The overall goal of this research project is to determine the relationships between the catalytic function and three-dimensional structure of human alcohol dehydrogenases (ADH). There are as many as nine different genetically encoded subunits of alcohol dehydrogenase which combine to form more than 20 different active dimeric isoenzymes. We have shown that the kinetic properties of these isoenzymes differ widely. In order to investigate structure-function relationships among these isoenzymes, we devised methods to express recombinant human alcohol dehydrogenases in E. coli and purify them to homogeneity. Recombinant beta1/beta1 was crystallized and its X-ray structure (the first of a human alcohol dehydrogenase) reveals differences from the horse liver enzyme in the coenzyme binding site, which explains the tighter binding of coenzyme to beta1/beta1 versus the horse liver enzyme. We will determine the X-ray structures of isoenzymes with different catalytic properties from beta1/beta1, for example, alpha/alpha, pi/pia, chi/chi, and the eta-like stomach enzyme. We are particularly interested in determining X-ray structures in enzyme substrate complexes which will elucidate the unique catalytic properties of ADH isoenzymes, for example, the crystallization of chichi with s-formylglutathione and NAD+ might yield important information regarding the mechanism of chichi as a formaldehyde dehydrogenase. We have created variants of beta1/beta1 by site-directed mutagenesis, expressed the variant enzymes in E. coli, and purified them to homogeneity. Using these methods we will examine structure-function relationships for the coenzyme and alcohol binding sites for ADH. Based on known X-ray structures, we will make specific mutations in these sites and examine the steady-state and stopped-flow kinetic properties of these mutants to establish the functional roles of these amino acids. We will use intrinsic Trp fluorescence of beta1/beta1 or fluorescence of mutants with Trp residues inserted or deleted in specific regions to probe the substrate and inhibitor specificity for the substrate-induced conformational change in the apo- versus NAD+ trifluoroethanol-enzyme complexes. The enzymatic properties of ADH play a key role in determining the pharmacokinetics of ethanol elimination in man. These studies should contribute to the basic understanding of the relative contribution of the various isoenzymes of human ADH to ethanol metabolism. They should also contribute to the basic biochemical knowledge about the structure and catalytic mechanisms of the human alcohol dehydrogenases.

Agency
National Institute of Health (NIH)
Institute
National Institute on Alcohol Abuse and Alcoholism (NIAAA)
Type
Research Project (R01)
Project #
2R01AA007117-06
Application #
3567366
Study Section
Biochemistry, Physiology and Medicine Subcommittee (ALCB)
Project Start
1987-02-01
Project End
1997-01-31
Budget Start
1992-02-01
Budget End
1993-01-31
Support Year
6
Fiscal Year
1992
Total Cost
Indirect Cost
Name
Indiana University-Purdue University at Indianapolis
Department
Type
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
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
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; 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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