Site-directed mutagenesis will be used to substitute amino acid residues in these enzymes, and steady-state and transient kinetics, x-ray crystallography, and other methods will be used to study these variants. Amino acid residues in the proton relay system, and the environment of the catalytic zinc, will be studied. Protein flexibility and hydrogen tunneling will be studied in the liver enzyme. "Extraneous" structural elements, including the structural zinc loop and other loops, will be deleted. The origins of the oligomeric structures will be explored. Fluorescence properties of the horse liver enzyme will be determined by x-ray crystallography. %%% Alcohol dehydrogenases from horse liver and yeast have been studied extensively. The objectives of the proposed research are to answer several outstanding questions about the catalytic mechanism of the enzyme, the evolution of specificity for substrates and coenzymes, and the role of the three dimensional structure in enzyme dynamics and catalysis. This research should increase our understanding of biocatalysis.
