5.3 Mutations
in the FG Loop
5.3.1 G121V
G121 in ecDHFR is one of the most thoroughly studied distal
residues and has been the subject of numerous kinetic and mutagenesis studies. It is universally conserved among all prokaryotic
DHFRs, which suggests that it plays a crucial role in the function of
DHFR [55]. G121 is located on the FG loop, 19 A ˚ from the active
site of the enzyme (Fig. 10) [80]. The substitution of Gly with Val
(G121V) has been the subject of numerous studies. Gekko et al.
demonstrated that the G121V mutant decreased the rate of steadystate catalysis 20-fold, concluding that amino acid substitutions at
position 121 significantly influence its enzymatic function
[55, 90]. The fact that enzyme function is affected by mutations
in residues far from the active site suggests global dynamics of the
protein play an important role in catalysis [90]. In a later study,
Cameron et al. analyzed the full kinetic scheme of G121V DHFR
and determined that the rate of hydride transfer decreased
170-fold. In addition, G121V was found to introduce a catalytically
significant conformational exchange preceding the hydride transfer
step, at a rate of 3.5 s
À1 [91]. In contrast to WT DHFR, the closed
conformation is energetically disfavored for G121V DHFR
[92]. As mentioned earlier, the Michaelis complex for ecDHFR is
in the closed conformation. The mutant remains in the occluded
conformation, which interferes with coupled loop movements and
impairs catalysis by destabilizing the closed Michaelis complex and
introducing an extra conformational exchange step into the kinetic
pathway [92].
In a study by Boehr et al. nuclear magnetic resonance relaxation
experiments were used to determine the mechanism by which the
G121V mutation effects DHFR kinetics and dynamics on the
picosecond to nanosecond and microsecond to millisecond timescales. The authors recorded the 15 N relaxation data for both wild
type and G121V DHFR with folate bound. They found that the
mutant ternary complex (G121V:NADPH:THF) adopts an
occluded conformation which is very similar to that of the wildtype ternary complex. However, this mutation causes substantial
changes in dynamics, restricting the amplitude and altering the
timescale of motions of residues in the FG loop and the Met20
loop. The effects of the G121V mutation are transmitted to distal
sites through subtle changes in the accessible conformational space
by molecular fluctuations on the picosecond to nanosecond timescales. Therefore, they conclude that their results are consistent
with theoretical experiments that suggest that long-range allostery
in DHFR arises from a redistribution of the conformational ensemble, with significant contributions from perturbations of the protein dynamics [93].
In another study, Mauldin et al. used NMR to investigate
whether the decrease in catalytic efficacy caused by the G121V
mutant was the result of changes in structure, flexibility, or both
[94]. Since the G121V mutants favor the occluded conformation,
206
Melanie Goldstein and Nina M. Goodey
in the FG Loop
5.3.1 G121V
G121 in ecDHFR is one of the most thoroughly studied distal
residues and has been the subject of numerous kinetic and mutagenesis studies. It is universally conserved among all prokaryotic
DHFRs, which suggests that it plays a crucial role in the function of
DHFR [55]. G121 is located on the FG loop, 19 A ˚ from the active
site of the enzyme (Fig. 10) [80]. The substitution of Gly with Val
(G121V) has been the subject of numerous studies. Gekko et al.
demonstrated that the G121V mutant decreased the rate of steadystate catalysis 20-fold, concluding that amino acid substitutions at
position 121 significantly influence its enzymatic function
[55, 90]. The fact that enzyme function is affected by mutations
in residues far from the active site suggests global dynamics of the
protein play an important role in catalysis [90]. In a later study,
Cameron et al. analyzed the full kinetic scheme of G121V DHFR
and determined that the rate of hydride transfer decreased
170-fold. In addition, G121V was found to introduce a catalytically
significant conformational exchange preceding the hydride transfer
step, at a rate of 3.5 s
À1 [91]. In contrast to WT DHFR, the closed
conformation is energetically disfavored for G121V DHFR
[92]. As mentioned earlier, the Michaelis complex for ecDHFR is
in the closed conformation. The mutant remains in the occluded
conformation, which interferes with coupled loop movements and
impairs catalysis by destabilizing the closed Michaelis complex and
introducing an extra conformational exchange step into the kinetic
pathway [92].
In a study by Boehr et al. nuclear magnetic resonance relaxation
experiments were used to determine the mechanism by which the
G121V mutation effects DHFR kinetics and dynamics on the
picosecond to nanosecond and microsecond to millisecond timescales. The authors recorded the 15 N relaxation data for both wild
type and G121V DHFR with folate bound. They found that the
mutant ternary complex (G121V:NADPH:THF) adopts an
occluded conformation which is very similar to that of the wildtype ternary complex. However, this mutation causes substantial
changes in dynamics, restricting the amplitude and altering the
timescale of motions of residues in the FG loop and the Met20
loop. The effects of the G121V mutation are transmitted to distal
sites through subtle changes in the accessible conformational space
by molecular fluctuations on the picosecond to nanosecond timescales. Therefore, they conclude that their results are consistent
with theoretical experiments that suggest that long-range allostery
in DHFR arises from a redistribution of the conformational ensemble, with significant contributions from perturbations of the protein dynamics [93].
In another study, Mauldin et al. used NMR to investigate
whether the decrease in catalytic efficacy caused by the G121V
mutant was the result of changes in structure, flexibility, or both
[94]. Since the G121V mutants favor the occluded conformation,
206
Melanie Goldstein and Nina M. Goodey