HUMAN ALDOSE REDUCTASE - SUBTLE EFFECTS REVEALED BY RAPID KINETIC-STUDIES OF THE C298A MUTANT ENZYME

Transient kinetic data for D-xylose reduction with NADPH and NADPD and for xylitol oxidation with NADP(+) catalyzed by recombinant C298A mutant human aldose reductase at pH 8 have been used to obtain estimates for each of the rate constants in the complete reaction mechanism as outlined for the wild-type enzyme in the preceding paper (Grimshaw et al., 1995a). Analysis of the resulting kinetic model shows that the nearly 9-fold increase in V-xylose/E(t) for C298A mutant enzyme relative to wild-type human aldose reductase is due entirely to an 8.7-fold increase in the rate constant for the conformational change that converts the tight (K-i NADP+ = 0.14 mu M) binary *E . NADP(+) complex to the weak (K-d NADP+ 6.8 mu M) E . NADP(+) complex from which NADP(+) is released. Evaluation of the rate expressions derived from the kinetic model for the various steady-state kinetic parameters reveals that the 37-fold increase in K-xylose seen for C298A relative to wild-type aldose reductase is largely due to this same increase in the net rate of NADP(+) release; the rate constant for xylose binding accounts for only a factor of 5.5. A similar 17-fold increase in the rate constant for the conformational change preceding NADPH release does not, however, result in any increase in V-xylitol/E(t), because hydride transfer is largely rate-limiting for reaction in this direction. By contrast, the rate constant for conformational clamping in the opposite direction (weak --> tight binary complex) is not greatly affected by the mutation, suggesting that Cys298 does not regulate the rate of closure of the nucleotide enfolding protein loop, but does stabilize the closed conformation. The rate of hydride transfer is reduced 2-fold in the C298A mutant, which, when combined with the increase in V-xylose/E(t), results in a small, but significant, primary deuterium isotope effect on turnover (V-D(xylose) = 1.07). These results demonstrate the utility of using the kinetic model developed for the wild-type enzyme to analyze transient kinetic data in order to ascribe changes in kinetic parameters(V/E(t), K-m, V-D, etc.) to changes in individual rate constants in the overall mechanism.