scatchard plot of ligand-receptor interaction.
Ritonavir
Structures of HIV protease inhibitors. NHtBu denotes an amino-tertiary
butyl and Ph denotes a phenyl group.
The total number of receptor binding sites, [TR], is equal to
the sum of the unbound receptor sites, [R], and the bound
receptor sites, [LR], Therefore,
[R] = [TR] - [LR]
(
6
.
1 0
)
Substituting for [R] in Equation (
6
.
8
) yields
Y
1
LRJ
a
[L] ([TR] —
[LR])
(
6
.
1 1
)
which on rearrangement gives
[LR]
i— ± = tfa([TR]-[LR])
[.LJ
(
6
.
1 2
)
where
[LR]
concentration of bound ligand
[L]
concentration of unbound ligand
A plot of [LR]/[L] versus [LR] yields a straight line and
is known as a
Scatchard plot
(Figure 6-14). The slope
of the line gives the value
(—Ka),
while the intersection
of the line with the abscissa yields the value for the to-
tal number of sites [TR], Comparing this relationship to
the Michaelis-Menten analysis of enzyme kinetics shows
that 1
/ Ka
(where 50% of the sites are occupied) is equiva-
lent to
Km
and the maximum concentration (or number) of
bound ligand sites is equivalent to Vmax • The Scatchard plot
is similar to the Eadie-Hofstee plot (Figure 6-5), but the
axes are reversed. These principles have been applied in
the assay of ligands in human biological fluids by the use
of either specific receptor proteins (radioreceptor assay)
or specific binding proteins, namely, antibodies (radioim-
munoassay (RIA)). In these assays, the unlabeled ligand
(the quantity of which is to be determined) competes with a
predetermined amount of added radioactive ligand to bind
a limited amount of specific receptor protein. This step
is followed by separation of protein-bound ligand from
the unbound (free) ligand and measurement of radioac-
tivity of each fraction. Separation of the protein-bound
ligand from the free ligand is accomplished by protein
precipitation, adsorption of the free form, chromatogra-
phy, or electrophoresis. Under standard conditions of time
and temperature, the amount of radioactivity found in the
protein-bound ligand fraction is inversely proportional to
the concentration of unlabeled ligand and the amount of
radioactivity found in the unbound (free) ligand fraction is
directly proportional to unlabeled ligand concentrations.
This procedure has been widely used for the assay of hor-
mones, vitamins, drugs, and other compounds. Since the
concentrations of hormones in body fluids are very low
(10
- 8
to 10
12
M), assay sensitivity is increased by lig-
ands with high specific radioactivity.
6.6 Mechanisms of Enzyme Action
The mechanism of a reaction catalyzed by an enzyme
is a detailed description of the chemical interactions oc-
curring among the substrates, enzymes, and cofactors.
