5 Effects of Distal Mutations and Evidence for Conformational Selection in DHFR
Over the last 30 years, the effect of mutations distal from the active
site on catalytic activity and inhibitor binding has become the focus
of many studies. The terms distal and allosteric are used to indicate
that an amino acid residue is located away from the active site
[80]. To understand the effects of distal mutations, it is necessary
to recognize the flexible nature of proteins. Enzymes are intrinsically flexible molecules and undergo conformational changes upon
ligand binding and throughout catalysis. A “network” of amino
acids located in and away from the active site may allosterically
regulate the protein and form “global protein dynamics” that are
crucial to enzyme catalysis [81, 82]. The term “global protein
dynamics” refers to the overall motions exhibited by all atoms in
the protein. Therefore, the effect of a distal mutation on catalysis
and ligand binding is transmitted indirectly, through the network
of amino acids comprising the protein [80]. The wealth of information available about DHFR makes it a great enzyme for studying
global protein dynamics. In this work, research and methods on the
role of distal residues and effect of distal mutations in DHFR are
outlined. We describe the application of these methods to address
several questions: (a) Do allosteric mutations effect the conformational motions associated with inhibitor binding? (b) Are the conformational motions inhibitor specific? (c) Do allosteric mutations
alter the conformational equilibrium of DHFR prior to inhibitor
binding? and (d) What is the significance of these effects on inhibitor binding and specificity?
Numerous DHFR mutagenesis studies have been published
which focus on the effect of distal residues of DHFR catalysis and
inhibitor binding. Several mechanisms have been proposed to
explain how distal mutations change enzyme function. One proposes that the effect is due to changes in the conformational equilibria between the different conformers. The equilibrium is
disturbed because some of the intermolecular contacts that stabilize
particular conformers have been altered by the mutation, which
emphasizes the delicate balance of the conformational ensemble
[80]. A second mechanism proposes that changes caused by a distal
mutation result in a different pattern of interactions with the rest of
the protein and that these changes are propagated throughout the
protein [80]. A third mechanism involves changes in protein conformational motion. In this case, the functional effect is due to
changes in flexibility and mobility, rather than structural changes in
the protein [80]. In this section, we will review several single and
multiple site mutation studies on the catalytic activity and inhibitor
binding of DHFR.
Distal Regions Regulate Dihydrofolate Reductase-Ligand Interactions
203
Over the last 30 years, the effect of mutations distal from the active
site on catalytic activity and inhibitor binding has become the focus
of many studies. The terms distal and allosteric are used to indicate
that an amino acid residue is located away from the active site
[80]. To understand the effects of distal mutations, it is necessary
to recognize the flexible nature of proteins. Enzymes are intrinsically flexible molecules and undergo conformational changes upon
ligand binding and throughout catalysis. A “network” of amino
acids located in and away from the active site may allosterically
regulate the protein and form “global protein dynamics” that are
crucial to enzyme catalysis [81, 82]. The term “global protein
dynamics” refers to the overall motions exhibited by all atoms in
the protein. Therefore, the effect of a distal mutation on catalysis
and ligand binding is transmitted indirectly, through the network
of amino acids comprising the protein [80]. The wealth of information available about DHFR makes it a great enzyme for studying
global protein dynamics. In this work, research and methods on the
role of distal residues and effect of distal mutations in DHFR are
outlined. We describe the application of these methods to address
several questions: (a) Do allosteric mutations effect the conformational motions associated with inhibitor binding? (b) Are the conformational motions inhibitor specific? (c) Do allosteric mutations
alter the conformational equilibrium of DHFR prior to inhibitor
binding? and (d) What is the significance of these effects on inhibitor binding and specificity?
Numerous DHFR mutagenesis studies have been published
which focus on the effect of distal residues of DHFR catalysis and
inhibitor binding. Several mechanisms have been proposed to
explain how distal mutations change enzyme function. One proposes that the effect is due to changes in the conformational equilibria between the different conformers. The equilibrium is
disturbed because some of the intermolecular contacts that stabilize
particular conformers have been altered by the mutation, which
emphasizes the delicate balance of the conformational ensemble
[80]. A second mechanism proposes that changes caused by a distal
mutation result in a different pattern of interactions with the rest of
the protein and that these changes are propagated throughout the
protein [80]. A third mechanism involves changes in protein conformational motion. In this case, the functional effect is due to
changes in flexibility and mobility, rather than structural changes in
the protein [80]. In this section, we will review several single and
multiple site mutation studies on the catalytic activity and inhibitor
binding of DHFR.
Distal Regions Regulate Dihydrofolate Reductase-Ligand Interactions
203