PMID: 2498330Jun 5, 1989Paper

Hydride transfer by dihydrofolate reductase. Causes and consequences of the wide range of rates exhibited by bacterial and vertebrate enzymes.

The Journal of Biological Chemistry
W A BeardR L Blakley

Abstract

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 formati...Continue Reading

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