The role of fast protein dynamics
in enzyme catalysis has been
of great interest in the past decade. Recent “heavy enzyme”
studies demonstrate that protein mass-modulated vibrations are linked
to the energy barrier for the chemical step of catalyzed reactions.
However, the role of fast dynamics in the overall catalytic mechanism
of an enzyme has not been addressed. Protein mass-modulated effects
in the catalytic mechanism of Escherichia coli dihydrofolate
reductase (ecDHFR) are explored by isotopic substitution (13C, 15N, and non-exchangeable 2H) of the wild-type
ecDHFR (l-DHFR) to generate a vibrationally perturbed
“heavy ecDHFR” (h-DHFR). Steady-state,
pre-steady-state, and ligand binding kinetics, intrinsic kinetic isotope
effects (KIEint) on the chemical step, and thermal unfolding
experiments of both l- and h-DHFR
show that the altered protein mass affects the conformational ensembles
and protein–ligand interactions, but does not affect the hydride
transfer at physiological temperatures (25–45 °C). Below
25 °C, h-DHFR shows altered transition state
(TS) structure and increased barrier-crossing probability of the chemical
step compared with l-DHFR, indicating temperature-dependent
protein vibrational coupling to the chemical step. Protein mass-modulated
vibrations in ecDHFR are involved in TS interactions at cold temperatures
and are linked to dynamic motions involved in ligand binding at physiological
temperatures. Thus, mass effects can affect enzymatic catalysis beyond
alterations in promoting vibrations linked to chemistry.
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