Kinetic characterization of yeast alcohol dehydrogenases. Amino acid residue 294 and substrate specificity.
A three-dimensional model of yeast alcohol dehydrogenase, based on the homologous horse liver enzyme, was used to compare the substrate binding pockets of the three isozymes (I, II, and III) from Saccharomyces cerevisiae and the enzyme from Schizosaccharomyces pombe. Isozyme I and the S. pombe enzyme have methionine at position 294 (numbered as in the liver enzyme, corresponding to 270 in yeast), whereas isozymes II and III have leucine. Otherwise the active sites of the S. cerevisiae enzymes are the same. All four wild-type enzymes were produced from the cloned genes. In addition, oligonucleotide-directed mutagenesis was used to change Met-294 in alcohol dehydrogenase I to leucine. The mechanisms for all five enzymes were predominantly ordered with ethanol (but partially random with butanol) at pH 7.3 and 30 degrees C. The wild-type alcohol dehydrogenases and the leucine mutant had similar kinetic constants, except that isozyme II had 10-20-fold smaller Michaelis and inhibition constants for ethanol. Thus, residue 294 is not responsible for this difference. Apparently, substitutions outside of the substrate binding pocket indirectly affect the interactions of the alcohol dehydrogenases with ethanol. Nevertheless, the substitution of methionine with leucine in the substrate binding site of alcohol dehydrogenase I produced a 7-10-fold increase in reactivity (V/Km) with butanol, pentanol, and hexanol. The higher activity is due to tighter binding of the longer chain alcohols and to more rapid hydrogen transfer.
D000429:Alcohol Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001483:Base Sequence; D001665:Binding Sites; D007527:Isoenzymes / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D010957:Plasmids; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013379:Substrate Specificity
D000596:Amino Acids; D007527:Isoenzymes; D000429:Alcohol Oxidoreductases