TY - JOUR
T1 - Protein mass-modulated effects in the catalytic mechanism of dihydrofolate reductase
T2 - Beyond promoting vibrations
AU - Wang, Zhen
AU - Singh, Priyanka
AU - Czekster, Clarissa M.
AU - Kohen, Amnon
AU - Schramm, Vern L.
PY - 2014/6/11
Y1 - 2014/6/11
N2 - 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.
AB - 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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U2 - 10.1021/ja501936d
DO - 10.1021/ja501936d
M3 - Article
C2 - 24820793
AN - SCOPUS:84902258731
SN - 0002-7863
VL - 136
SP - 8333
EP - 8341
JO - Journal of the American Chemical Society
JF - Journal of the American Chemical Society
IS - 23
ER -