Grimshaw C E, Bohren K M, Lai C J, Gabbay K H
Whittier Diabetes Program, Department of Medicine, University of California, San Diego, La Jolla 92093-0983, USA.
Biochemistry. 1995 Nov 7;34(44):14366-73. doi: 10.1021/bi00044a013.
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 Vxylose/Et 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 (Ki NADP+ = 0.14 microM) binary *E.NADP+ complex to the weak (Kd NADP+ = 6.8 microM) 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 Kxylose 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 Vxylitol/Et, because hydride transfer is largely rate-limiting for reaction in this direction.(ABSTRACT TRUNCATED AT 250 WORDS)
