HUMAN ALDOSE REDUCTASE - RATE CONSTANTS FOR A MECHANISM INCLUDING INTERCONVERSION OF TERNARY COMPLEXES BY RECOMBINANT WILD-TYPE ENZYME

We have used transient kinetic data for partial reactions of recombina nt human aldose reductase and simulations of progress curves for D-xyl ose reduction with NADPH and for xylitol oxidation with NADP(+) to est imate rate constants for the following mechanism at pH 8.0: E reversib le arrow E . NADPH reversible arrowE . NADPH reversible arrow*E . NAD PH . RCHO reversible arrowE . NADP(+). RCH(2)OH reversible arrow*E . NADP(+)reversible arrow E . NADP(+)reversible arrow E. The mechanism i ncludes kinetically significant conformational changes of the two bina ry E nucleotide complexes which correspond to the movement of a crysta llographically identified nucleotide-clamping loop involved in nucleot ide exchange. The magnitude of this conformational clamping is substan tial and results in a 100- and 650-fold lowering of the nucleotide dis sociation constant in the productive E . NADPH and *E . NADP(+) compl exes, 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 deuter ium isotope effect (Dk(burst) = 3.6-4.1), indicating that hydride tran sfer contributes significantly to rate limitation of the sequence of s teps 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-xyl ose (130 s(-1)) is only 5- to 10-fold lower than the corresponding rat e constant determined for NADH-dependent carbonyl reduction catalyzed by lactate or liver alcohol dehydrogenase. Hydride transfer from alcoh ol to NADP(+) (0.6 s(-1)), however, is at least 100- to 1000-fold slow er than NAD(+)-dependent alcohol oxidation mediated by these two enzym es, resulting in a bound-state equilibrium constant for aldose reducta se which greatly favors the forward reaction. The proposed kinetic mod el provides a basic set of rate constants for interpretation of kineti c results obtained with aldose reductase mutants generated for the pur pose of examining structure-function relationships of different compon ents of the native enzyme.