Triple Isotope Effects Support Concerted Hydride and Proton Transfer and Promoting Vibrations in Human Heart Lactate Dehydrogenase

Zhen Wang, Eric P. Chang, Vern L. Schramm

Research output: Contribution to journalArticle

13 Citations (Scopus)

Abstract

Transition path sampling simulations have proposed that human heart lactate dehydrogenase (LDH) employs protein promoting vibrations (PPVs) on the femtosecond (fs) to picosecond (ps) time scale to promote crossing of the chemical barrier. This chemical barrier involves both hydride and proton transfers to pyruvate to form l-lactate, using reduced nicotinamide adenine dinucleotide (NADH) as the cofactor. Here we report experimental evidence from three types of isotope effect experiments that support coupling of the promoting vibrations to barrier crossing and the coincidence of hydride and proton transfer. We prepared the native (light) LDH and a heavy LDH labeled with 13C, 15N, and nonexchangeable 2H (D) to perturb the predicted PPVs. Heavy LDH has slowed chemistry in single turnover experiments, supporting a contribution of PPVs to transition state formation. Both the [4-2H]NADH (NADD) kinetic isotope effect and the D2O solvent isotope effect were increased in dual-label experiments combining both NADD and D2O, a pattern maintained with both light and heavy LDHs. These isotope effects support concerted hydride and proton transfer for both light and heavy LDHs. Although the transition state barrier-crossing probability is reduced in heavy LDH, the concerted mechanism of the hydride-proton transfer reaction is not altered. This study takes advantage of triple isotope effects to resolve the chemical mechanism of LDH and establish the coupling of fs-ps protein dynamics to barrier crossing.

Original languageEnglish (US)
Pages (from-to)15004-15010
Number of pages7
JournalJournal of the American Chemical Society
Volume138
Issue number45
DOIs
StatePublished - Nov 16 2016

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Proton transfer
Vibration
L-Lactate Dehydrogenase
Hydrides
Isotopes
Protons
NAD
Proteins
Light
Experiments
Pyruvic Acid
Oxidoreductases
Labels
Lactic Acid
Sampling
Kinetics

ASJC Scopus subject areas

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

Cite this

Triple Isotope Effects Support Concerted Hydride and Proton Transfer and Promoting Vibrations in Human Heart Lactate Dehydrogenase. / Wang, Zhen; Chang, Eric P.; Schramm, Vern L.

In: Journal of the American Chemical Society, Vol. 138, No. 45, 16.11.2016, p. 15004-15010.

Research output: Contribution to journalArticle

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abstract = "Transition path sampling simulations have proposed that human heart lactate dehydrogenase (LDH) employs protein promoting vibrations (PPVs) on the femtosecond (fs) to picosecond (ps) time scale to promote crossing of the chemical barrier. This chemical barrier involves both hydride and proton transfers to pyruvate to form l-lactate, using reduced nicotinamide adenine dinucleotide (NADH) as the cofactor. Here we report experimental evidence from three types of isotope effect experiments that support coupling of the promoting vibrations to barrier crossing and the coincidence of hydride and proton transfer. We prepared the native (light) LDH and a heavy LDH labeled with 13C, 15N, and nonexchangeable 2H (D) to perturb the predicted PPVs. Heavy LDH has slowed chemistry in single turnover experiments, supporting a contribution of PPVs to transition state formation. Both the [4-2H]NADH (NADD) kinetic isotope effect and the D2O solvent isotope effect were increased in dual-label experiments combining both NADD and D2O, a pattern maintained with both light and heavy LDHs. These isotope effects support concerted hydride and proton transfer for both light and heavy LDHs. Although the transition state barrier-crossing probability is reduced in heavy LDH, the concerted mechanism of the hydride-proton transfer reaction is not altered. This study takes advantage of triple isotope effects to resolve the chemical mechanism of LDH and establish the coupling of fs-ps protein dynamics to barrier crossing.",
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