The mechanism of enzyme-catalyzed reduced nicotinamide adenine dinucleotide-dependent reductions. Substituent and isotope effects in the yeast alcohol dehydrogenase reaction.

Yeast alcohol dehydrogenase has been found to catalyze the reduction of a series of para-substituted benzaldehydes at markedly different rates. By steady state kinetic analysis, values of kB and ko have been obtained for yeast alcohol dehydrogenase reduction of $-Br-, p-Cl-, H-, &CH3, &CH80benzaldehyde by reduced nicotinamide adenine dinucleotide (NADH) and reduced nicotinamide adenine dinucleotide with deuterium in the nicotinamide 4-A position (NADD). Hammett plots of log ka and log kD versus u+ are linear with p = 2.1’7 and 2.24, respectively. Since the p values are almost the same for both NADH and NADD reduction, the difference in the intercept of the two Hammett plots gives the value of the kinetic isotope effect, k=: kn = 3.6. The significance of the sign and magnitude of these p values is discussed and shown to be consistent with a direct hydride transfer. The p value for the equilibrium conversion of para-substituted benzaldehydes to their corresponding alcohols was determined to be p = 1.5. The difference between the kinetically determined p and the p for K,, may reflect an enhanced polarization of the carbonyl of the substrate in forming the ternary complex. Consistent with this hypothesis, the association constants of substituted benzaldehydes for binary complex, calculated from the observed isotope effects on the limiting Michaelis constants, were found to increase with electron-donating group (p = -0.85).