which a ligand recognizes its target is a crucial prerequisite for
rationally designing novel and effective drugs and therapeutics
[1]. By designing small molecules that target a specific conformation, it may be possible to design more potent or selective drugs
that bind to the preferred conformer of the target enzyme. This
would in turn shift the equilibrium toward this state, redistributing
the conformational ensemble so that the favored state predominates in solution [4]. Therefore, the innate flexibility and conformational dynamics of proteins and enzymes may be exploited to
improve the efficacy of rational drug design.
In this paper, we use the enzyme dihydrofolate reductase
(DHFR) to illustrate how the conformational dynamics influence
substrate, cofactor, and inhibitor binding. DHFR has become an
important model system for investigating the link between protein
dynamics and catalytic function for several reasons. As we discuss
the many studies on the link between DHFR structure, motions,
and catalysis, we provide an overview of the methods used in these
studies. DHFR is a known drug target for inhibiting DNA synthesis
in rapidly proliferating cancer cells and microbial infections
[21, 22] and a wealth of enzymological studies that have focused
on DHFR make it a unique system for investigating the role of
dynamics in catalysis [18, 21].
2 Dihydrofolate Reductase
2.1 Kinetic
Mechanism
and Structure
DHFR kinetics have been extensively reviewed [18, 21, 23]. A
notable feature of the catalytic cycle is that hydride transfer, the
chemical step of the reaction, is not immediately followed by the
release of product. Instead, following hydride transfer, the oxidized
NADP
+ cofactor is released and NADPH rebinds before product
release. Thus, free enzyme is not generated under physiological
conditions and the enzyme remains “primed” for the next round
of catalysis [18, 23]. The coordination of substrate binding and
product release is maintained via a synergistic interaction between
the substrate and cofactor in the binding site, in which the off-rates
of NADP
+ increase in the presence of bound product and the
off-rates of product (THF) increase in the presence of the reduced
cofactor, NADPH [18, 24].
DHFR displays a high degree of structural homology in different species, despite low sequence homology [18, 25]. The first
X-ray crystal structure of a DHFR was of the Escherichia coli
enzyme (ecDHFR) and was published almost 40 years ago
[26]. The ecDHFR structure has been extensively discussed elsewhere [26, 27]. The major subdomain is dominated by a set of
three flexible loops that are located on the ligand binding face that
surround the active site. The loops are designated Met20 (residues
9-24), F-G (residues 116-132), and G-H (residues 142-150).
188
Melanie Goldstein and Nina M. Goodey
rationally designing novel and effective drugs and therapeutics
[1]. By designing small molecules that target a specific conformation, it may be possible to design more potent or selective drugs
that bind to the preferred conformer of the target enzyme. This
would in turn shift the equilibrium toward this state, redistributing
the conformational ensemble so that the favored state predominates in solution [4]. Therefore, the innate flexibility and conformational dynamics of proteins and enzymes may be exploited to
improve the efficacy of rational drug design.
In this paper, we use the enzyme dihydrofolate reductase
(DHFR) to illustrate how the conformational dynamics influence
substrate, cofactor, and inhibitor binding. DHFR has become an
important model system for investigating the link between protein
dynamics and catalytic function for several reasons. As we discuss
the many studies on the link between DHFR structure, motions,
and catalysis, we provide an overview of the methods used in these
studies. DHFR is a known drug target for inhibiting DNA synthesis
in rapidly proliferating cancer cells and microbial infections
[21, 22] and a wealth of enzymological studies that have focused
on DHFR make it a unique system for investigating the role of
dynamics in catalysis [18, 21].
2 Dihydrofolate Reductase
2.1 Kinetic
Mechanism
and Structure
DHFR kinetics have been extensively reviewed [18, 21, 23]. A
notable feature of the catalytic cycle is that hydride transfer, the
chemical step of the reaction, is not immediately followed by the
release of product. Instead, following hydride transfer, the oxidized
NADP
+ cofactor is released and NADPH rebinds before product
release. Thus, free enzyme is not generated under physiological
conditions and the enzyme remains “primed” for the next round
of catalysis [18, 23]. The coordination of substrate binding and
product release is maintained via a synergistic interaction between
the substrate and cofactor in the binding site, in which the off-rates
of NADP
+ increase in the presence of bound product and the
off-rates of product (THF) increase in the presence of the reduced
cofactor, NADPH [18, 24].
DHFR displays a high degree of structural homology in different species, despite low sequence homology [18, 25]. The first
X-ray crystal structure of a DHFR was of the Escherichia coli
enzyme (ecDHFR) and was published almost 40 years ago
[26]. The ecDHFR structure has been extensively discussed elsewhere [26, 27]. The major subdomain is dominated by a set of
three flexible loops that are located on the ligand binding face that
surround the active site. The loops are designated Met20 (residues
9-24), F-G (residues 116-132), and G-H (residues 142-150).
188
Melanie Goldstein and Nina M. Goodey