Transient-state and steady-state kinetic studies of the mechanism of NADH-dependent aldehyde reduction catalyzed by xylose reductase from the yeast Candida tenuis

Microbial xylose reductase, a representative aldo-keto reductase of primary sugar metabolism, catalyzes the NAD(P)H-dependent reduction of D-xylose wi th a turnover number approximately 100 times that of human aldose reductase for the same reaction. To determine the mechanistic basis for that physiol ogically relevant difference and pinpoint features that are unique to the m icrobial enzyme among other aldo/keto reductases, we carried out stopped-fl ow studies with wild-type xylose reductase from the yeast Candida tenuis. A nalysis of transient kinetic data for binding of NAD(+) and NADH, and reduc tion of D-xylose and oxidation of xylitol at pH 7.0 and 25 degreesC provide d estimates of rate constants for the following mechanism: E + NADH reversi ble arrow E.NADH reversible arrow *E.NADH + D-xylose reversible arrow *E.NA DH"D-xylose reversible arrow *E"NAD(+).xylitol reversible arrow *E.NAD(+) r eversible arrow E.NAD(+) reversible arrow E + NAD+. The net rate constant o f dissociation of NAD+ is similar to 90% rate limiting for k(cat) of D-xylo se reduction. It is controlled by the conformational change which precedes nucleotide release and whose rate constant of 40 s(-1) is 200 times that of completely rate-limiting *E.NADP(+) --> E.NADP(+) step in aldehyde reducti on catalyzed by human aldose reductase [Grimshaw, C. E., et al. (1995) Bioc hemistry 34, 14356-14365]. Hydride transfer from NADH occurs with a rate co nstant of approximately 170 s(-1). In reverse reaction, the *E.NADH --> E.N ADH step takes place with a rate constant of 15 s(-1), and the rate constan t of ternary-complex interconversion (3.8 s(-1)) largely determines xylitol turnover (0.9 s(-1)). The bound-state equilibrium constant for C. tenuis x ylose reductase is estimated to be similar to 45 (= 170/3.8), thus greatly favoring aldehyde reduction. Formation of productive complexes, *E.NAD(+) a nd *E.NADH, leads to a 7- and 9-fold decrease of dissociation constants of initial binary complexes, respectively, demonstrating that 12-fold differen tial binding of NADH (K-i = 16 muM) vs NAD(+) (K-i = 195 muM) chiefly refle cts difference in stabilities of E.NADH and E.NAD(+). Primary deuterium iso tope effects on k(cat) and k(cat)/K-xylose were, respectively, 1.55 +/- 0.0 9 and 2.09 +/- 0.31 in H2O, and 1.26 +/- 0.06 and 1.58 +/- 0.17 in D2O. No deuterium solvent isotope effect on k(cat)/K-xylose was observed. When deut eration of coenzyme selectively slowed the hydride transfer step, (D2O)(k(c at)/K-xylose) was inverse (0.89 +/- 0.14). The isotope effect data suggest a chemical mechanism of carbonyl reduction by xylose reductase in which tra nsfer of hydride ion is a partially rate-limiting step and precedes the pro ton-transfer step.