The overall goal is to understand the mechanisms by which liver alcohol dehydrogenases (ADH) regulate the metabolism of alcohol. Since ADH catalyzes the rate-limiting step in ethanol metabolism, we propose that the enzymatic properties of ADH play a key role in determining the pharmacokinetics of ethanol elimination. Studies in our laboratory and elsewhere have indicated that there are as many as 8 different genetically determined ADH subunits which combine to form more than 20 different active dimeric isoenzymes. It has been shown that ADH isoenzymes exhibit widely different kinetic properties and recent sequence analysis of ADH subunits and genes has identified some of the substitutions responsible for these differences.
Our aim i s to systematically examine the function of amino acids in the active site of human liver B1B1 ADH by site-directed mutagenesis. We will create mutagenized forms of the B1 cDNA, which we have recently cloned, by annealing synthetic oligonucleotides containing a mismatched sequence to the cDNA. Mutagenized forms of B1B1 containing the substituted amino acid encoded by the mismatched oligonucleotide will be expressed in yeast. We will examine the steady-state and stopped-flow kinetic properties and substrate specificities of the mutagenized ADHs. By these methods, we will examine structure-function relationships for basic residues which bind the pyrophosphate of coenzyme, residues that participate in the putative alcohol-proton relay system, residues in the alcohol binding pocket, residues which undergo a conformational change upon binding coenzyme, and residues which bind the noncatalytic zinc atom and determine the structure of this site. We will also examine the specificity of formation of complexes of ADH isoenzymes with glyceraldehyde-3-phosphate dehydrogenase and investigate the proposed direct intermolecular transfer of NADH between these dehydrogenases. The study of structure-function relationships in the human liver B1B1 ADH should contribute substantially to the fundamental understanding of the mechanism of catalysis by ADH and to the role that human liver ADH isoenzymes may play in the regulation of alcohol metabolism.

Agency
National Institute of Health (NIH)
Institute
National Institute on Alcohol Abuse and Alcoholism (NIAAA)
Type
Research Project (R01)
Project #
5R01AA007117-05
Application #
3110728
Study Section
Biochemistry, Physiology and Medicine Subcommittee (ALCB)
Project Start
1987-02-01
Project End
1992-01-31
Budget Start
1991-02-01
Budget End
1992-01-31
Support Year
5
Fiscal Year
1991
Total Cost
Indirect Cost
Name
Indiana University-Purdue University at Indianapolis
Department
Type
Schools of Medicine
DUNS #
005436803
City
Indianapolis
State
IN
Country
United States
Zip Code
46202
Bosron, W F; Ehrig, T; Li, T K (1993) Genetic factors in alcohol metabolism and alcoholism. Semin Liver Dis 13:126-35
Stone, C L; Hurley, T D; Amzel, L M et al. (1993) Kinetics of a glycine for Arg-47 human alcohol dehydrogenase mutant can be explained by Lys-228 recruitment into the pyrophosphate binding site. Adv Exp Med Biol 328:429-37
Stone, C L; Bosron, W F; Dunn, M F (1993) Amino acid substitutions at position 47 of human beta 1 beta 1 and beta 2 beta 2 alcohol dehydrogenases affect hydride transfer and coenzyme dissociation rate constants. J Biol Chem 268:892-9
Hurley, T D; Bosron, W F (1992) Human alcohol dehydrogenase: dependence of secondary alcohol oxidation on the amino acids at positions 93 and 94. Biochem Biophys Res Commun 183:93-9
Ehrig, T; Muhoberac, B B; Hurley, T D et al. (1992) Tryptophan fluorescence quenching by alkaline pH and ternary complex formation in human beta 1 beta 1 and horse EE alcohol dehydrogenases. FEBS Lett 300:283-5
Ehrig, T; Hurley, T D; Edenberg, H J et al. (1991) General base catalysis in a glutamine for histidine mutant at position 51 of human liver alcohol dehydrogenase. Biochemistry 30:1062-8
Hurley, T D; Ehrig, T; Edenberg, H J et al. (1991) Characterization of human alcohol dehydrogenases containing substitutions at amino acids 47 and 51. Adv Exp Med Biol 284:271-5
Hurley, T D; Bosron, W F; Hamilton, J A et al. (1991) Structure of human beta 1 beta 1 alcohol dehydrogenase: catalytic effects of non-active-site substitutions. Proc Natl Acad Sci U S A 88:8149-53
Hurley, T D; Edenberg, H J; Bosron, W F (1990) Expression and kinetic characterization of variants of human beta 1 beta 1 alcohol dehydrogenase containing substitutions at amino acid 47. J Biol Chem 265:16366-72
Ehrig, T; Bosron, W F; Li, T K (1990) Alcohol and aldehyde dehydrogenase. Alcohol Alcohol 25:105-16

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