Lactate dehydrogenase catalyzes the reduction of pyruvate by facilitating the direct, simultaneous transfer of a proton and two electrons from the coenzyme, NADH, to the carbonyl carbon of pyruvate. This hydride transfer occurs with a high degree of stereochemical fidelity relative to nonenzymic reactions (< 1 error in 108 transfers). Because many other dehydrogenases may operate in a similar manner, the proposed research serves as a model for future explorations of how dehydrogenases accomplish large rate accelerations of chemical reactions. Both chemical kinetics and Raman spectroscopy will be used to determine changes in the chemical bonds of both the substrate and coenzyme upon binding to the enzyme, and these changes will be related to the structure, which is known, of the enzyme with substrate and coenzyme bound. These studies will partially involve the synthesis of isotopically labeled substrates and coenzymes so that Raman bands can be assigned to the molecular motions of these molecules when they are both free in solution and bound to enzyme. These studies also will be extended to mutant forms of lactate and to other dehydrogenases to produce a more general picture of the interactions that occur. A combination of these studies with future studies involving additional structure determinations and energy evaluations by computational methods will increase our understanding of the molecular basis of why enzymes are such extraordinary catalysts with such unerring fidelity.
