We have used transient kinetic data for partial reactions of recombinant human aldose reductase and simulations of progress curves for D-xylose reduction with NADPH and for xylitol oxidation with NADP+ to estimate rate constants for the following mechanism at pH 8.0: E<-->E.NADPH<-->*E.NADPH<-->*E.NADPH.RCHO<-->*E.NADP+.RCH2OH <-->*E.NADP+<--> E.NADP+<-->E. The mechanism includes kinetically significant conformational changes of the two binary E.nucleotide complexes which correspond to the movement of a crystallographically identified nucleotide-clamping loop involved in nucleotide exchange. The magnitude of this conformational clamping is substantial and results in a 100- and 650-fold lowering of the nucleotide dissociation constant in the productive *E.NADPH and *E.NADP+ complexes, respectively. The transient reduction of D-xylose displays burst kinetics consistent with the conformational change preceding NADP+ release (*E.NADP+-->E.NADP+) as the rate-limiting step in the forward direction. The maximum burst rate also displays a large deuterium isotope effect (Dkburst = 3.6-4.1), indicating that hydride transfer contributes significantly to rate limitation of the sequence of steps up to and including release of xylitol. In the reverse reaction, no burst of NADPH production is observed because the hydride transfer step is overall 85% rate-limiting. Even so, the conformational change preceding NADPH release (*E.NADPH-->E.NADPH) still contributes 15% to the rate limitation for reaction in this direction. The estimated rate constant for hydride transfer from NADPH to the aldehyde of D-xylose (130 s-1) is only 5- to 10-fold lower than the corresponding rate constant determined for NADH-dependent carbonyl reduction catalyzed by lactate or liver alcohol dehydrogenase. Hydride transfer from alcohol to NADP+ (0.6 s-1), however, is at least 100- to 1000-fold slower than NAD(+)-dependent alcohol oxidation mediated by these two enzymes, resulting in a bound-state equilibrium constant for aldose reductase which greatly favors the forward reaction. The proposed kinetic model provides a basic set of rate constants for interpretation of kinetic results obtained with aldose reductase mutants generated for the purpose of examining structure-function relationships of different components of the native enzyme.

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Detailed analyses of the pH variation of kinetic parameters for the forward aldehyde reduction and reverse alcohol oxidation reactions mediated by recombinant human aldose reductase, for inhibitor binding, and for kinetic isotope effects on aldehyde reduction have revealed that the pK value for the active site acid-base catalyst group Tyr48 is quite sensitive to the oxidation state of the bound nucleotide (NADPH or NADP+) and to the presence or absence of the Cys298 sulfhydryl moiety. Thus, the Tyr48 residue of C298A mutant enzyme displays a pK value that ranges from 7.6 in the productive *E.NADP+ complex that binds and reacts with alcohols to 8.7 in the productive *E.NADPH complex that binds and reacts with aldehyde substrates. For wild-type enzyme, Tyr48 in the latter complex displays a lower pK value of about 8.25. Assignment of the pK values was facilitated by the recognition and quantitation of the degree of stickiness of several aldehyde substrates in the forward reaction. The unusual pH dependence for Valdehyde/Et and DValdehyde, which decrease roughly 20-fold through a wave and remain constant at high pH, respectively, is shown to arise from the pH-dependent decrease in the net rate of NADP+ release. The results described are fully consistent with the chemical mechanism for aldose reductase catalysis proposed previously (Bohren et al., 1994) and, furthermore, establish that binding of the spirohydantoin class of aldose reductase inhibitors, e.g., sorbinil, occurs via a reverse protonation scheme in which the ionized inhibitor binds preferentially to the *E.NADP+ complex with Tyr48 present as the protonated hydroxyl form. The latter finding allows us to propose a unified model for high-affinity aldose reductase inhibitor binding that focuses on the transition state-like nature of the *E-Tyr48-OH.NADP+.inhibitor- complex.

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)

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