; R o o t E n t r y F : @ C o m p O b j b W o r d D o c u m e n t # O b j e c t P o o l : : F Microsoft Word 6.0 Document MSWordDoc Word.Document.6 ; Oh +' 0 $ H l D h R:WWUSERTEMPLATENORMAL.DOT marcia steinberg marcia steinberg @ P: @ e = e # j j j j j j j O L # 1 T 4 O j O j j j j ~ j j j j 4 9506831 Plapp Alcohol dehydrogenases from horse liver and yeast have been studied extensively, but the origin of the catalytic activity and the function of the whole protein in catalytic dynamics is not fully understood. Roles for some amino acid residues at the active sites have been proposed from knowledge of the three dimensional structures, chemical modification, pH dependencies, and site directed mutagenesis, but contributions to catalysis should be quantitatively evaluated. Although enzymologists consider that residues at the active site are most critical for catalysis, emerging data suggest that amino acid residues distant from the active site contribute indirectly to catalytic power. Moreover, conformational change s can affect catalysis in unknown ways. Alcohol dehydrogenases offer a good system for studying these and other aspects of dynamics and catalysis. Three dimensional structures of the liver enzyme in two conformational states have been solved, and amino acid sequences for more than 60 alcohol dehydrogenases are available. Rate and dissociation constants for each step in the mechanism can be estimated from steady state and transient data and can quantitatively assess the effects of structural changes. The objectives of the proposed research are to answer questions about (1) the pH dependence of the catalytic mechanism, (2) the contribution of protein dynamics and conformational changes to catalysis and hydrogen tunneling, and (3) the tertiary and quaternary structures of various states of the horse and yeast enzymes. Site directed mutagenesis will be used to substitute amino acid residues, and steady state and transient kinetics, x ray crystallography, NMR, and fluorescence will be used to study these variants. Amino acid residues in the proton relay system and other residues at or distant from the active site will be substituted in order to study the origin of the pH dependencies for coenzyme binding and substrate interconversion steps. Residues near the coenzyme and substrate binding sites will be studied, as will some residues that contribute to the overall electrostatic potential of the protein. Protein flexibility and hydrogen tunneling will be studied in the liver enzyme with substitutions of residues in the interiors of the domains and at hinge regions, and by deletion of Q loops or other "extraneous" structural elements. Dynamic fluorescence properties of the horse liver enzyme with substituted tryptophan residues will be assessed. Three dimensional structures of complexes of the tetrameric yeast alcohol dehydrogenase and of potentially new conformational states of the dimeric horse liver enzyme will be determined by x ray crystallography. This research should increase our understanding of biocatalysis. %%% Alcohol dehydrogenase is the enzyme required for the production of ethanol by fermentation in yeast and for the removal of alcohols by metabolism in man. The enzyme accelerates the rate of dehydrogenation of alcohols with a coenzyme, but the catalytic mechanism is not yet fully understood. Enzymes are large proteins that have a smaller active site region with several amino acid residues that directly bind the alcohol and the coenzyme. Other amino acid residues outside of the active site provide a framework for the catalytic process and indirectly affect the rate of the reaction by controlling protein dynamics. Three dimensional structures of the horse and human liver enzymes have been determined, and now the structure of the yeast enzyme will be determined. The horse liver enzyme changes conformation during catalysis, which may facilitate the transfer of hydrogen by a "tunneling" process. The roles of the various amino acid residues in the horse and yeast enzymes will be studied by "protein engineering", which uses site directed mutagenesis to change individual amino acids, and by steady state and rapid reaction kinetics, X ray crystallography, and other spectroscopic methods that can detect the effects of the mutations. These studies will increase our understanding of biocatalysis. *** @ @ ; S u m m a r y I n f o r m a t i o n (
