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 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 k_off = V_M. 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, pK_a = 6.5, reflecting ionization of a single group in the active site. This scheme accounts for the apparent pK_a= 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.