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 mut ant human aldose reductase at pH 8 have been used to obtain estimates for each of the rate constants in the complete reaction mechanism as o utlined for the wild-type enzyme in the preceding paper (Grimshaw et a l., 1995a). Analysis of the resulting kinetic model shows that the nea rly 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 inc rease 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 re leased. Evaluation of the rate expressions derived from the kinetic mo del for the various steady-state kinetic parameters reveals that the 3 7-fold increase in K-xylose seen for C298A relative to wild-type aldos e reductase is largely due to this same increase in the net rate of NA DP(+) 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 t he conformational change preceding NADPH release does not, however, re sult in any increase in V-xylitol/E(t), because hydride transfer is la rgely rate-limiting for reaction in this direction. By contrast, the r ate constant for conformational clamping in the opposite direction (we ak --> tight binary complex) is not greatly affected by the mutation, suggesting that Cys298 does not regulate the rate of closure of the nu cleotide enfolding protein loop, but does stabilize the closed conform ation. The rate of hydride transfer is reduced 2-fold in the C298A mut ant, which, when combined with the increase in V-xylose/E(t), results in a small, but significant, primary deuterium isotope effect on turno ver (V-D(xylose) = 1.07). These results demonstrate the utility of usi ng the kinetic model developed for the wild-type enzyme to analyze tra nsient 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.