How small molecules bind to proteins is of central importance and an important example of this concerns the mechanisms of enzymatic catalysis. Enzymes catalyze specific chemical reactions many, many orders of magnitude better than man-made catalysis; also, a particular enzyme has been designed to work on a generally specific set of reactants. These properties are defined by the usually non-covalent interactions between an enzyme and its particular substrates. Raman vibrational spectroscopy of bound substrates yields especially useful information on these interactions in that the bond orders of specific molecular coordinates can be determined through measurements of force constants. From this, a picture of how the substrate binds to protein is possible. Special technical advances in Raman difference spectroscopy have been accomplished during the course of this work that permit measurement of the Raman spectrum of a small molecule when embedded within a large protein. A number of approaches are contemplated in the present proposal to improve the sensitivity of this technique. It is proposed to investigate the NAD-linked dehydrogenases, an important class of enzymes throughout metabolism. Lactate dehydrogenase will be studied in depth, with a particular examination of the two coordinates key to catalysis, the substrate's carbonyl stretch and NAD's C4-H stretch. Mutants of this and other dehydrogenases will be examined to see how key residues affect these and other coordinates in a combined vibrational-molecular biology approach. The mutants will also be used in studies aimed at assessing the feasibility of Raman measurements of single protein residues and how specific residue changes affect protein structure using Raman difference spectroscopy. This could be a particularly powerful approach to a very difficult problem. Measurements on a selected set of dehydrogenases should be decisive on a decades old problem of whether or not there is a mechanistic imperative to the stereospecificity observed in the dehydrogenases. The binding of adenine to a selected set of dehydrogenases will be studied because of the importance of (particularly purine) nucleotide binding to proteins to allosteric control and biochemical energetics. In these and other studies, isotopically labelled compounds will be available.