The combined application of both substituent and isotope effects provides a unique probe for studying ground state and transition state interactions between enzymes and substrates. It is the objective of this proposal to study the mechanism of action of enzymes which catalyze hydrogen abstraction reactions and are characterized by a broad substrate specificity, so that substituent and isotope effects can be studied together in each enzyme system. Kinetic studies have been carried out on the yeast alcohol dehydrogenase catalyzed reduction of substituted benzaldehydes by NADH(D), demonstrating the validity of this approach to the study of enzyme catalysis. Continuing investigations on the nature of ground state and transition state interactions in the yeast alcohol dehydrogenase (YADH) system will include studies on the YADH catalyzed oxidation of protonated and deuterated benzyl alcohols by NAD ion the binding of substituted benzaldehydes and benzyl alcohols to YADH, and the inactivation of YADH by an active-site directed reagent (styrene oxide, an analogue of benzyl alcohol). Additional enzyme systems for which substituent and isotope effect studies are planned are benzyl alcohol dehydrogenase, phenyl pyruvate tautomerase, mandelate racemase and Beta- dopamine hydroxylase.