COOPERATIVITY IN ALCOHOL DEHYDROGENASE

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The overall goal of this project is to determine the role of cooperativity in liver alcohol dehydrogenase. Liver alcohol dehydrogenase is a rate-limiting factor in ethanol metabolism, underscoring its importance in ethanol elimination. Horse and human liver alcohol dehydrogenases exist as a mixture of homo-and heterodimeric isoenzymes. Apparent cooperativity has been observed in many of these isoenzymes, specifically for the gamma2gamma2 human enzyme and the heterodimeric isoenzymes from both horse and human liver tissues. The steady-state and transient kinetics of recombinant gamma2gamma2 human enzyme will be determined in an effort to understand the observed cooperativity. Along with these studies, the binding of substrates and coenzymes to gamma2gamma2 human enzyme will be measured using epr and fluorescence spectroscopy. Site-directed mutagenesis will be used to disrupt coenzyme binding to determine if the conformational change associated with coenzyme binding to gamma2gamma2 is involved in mediating the subunit interactions. Cooperativity in the alphagamma2 human liver isoenzyme and in horse liver heterodimers consisting of a wild-type and a mutant subunit will also be studied. The results of this research will contribute to the current understanding of ethanol metabolism in man.