Alcohol dehydrogenases from horse liver and yeast have been studied extensively. The three-dimensional structures of the horse liver enzyme and several complexes with substrates and ligands are known, and the genes for four yeast alcohol dehydrogenases have been cloned in plasmids. Thus, answers can now be obtained to several outstanding questions about the catalytic mechanism of the enzyme, the correlation of kinetic characteristics with the structure and function, and the involvement of the tertiary and quaternary structures in activity. """"""""Site-specific mutagenesis"""""""" will be used to prepare variants of alcohol dehydrogenases for the following studies. The importance of the proton relay system, which includes His-51 and Ser-48 in the liver enzyme, will be investigated by changing these residues to ones that cannot participate in the proton relay. Amino acid residues that contribute to the environment of the catalytic zinc ion will be substituted, as will amino acids involved in coenzyme binding. The size of the substrate binding pocket will be increased or decreased and the effects on the substrate and rate enhancement specificity will be determined. The kinetics of the enzymes under physiological conditions in vitro will be related to the flux in vivo and to the growth rates of yeast. An attempt will be made to change the specificity of the enzyme for coenzyme and substrate by, for instance, making substitutions that will allow the enzyme to bind NADP as a coenzyme and L-lactate as a substrate. The role of the structural zinc in activity will be examined by removing residues that bind the zinc. Residues in the postulated contact regions between two dimers of the tetrameric yeast enzyme will be altered in an attempt to prepare a dimeric yeast enzyme like the liver form. Extraneous loops or regions of the molecule will be removed in an attempt to make a minimal catalytic unit. A yeast alcohol dehydrogenase will be crystallized for determination of the structure by x-ray crystallography. The cDNA for horse liver alcohol dehydrogenase will be cloned for the same kinds of studies.

Agency
National Institute of Health (NIH)
Institute
National Institute on Alcohol Abuse and Alcoholism (NIAAA)
Type
Research Project (R01)
Project #
5R01AA006223-06
Application #
3109434
Study Section
Alcohol Biomedical Research Review Committee (ALCB)
Project Start
1983-12-01
Project End
1991-11-30
Budget Start
1988-12-01
Budget End
1989-11-30
Support Year
6
Fiscal Year
1989
Total Cost
Indirect Cost
Name
University of Iowa
Department
Type
Schools of Medicine
DUNS #
041294109
City
Iowa City
State
IA
Country
United States
Zip Code
52242
Pal, Suresh; Park, Doo-Hong; Plapp, Bryce V (2009) Activity of yeast alcohol dehydrogenases on benzyl alcohols and benzaldehydes: characterization of ADH1 from Saccharomyces carlsbergensis and transition state analysis. Chem Biol Interact 178:16-23
Plapp, B V (1994) Control of alcohol metabolism. EXS 71:311-22
Ramaswamy, S; Kratzer, D A; Hershey, A D et al. (1994) Crystallization and preliminary crystallographic studies of Saccharomyces cerevisiae alcohol dehydrogenase I. J Mol Biol 235:777-9
Bahnson, B J; Park, D H; Kim, K et al. (1993) Unmasking of hydrogen tunneling in the horse liver alcohol dehydrogenase reaction by site-directed mutagenesis. Biochemistry 32:5503-7
Green, D W; Sun, H W; Plapp, B V (1993) Inversion of the substrate specificity of yeast alcohol dehydrogenase. J Biol Chem 268:7792-8
Plapp, B V; Green, D W; Sun, H W et al. (1993) Substrate specificity of alcohol dehydrogenases. Adv Exp Med Biol 328:391-400
Park, D H; Plapp, B V (1992) Interconversion of E and S isoenzymes of horse liver alcohol dehydrogenase. Several residues contribute indirectly to catalysis. J Biol Chem 267:5527-33
Light, D R; Dennis, M S; Forsythe, I J et al. (1992) Alpha-isoenzyme of alcohol dehydrogenase from monkey liver. Cloning, expression, mechanism, coenzyme, and substrate specificity. J Biol Chem 267:12592-9
Sun, H W; Plapp, B V (1992) Progressive sequence alignment and molecular evolution of the Zn-containing alcohol dehydrogenase family. J Mol Evol 34:522-35
Fan, F; Lorenzen, J A; Plapp, B V (1991) An aspartate residue in yeast alcohol dehydrogenase I determines the specificity for coenzyme. Biochemistry 30:6397-401

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