CATALYSIS BY YEAST ALCOHOL DEHYDROGENASE Bryce V. Plapp, Axel J. Ganzhorn, Robert M. Gould, David W. Green, Tobias Jacobi, Edda Warth, and Darla Ann Kratzer Department of Biochemistry, The University of Iowa Iowa City, IA 52242 INTRODUCTION The structure and mechanism of alcohol dehydrogenases have been extensively studied (Branden et al., 1975; Klinman, 1981; Pettersson, 1987). The three-dimensional structures of the horse liver enzyme in several ternary complexes have been solved at high resolution (Eklund et al., 1981, 1982). Amino acid sequences for more than 22 NAD+-dependent alcohol dehydrogenases from 11 animal, plant and fungal species are known. Comparison of these sequences raises many questions about the structure-function relationships in these enzymes. How do the amino acid residues at the active site participate in catalysis? What is the basis of substrate specificity? What was selected for during the evolution of the different enzymes? Since these enzymes are homologous, it is possible to build a model of yeast alcohol dehydrogenase I (ADH I: constitutive, cytoplasmic) based upon the three-dimensional structure of the horse liver enzyme. Figure 1 is a representation of the active site of the yeast enzyme. It shows that many amino acid residues contact the substrate and can participate in catalysis. We should like to know how each amino acid residue contributes. Some clues to the roles of these residues come from the inspection of the structure, from chemical modification studies, and from examining amino acid substitutions in this family of alcohol dehydrogenases. Table 1 shows the substitutions that are known to occur in these alcohol dehydrogenases. Many of the residues are strictly conserved, whereas others only have conservative changes, and other positions have many substitutions. In particular, substitutions within the substrate binding pocket apparently give rise to the enzymes with greatly different specificities for substrates. For instance, the yeast enzyme is most active on ethanol, and the model in Figure 1 shows that the active site has two tryptophan residues, which could greatly restrict the access of larger substrates. In contrast, the horse liver enzyme is active on substrates such as cyclohexanol, apparently because it has the smaller leucine and phenylalanine residues. Other enzyme variants have residues with smaller side chains and are active on molecules as large as steroid alcohols. Using site-directed mutagenesis and steady-state kinetic studies, we have explored the roles of some of these residues. Our results provide some information about the general principles concerning the function of the residues in catalysis. In addition, the work provides some surprising results that lead to new questions about enzyme structure and function. Enzymology and Molecular Biology of Carbonyl Metabolism 3 Edited by H. Weiner et al., Plenum Press, New York, 1990

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