Acid-Base Catalysis in the Yeast Alcohol Dehydrogenase Reaction*

The effect of pH on steady state kinetic parameters for the yeast alcohol dehydrogenase-catalyzed reduction of aldehydes and oxidation of alcohols has been studied. The oxidation of p-CH3 benzyl alcohol-l, l-h2 and -l,l-dz by NAD+ was found to be characterized by large deuterium isotope effects (&J&n = 4.1 f 0.1) between pH 7.5 and 9.5, indicating a rate-limiting hydride transfer step in this pH range; a plot of &at uersus pH could be fit to a theoretical . . trtratron curve, pK = 8.25, where koat increases with increasing PH. The Michaelis constant for P-CH3 benzyl alcohol was independent of pH. The reduction of @-CHa benzaldehyde by NADH and reduced nicotinamide adenine dinucleotide with deuterium in the 4-A position (NADD) could not be studied below pH 8.5 due to substrate inhibition; however, between pH 8.5 and 9.5, kcat was found to decrease with increasing pH and to be characterized by significant isotope effects (kH/kD = 3.3 + 0.3). In the case of acetaldehyde reduction by NADH and NADD, isotope effects were found to be small and essentially invariant (kH/kD = 2.0 + 0.4) between pH 7.2 and 9.5, suggesting a partially rate-limiting hydride transfer step for this substrate; a plot of kcstlK’a (where K’b is the Michaelis constant for acetaldehyde) versus pH could be fit to a titration curve, pK = 8.25. The titration curve for acetaldehyde reduction has the same pK but is opposite in direction to that observed for &CH3 benzyl alcohol oxidation. The data presented in this paper indicate a dependence on different enzyme forms for aldehyde reduction and alcohol oxidation and are consistent with a single active site side chain, pK = 8.25, which functions in acid-base catalysis of the hydride transfer step.