ProteinMass-Modulated Effects in the Catalytic Mechanismof Dihydrofolate Reductase: Beyond Promoting Vibrations

摘要

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). Below25 °C, h-DHFR shows altered transition state(TS) structure and increased barrier-crossing probability of the chemicalstep compared with l-DHFR, indicating temperature-dependentprotein vibrational coupling to the chemical step. Protein mass-modulatedvibrations in ecDHFR are involved in TS interactions at cold temperaturesand are linked to dynamic motions involved in ligand binding at physiologicaltemperatures. Thus, mass effects can affect enzymatic catalysis beyondalterations in promoting vibrations linked to chemistry.