SPECIFICITY OF ALCOHOL DEHYDROGENASES

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The overall goal of this research is to determine the specificities of liver alcohol dehydrogenases for substrates and inhibitors in order to elucidate the role of the enzymes in the metabolism of alcohols and to develop inhibitors or activators that would change the rates of metabolism. Liver alcohol dehydrogenase is a rate-limiting factor in alcohol metabolism, and amethystic agents would be useful to treat acute and chronic alcoholism. Thus, inhibitors could be used to prevent poisoning by methanol or ethylene glycol and to maintain the redox state of the cell so that metabolism of other substrates is not altered. Activators could be used t induce sobriety in intoxicated individuals, and perhaps to discourage drinking. The specificities of horse liver isoenzymes and the primate alpha-isoenzyme, which have high activity on ethanol and good activities on branched, secondary, cyclic and steroidal alcohols will be determined by steady-state and transient kinetic methods that provide rate constants for binding of substrates and for hydrogen transfer. The results will be correlated with the structures of enzymes so that specificities for potential substrates or different isoenzymes, such as the human Class I enzymes, can be predicted. These studies should provide insight into the physiological roles of the enzymes. New inhibitors with sulfoxide, amide and formamide functional groups, and structures that bind stereospecifically will be synthesized and evaluated. Three dimensional structures of complexes of the enzyme with substrates and inhibitors will be determined at high resolution by x-ray crystallography. Further tests of the determinants of specificity will use site-directed mutagenesis to alter substrate binding sites. Compounds that could activate the enzyme in a couple exchange reaction, which bypasses the rate-limiting reoxidation of coenzyme, will be prepared and evaluated. The metabolism of various alcohols will be described by kinetic simulations so that the effects of ethanol on the physiological state can be described quantitatively.