Transition-state structure in the yeast alcohol dehydrogenase reaction: the magnitude of solvent and alpha-secondary hydrogen isotope effects.

Solvent and alpha-secondary isotope effects have been measured in the yeast alcohol dehydrogenase reaction, under conditions of a rate-limiting transfer of hydrogen between coenzyme and substrate. Determination of catalytic constants (at saturating concentrations of substrate and coenzyme) in H2O and D2O as a function of pH(D) has allowed the separation of solvent effects on pKa from kcat: delta pKa = pKD--pKH = 0.02--0.21, kH2O/kD2O = 1.20 +/- 0.09 in the direction of p-methoxybenzyl alcohol oxidation, and kH2O/kD2O = 0.50 +/- 0.05 and 0.58 +/- 0.06 for p-methoxybenzaldehyde reducation by NADH and [4-2H]NADH. The small effect of D2O on pKa, which contrasts with the common observation that delta pKa congruent to 0.4--0.6, is tentatively assigned to ionization of an active-site ZnOH2. The near absence of an isotope effect on kcat in the direction of alcohol oxidation rules out a mechanism involving concerted catalysis by an active-site base of hydride transfer. In the direction of aldehyde reduction, the observation of inverse isotope effects on kcat is concluded to reflect displacement of zinc-bound water by substrate to form an inner-sphere complex, subsequent to the E.S complex. Equilibrium alpha-secondary isotope effects, measured as a frame of reference for kinetic values, indicate KH/KT = 1.33 +/- 0.05 and 1.34 +/- 0.09 for the oxidation of [1(S)-3H]benzyl alcohol and p-methoxy[1(S)-3H]benzyl alcohol, respectively. Kinetic alpha-secondary isotope effects are within experimental error of equilibrium values, kH/kT = 1.34 +/- 0.07 and 1.38 +/- 0.02 for [1(S)-3H]benzyl alcohol and p-methoxy[1(S)-3H]benzyl alcohol oxidation, respectively. The near identity of kinetic and equilibrium alpha-secondary isotope effects in the direction of alcohol oxidation implicates a transition-state structure which resembles aldehyde with regard to bond hybridization properties. This result contrasts sharply with previously reported structure--reactivity correlations, which implicate a transition-state structure resembling alcohol with regard to charge properties. The significance of these findings to the mechanism of NAD(P)H-dependent redox reactions is discussed.