analyzed at 1 atm and 310 K until volumetric fluctuations were
stabilized and the desired average pressure was maintained. The
DHF-bound WT DHFR structure (PDB code 1rx2) was used in
the molecular dynamics simulations. In addition, experimental
values for k cat , K m , and K I values were measured. Isothermal titration calorimetry (ITC) measurements were conducted that distinguish enthalpic and entropic contributions to TMP binding
[98]. This thorough study provides insight into the binding kinetics and dynamics of TMP to WT DHFR and the L28R mutant,
which confers resistance to TMP [98]. L28R is a common TMP
resistance-conferring mutation [98]. In WT ecDHFR, L28 does
not exhibit any hydrogen bond interactions with DHF (Fig. 11).
However, for the L28R mutant, sample donor–acceptor distances
between the methyl group of R28 and DHF indicate the presence
of hydrogen bonds between the p-aminobenzoyl glutamate tail of
DHF and the α-amino group of R28 (Fig. 11) [98]. This interaction stabilizes the DHF-bound complex and provides a unique
mechanism of resistance. Typically, mutations that confer resistance
to a competitive inhibitor make enzymes more promiscuous and
decrease affinity for both inhibitor and the natural substrate. For
the L28R mutant, an increase in DHF affinity and a decrease in
TMP affinity are observed (Table 3). While this also results in
slower product release and catalytic rate for the L28R mutant, the
WT:L28
HD13
–DHF
OE2
L28R:R28
HH11
–DHF
OE2
0
0
5
10
15
0
5
10
15
30
60
90
120
150
180
210
0
3 0
distance (Å)
distance (Å)
60
90
120
150
180
210
Fig. 11 Comparison of WT DHFR:DHF and L28R:DHF complexes. In the WT
enzyme, L28 does not form hydrogen bond interactions with the substrate. In the
mutant enzyme, R28 interacts with the p-aminobenzoyl glutamate tail via its side
chain further stabilizing the protein–ligand interaction and altering the binding
conformation of DHF in the binding site. (This figure was reproduced from Fig. 4
in ref. 98)
Distal Regions Regulate Dihydrofolate Reductase-Ligand Interactions
209
stabilized and the desired average pressure was maintained. The
DHF-bound WT DHFR structure (PDB code 1rx2) was used in
the molecular dynamics simulations. In addition, experimental
values for k cat , K m , and K I values were measured. Isothermal titration calorimetry (ITC) measurements were conducted that distinguish enthalpic and entropic contributions to TMP binding
[98]. This thorough study provides insight into the binding kinetics and dynamics of TMP to WT DHFR and the L28R mutant,
which confers resistance to TMP [98]. L28R is a common TMP
resistance-conferring mutation [98]. In WT ecDHFR, L28 does
not exhibit any hydrogen bond interactions with DHF (Fig. 11).
However, for the L28R mutant, sample donor–acceptor distances
between the methyl group of R28 and DHF indicate the presence
of hydrogen bonds between the p-aminobenzoyl glutamate tail of
DHF and the α-amino group of R28 (Fig. 11) [98]. This interaction stabilizes the DHF-bound complex and provides a unique
mechanism of resistance. Typically, mutations that confer resistance
to a competitive inhibitor make enzymes more promiscuous and
decrease affinity for both inhibitor and the natural substrate. For
the L28R mutant, an increase in DHF affinity and a decrease in
TMP affinity are observed (Table 3). While this also results in
slower product release and catalytic rate for the L28R mutant, the
WT:L28
HD13
–DHF
OE2
L28R:R28
HH11
–DHF
OE2
0
0
5
10
15
0
5
10
15
30
60
90
120
150
180
210
0
3 0
distance (Å)
distance (Å)
60
90
120
150
180
210
Fig. 11 Comparison of WT DHFR:DHF and L28R:DHF complexes. In the WT
enzyme, L28 does not form hydrogen bond interactions with the substrate. In the
mutant enzyme, R28 interacts with the p-aminobenzoyl glutamate tail via its side
chain further stabilizing the protein–ligand interaction and altering the binding
conformation of DHF in the binding site. (This figure was reproduced from Fig. 4
in ref. 98)
Distal Regions Regulate Dihydrofolate Reductase-Ligand Interactions
209