Hydride Transfer in DHFR by Transition Path Sampling, Kinetic Isotope Effects, and Heavy Enzyme Studies

Zhen Wang, Dimitri Antoniou, Steven D. Schwartz, Vern L. Schramm

Research output: Contribution to journalArticle

24 Scopus citations

Abstract

Escherichia coli dihydrofolate reductase (ecDHFR) is used to study fundamental principles of enzyme catalysis. It remains controversial whether fast protein motions are coupled to the hydride transfer catalyzed by ecDHFR. Previous studies with heavy ecDHFR proteins labeled with 13C, 15N, and nonexchangeable 2H reported enzyme mass-dependent hydride transfer kinetics for ecDHFR. Here, we report refined experimental and computational studies to establish that hydride transfer is independent of protein mass. Instead, we found the rate constant for substrate dissociation to be faster for heavy DHFR. Previously reported kinetic differences between light and heavy DHFRs likely arise from kinetic steps other than the chemical step. This study confirms that fast (femtosecond to picosecond) protein motions in ecDHFR are not coupled to hydride transfer and provides an integrative computational and experimental approach to resolve fast dynamics coupled to chemical steps in enzyme catalysis.

Original languageEnglish (US)
Pages (from-to)157-166
Number of pages10
JournalBiochemistry
Volume55
Issue number1
DOIs
StatePublished - Jan 19 2016

ASJC Scopus subject areas

  • Biochemistry

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