Hydride Transfer by Dihydrofolate Reductase

Transient and steady-state kinetics have been examined for dihydrofolate reductase (DHFR) from a number of sources. Rates of hydride transfer at pH 7.65 cover a wide range, from 7 s-1 for DHFR from a strain of Lactobacillus casei (LCDHFR1) to 3000 s-1 for recombinant human DHFR (rHDHFR). In all cases as the pH is increased from 7 to 10, Vmax for the steady-state reaction decreases, and DVmax, the primary isotope effect, increases. This indicates a decrease in the rate of hydride transfer with increasing pH. The cross-over points, at which rates of product release and hydride transfer become equal, were calculated to occur at DVmax = 2.34. The higher the rate of hydride transfer at pH 7.65, the higher the pH of the cross-over point. For LCDHFR1 the low rate of hydride transfer results in this process being partially rate-limiting for the steady-state reaction even at pH 5, with a cross-over point at about pH 7. At pH 7.65 the burst phase associated with the initial conversion of enzyme-bound substrates to enzyme-bound products has an isotope effect of 3 or higher for LCDHFR and for DHFR from Escherichia coli (ECDHFR). In contrast, the vertebrate DHFRs (bovine, BDHFR; chicken, CDHFR; and rHDHFR) exhibit a burst of product formation which is only partially limited by hydride transfer at this pH (Dkb: 2.3, 2.2, and 2.1, respectively). An obligatory isomerization of the ternary substrate complex or of the ternary product complex is postulated to be partially rate-limiting for the vertebrate enzymes. At pH 5 LCDHFR1 and ECDHFR also exhibit evidence of such a rate-limiting obligatory conformational transition of the substrate or product ternary complex.