Protein mass-modulated effects in the catalytic mechanism of dihydrofolate reductase: Beyond promoting vibrations

Zhen Wang, Priyanka Singh, Clarissa M. Czekster, Amnon Kohen, Vern L. Schramm

Research output: Contribution to journalArticle

40 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)8333-8341
Number of pages9
JournalJournal of the American Chemical Society
Volume136
Issue number23
DOIs
StatePublished - Jun 11 2014

Fingerprint

Tetrahydrofolate Dehydrogenase
Vibration
Proteins
Escherichia coli
Enzymes
Ligands
Catalysis
Temperature
Kinetics
Energy barriers
Oxidoreductases
Hydrides
Isotopes
Substitution reactions
Hot Temperature

ASJC Scopus subject areas

  • Chemistry(all)
  • Catalysis
  • Biochemistry
  • Colloid and Surface Chemistry

Cite this

Protein mass-modulated effects in the catalytic mechanism of dihydrofolate reductase : Beyond promoting vibrations. / Wang, Zhen; Singh, Priyanka; Czekster, Clarissa M.; Kohen, Amnon; Schramm, Vern L.

In: Journal of the American Chemical Society, Vol. 136, No. 23, 11.06.2014, p. 8333-8341.

Research output: Contribution to journalArticle

Wang, Zhen ; Singh, Priyanka ; Czekster, Clarissa M. ; Kohen, Amnon ; Schramm, Vern L. / Protein mass-modulated effects in the catalytic mechanism of dihydrofolate reductase : Beyond promoting vibrations. In: Journal of the American Chemical Society. 2014 ; Vol. 136, No. 23. pp. 8333-8341.
@article{0b06bc1338114a03a00021de944ec378,
title = "Protein mass-modulated effects in the catalytic mechanism of dihydrofolate reductase: Beyond promoting vibrations",
abstract = "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.",
author = "Zhen Wang and Priyanka Singh and Czekster, {Clarissa M.} and Amnon Kohen and Schramm, {Vern L.}",
year = "2014",
month = "6",
day = "11",
doi = "10.1021/ja501936d",
language = "English (US)",
volume = "136",
pages = "8333--8341",
journal = "Journal of the American Chemical Society",
issn = "0002-7863",
publisher = "American Chemical Society",
number = "23",

}

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.

UR - http://www.scopus.com/inward/record.url?scp=84902258731&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84902258731&partnerID=8YFLogxK

U2 - 10.1021/ja501936d

DO - 10.1021/ja501936d

M3 - Article

C2 - 24820793

AN - SCOPUS:84902258731

VL - 136

SP - 8333

EP - 8341

JO - Journal of the American Chemical Society

JF - Journal of the American Chemical Society

SN - 0002-7863

IS - 23

ER -