Effects of the Donor–Acceptor
Distance and
Dynamics on Hydride Tunneling in the Dihydrofolate Reductase Catalyzed
Reaction
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Abstract
A significant contemporary question in enzymology involves
the
role of protein dynamics and hydrogen tunneling in enhancing enzyme
catalyzed reactions. Here, we report a correlation between the donor–acceptor
distance (DAD) distribution and intrinsic kinetic isotope effects
(KIEs) for the dihydrofolate reductase (DHFR) catalyzed reaction.
This study compares the nature of the hydride-transfer step for a
series of active-site mutants, where the size of a side chain that
modulates the DAD (I14 in E. coli DHFR)
is systematically reduced (I14V, I14A, and I14G). The contributions
of the DAD and its dynamics to the hydride-transfer step were examined
by the temperature dependence of intrinsic KIEs, hydride-transfer
rates, activation parameters, and classical molecular dynamics (MD)
simulations. Results are interpreted within the framework of the Marcus-like
model where the increase in the temperature dependence of KIEs arises
as a direct consequence of the deviation of the DAD from its distribution
in the wild type enzyme. Classical MD simulations suggest new populations
with larger average DADs, as well as broader distributions, and a
reduction in the population of the reactive conformers correlated
with the decrease in the size of the hydrophobic residue. The more
flexible active site in the mutants required more substantial thermally
activated motions for effective H-tunneling, consistent with the hypothesis
that the role of the hydrophobic side chain of I14 is to restrict
the distribution and dynamics of the DAD and thus assist the hydride-transfer.
These studies establish relationships between the distribution of
DADs, the hydride-transfer rates, and the DAD’s rearrangement
toward tunneling-ready states. This structure–function correlation
shall assist in the interpretation of the temperature dependence of
KIEs caused by mutants far from the active site in this and other
enzymes, and may apply generally to C–H→C transfer reactions