they used MTX and the reduced cofactor NADPH to create a
model of the transition state. The high affinity of MTX for DHFR
effectively “locks” the enzyme in the closed conformation in WT
ecDHFR; thus, they reasoned that it may do the same for the
mutant enzyme. This allowed them to isolate the mutant in the
closed conformation, which is the catalytically relevant conformation of DHFR. Using
15 H and
2 H NMR spin relaxation experiments, they observed that the most dramatic effect of the G121V
mutation involves changes in the dynamics of the FG and Met20
loops on the μs–ms timescale. In the WT DHFR:NADPH:MTX
complex, loop motion is suppressed so that the complex favors the
closed conformation. However, in the mutant complex, the FG and
Met20 loops undergo fluctuations from the closed conformation.
These dynamic fluctuations serve to decrease the population of
conformers having the correct active site conformation for catalysis,
providing an explanation for the decrease in catalytic activity
observed for the G121V mutant [94].
5.3.2 ΔG121
The effect of deletion and insertion mutations in the FG loop has
also been studied to probe the role of distal residues in DHFR
catalysis. Deletion of the G121 residue (ΔG121) results in
decreased binding to NADPH by 20-fold, as well as a 550-fold
decrease in the hydride transfer rate [80, 95]. Additionally, ΔG121
requires conformational changes dependent on the initial binary
complex to attain the Michaelis complex. Insertion mutants also
displayed a significant decrease in substrate and cofactor binding.
However, the insertion of a glycine residue into a modified FG loop
eliminated the conformational changes required to attain the
Michaelis complex seen in the ΔG121 mutant [95]. These observations suggest that the FG loop plays a role in the formation of
liganded complexes and proper orientation of substrate and cofactor during catalysis. Through a transient interaction with the
Met20 loop, alterations to the FG loop can coordinate proximal
and distal effects on ligand binding and catalysis that implicate a
variety of enzyme conformations involved in the catalytic
cycle [95].
5.4 Mutations
in the GH Loop
5.4.1 S148
S148 is located in the GH loop (residues 142-149) of ecDHFR,
18 A ˚ away from the active site. This residue is involved in hydrogen
bonding interactions that modulate the conformation of the Met20
loop [80, 95]. To gauge the importance of these hydrogen bonding
interactions, S148 was replaced by Asp, Ala, and Lys. These mutations increased the affinity for the NADPH cofactor, but significantly decreased the affinity of the enzyme for DHF (Table 2).
Further analysis revealed that these mutations predominantly
effected the ligand release rates. Mutations at residue 148 altered
the preferred catalytic pathway by introducing branches at key
intermediates [80].
Distal Regions Regulate Dihydrofolate Reductase-Ligand Interactions
207
model of the transition state. The high affinity of MTX for DHFR
effectively “locks” the enzyme in the closed conformation in WT
ecDHFR; thus, they reasoned that it may do the same for the
mutant enzyme. This allowed them to isolate the mutant in the
closed conformation, which is the catalytically relevant conformation of DHFR. Using
15 H and
2 H NMR spin relaxation experiments, they observed that the most dramatic effect of the G121V
mutation involves changes in the dynamics of the FG and Met20
loops on the μs–ms timescale. In the WT DHFR:NADPH:MTX
complex, loop motion is suppressed so that the complex favors the
closed conformation. However, in the mutant complex, the FG and
Met20 loops undergo fluctuations from the closed conformation.
These dynamic fluctuations serve to decrease the population of
conformers having the correct active site conformation for catalysis,
providing an explanation for the decrease in catalytic activity
observed for the G121V mutant [94].
5.3.2 ΔG121
The effect of deletion and insertion mutations in the FG loop has
also been studied to probe the role of distal residues in DHFR
catalysis. Deletion of the G121 residue (ΔG121) results in
decreased binding to NADPH by 20-fold, as well as a 550-fold
decrease in the hydride transfer rate [80, 95]. Additionally, ΔG121
requires conformational changes dependent on the initial binary
complex to attain the Michaelis complex. Insertion mutants also
displayed a significant decrease in substrate and cofactor binding.
However, the insertion of a glycine residue into a modified FG loop
eliminated the conformational changes required to attain the
Michaelis complex seen in the ΔG121 mutant [95]. These observations suggest that the FG loop plays a role in the formation of
liganded complexes and proper orientation of substrate and cofactor during catalysis. Through a transient interaction with the
Met20 loop, alterations to the FG loop can coordinate proximal
and distal effects on ligand binding and catalysis that implicate a
variety of enzyme conformations involved in the catalytic
cycle [95].
5.4 Mutations
in the GH Loop
5.4.1 S148
S148 is located in the GH loop (residues 142-149) of ecDHFR,
18 A ˚ away from the active site. This residue is involved in hydrogen
bonding interactions that modulate the conformation of the Met20
loop [80, 95]. To gauge the importance of these hydrogen bonding
interactions, S148 was replaced by Asp, Ala, and Lys. These mutations increased the affinity for the NADPH cofactor, but significantly decreased the affinity of the enzyme for DHF (Table 2).
Further analysis revealed that these mutations predominantly
effected the ligand release rates. Mutations at residue 148 altered
the preferred catalytic pathway by introducing branches at key
intermediates [80].
Distal Regions Regulate Dihydrofolate Reductase-Ligand Interactions
207