title: Carboxyl groups near the active site zinc contribute to catalysis in yeast alcohol dehydrogenase.
abstract: The importance of carboxyl groups near the active site zinc for the catalytic function of alcohol dehydrogenase I from Saccharomyces cerevisiae was examined by directed mutagenesis and steady state kinetics. Asp-49 was changed to asparagine and Glu-68 to glutamine (residue numbering as for horse liver enzyme). The catalytic efficiencies (V/Km) for ethanol oxidation and acetaldehyde reduction were decreased by factors of 1000 with the Asn-49 mutant and 100 with the Gln-68 enzyme. For the Asn-49 mutant, dissociation constants for coenzymes increased 7-fold, and Michaelis and inhibition constants for substrates and substrate analogs increased by factors of 20-50. The turnover numbers were reduced 50-fold for ethanol oxidation and 15-fold for acetaldehyde reduction. Product and dead-end inhibition studies and kinetic isotope effects showed that the mechanism with NAD+ and ethanol was rapid equilibrium random, in contrast to the ordered mechanism of wild-type enzyme. Alcohol dehydrogenase I and the Asn-49 mutant had similar CD spectra and 2 zinc atoms/subunit, but slightly different UV absorption and fluorescence spectra. The Gln-68 mutant resembled the wild-type enzyme in most kinetic constants, but the turnover number for ethanol oxidation decreased 35-fold, and Kd for NAD+ and Km for acetaldehyde increased by factors of 4 and 50, respectively. The pK values for V1 and V1/Km for ethanol oxidation were shifted from 7.7 (wild-type) to 6.8 in the Gln-68 and 6.2 in the Asn-49 mutant. The altered electrostatic environment near the active site zinc apparently decreases activities by hindering isomerizations of enzyme-substrate complexes.
authors.affiliation: Department of Biochemistry, The University of Iowa, Iowa City 52242.
authors.forename: A J
authors.initials: AJ
authors.lastname: Ganzhorn
authors.forename: B V
authors.initials: BV
authors.lastname: Plapp
mesh_terms: D000426:Alcohol Dehydrogenase / Q000378:metabolism*; D000465:Algorithms; D001665:Binding Sites; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009243:NAD / Q000378:metabolism; D010449:Peptide Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013329:Structure-Activity Relationship; D015032:Zinc / Q000378:metabolism*
chemical_list: D009243:NAD; D000426:Alcohol Dehydrogenase; D015032:Zinc