Biomedical Engineering Reference
In-Depth Information
activity, are often uniquely well-suited for interactions with small molecule drugs. Thus, the very
nature of the chemistry of enzyme catalysis makes these proteins highly vulnerable to inactivation
by small molecule inhibitors that have the physicochemical characteristics of oral drugs.
Enzyme catalysis involves the conversion of a natural ligand (the substrate) into a different
chemical species (the product), most often through a process of chemical bond breaking and
formation steps. The chemical transformation of substrate to product almost always involves the
formation of a sequential series of intermediate chemical species along the reaction pathway.
Paramount in this reaction pathway is the formation of a short-lived, high-energy species referred
to as the transition state. To facilitate this sequential process of intermediate species formation, the
ligand-binding pocket(s) of enzymes must undergo specii c conformational changes that induce
strains at correct locations and align molecular orbitals to augment the chemical reactivity of the
appropriate functionalities on the substrate molecule(s), at dei ned moments during the reaction
cycle. The bases of mechanistic enzymology include understanding the chemical nature of the
various intermediate species formed, and their interactions with those elements of the enzyme-
binding pocket that facilitate chemical transformations. When these studies are coupled with
structural biology methods, such as x-ray crystallography and multidimensional nuclear magnetic
resonance (NMR) spectroscopy, a rich understanding of the structure-activity relationships
(SAR) that attend enzyme catalysis can be obtained. What is germane to the present discussion
is that this structural and mechanistic understanding can be exploited to discover and design
small molecule inhibitors—mimicking key structural features of reaction intermediates—that
form high-afi nity interactions with specii c conformational states of the ligand-binding pocket
of the target enzyme. In this chapter, we describe the application of mechanistic and structural
enzymology to drug discovery efforts with an emphasis on the evolution of structural changes
that attend catalysis and the exploitation of these various conformational forms for high-afi nity
inhibitor development.
11.2 MODES OF INHIBITOR INTERACTION WITH ENZYMES
The simplest enzyme-catalyzed reaction one can envisage is that of a single substrate (S) being
converted by the enzyme (E) to a single product (P). This reaction can be summarized by the
following equation:
k
¾¾®
1
k
E
+
S
ES
¾¾®
cat
E
+
P
(11.1)
¬¾¾
k
2
As summarized by Equation 11.1, enzyme and substrate combine to form a reversible initial encounter
complex (ES) that is governed by a forward rate constant for association (
k
1
) and a reverse rate
constant for dissociation (
k
2
). The equilibrium dissociation constant for the ES complex is given the
symbol
K
S
and is mathematically equivalent to the ratio of the rate constants
k
2
/
k
1
. Subsequent to initial
complex formation, a series of chemical steps ensue that are collectively quantii ed by the cumulative
rate constant
k
cat
. Thus,
k
cat
is not a microscopic rate constant, but rather summarizes all of the
intermediate states that must be formed during the chemical transformation of substrate to product
(see Section 11.3 for more details on the individual intermediate steps that may contribute to
k
cat
).
Three modes of inhibitor interaction with an enzyme target can be dei ned, based on their effects
on the catalytic steps summarized in Equation 11.1. Competitive inhibitors bind to the free enzyme
in a manner that blocks the binding of substrate so that they increase the apparent value of
K
S
, but
have no effect on the apparent value of
k
cat
. Noncompetitive inhibitors can bind to both the free
enzyme and to the ES complex (or intermediate species that follow the formation of the ES complex).
Such inhibitors can have some effect on the value of
K
S
but show the greatest effect on
k
cat
, as they
inhibit by blocking catalytic steps subsequent to substrate binding. Finally, uncompetitive inhibitors
have no afi nity for the free enzyme and only bind subsequent to the formation of the ES complex.
These inhibitors decrease the apparent value of
K
S
(i.e., increasing the apparent afi nity of the
