Construction and Evaluation of the Kinetic Scheme Associated with Dihydrofolate Reductase from Escherichia coli

Carol A. Fierke, Kenneth A. Johnson, Stephen Benkovic

Research output: Contribution to journalArticle

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Abstract

A kinetic scheme is presented for Escherichia coli dihydrofolate reductase that predicts steady-state kinetic parameters and full time course kinetics under a variety of substrate concentrations and pHs. This scheme was derived from measuring association and dissociation rate constants and presteady-state transients by using stopped-flow fluorescence and absorbance spectroscopy. The binding kinetics suggest that during steady-state turnover product dissociation follows a specific, preferred pathway in which tetrahydrofolate (H4F) dissociation occurs after NADPH replaces NADP+ in the ternary complex. This step, H4F dissociation from the E•NADPH.H4F ternary complex, is proposed to be the rate-limiting step for steady-state turnover at low pH because koff = VM. The rate constant for hydride transfer from NADPH to dihydrofolate (H2F), measured by pre-steady-state transients, has a deuterium isotope effect of 3 and is rapid, Khyd= 950 s-1, essentially irreversible, K = 1700, and pH dependent, pKa = 6.5, reflecting ionization of a single group in the active site. This scheme accounts for the apparent pKa= 8.4 observed in the steady state as due to a change in the rate-determining step from product release at low pH to hydride transfer above pH 8.4. This kinetic scheme is a necessary background to analyze the effects of single amino acid substitutions on individual rate constants.

Original languageEnglish (US)
Pages (from-to)4085-4092
Number of pages8
JournalBiochemistry
Volume26
Issue number13
DOIs
StatePublished - Jan 1 1987

All Science Journal Classification (ASJC) codes

  • Biochemistry

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