Construction and evaluation of the kinetic scheme associated with dihydrofolate reductase from Escherichia coli.
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 pre-steady-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 X NADPH X 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, Keq = 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.
Fierke
C A
CA
Department of Chemistry, Pennsylvania State University, University Park 16802.
Johnson
K A
KA
Benkovic
S J
SJ
D001665:Binding Sites; D001667:Binding, Competitive; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000201:enzymology*; D005069:Evaluation Studies as Topic; D005492:Folic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D009154:Mutation; D009249:NADP / Q000494:pharmacology; D013050:Spectrometry, Fluorescence / Q000379:methods; D013762:Tetrahydrofolate Dehydrogenase / Q000493:pharmacokinetics*
C010920:dihydrofolate; D009249:NADP; D005492:Folic Acid; D013762:Tetrahydrofolate Dehydrogenase