Carboxyl groups near the active site zinc contribute to catalysis in yeast alcohol dehydrogenase.
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.
Ganzhorn
A J
AJ
Department of Biochemistry, The University of Iowa, Iowa City 52242.
Plapp
B V
BV
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*
D009243:NAD; D000426:Alcohol Dehydrogenase; D015032:Zinc