title: An aspartate residue in yeast alcohol dehydrogenase I determines the specificity for coenzyme.
abstract: In the three-dimensional structures of enzymes that bind NAD or FAD, there is an acidic residue that interacts with the 2'- and 3'-hydroxyl groups of the adenosine ribose of the coenzyme. The size and charge of the carboxylate might repel the binding of the 2'-phosphate group of NADP and explain the specificity for NAD. In the NAD-dependent alcohol dehydrogenases, Asp-223 (horse liver alcohol dehydrogenase sequence) appears to have this role. The homologous residue in yeast alcohol dehydrogenase I (residue 201 in the protein sequence) was substituted with Gly, and the D223G enzyme was expressed in yeast, purified, and characterized. The wild-type enzyme is specific for NAD. In contrast, the D223G enzyme bound and reduced NAD+ and NADP+ equally well, but, relative to wild-type enzyme, the dissociation constant for NAD+ was increased 17-fold, and the reactivity (V/K) on ethanol was decreased to 1%. Even though catalytic efficiency was reduced, yeast expressing the altered or wild-type enzyme grew at comparable rates, suggesting that equilibration of NAD and NADP pools is not lethal. Asp-223 participates in binding NAD and in excluding NADP, but it is not the only residue important for determining specificity for coenzyme.
authors.affiliation: Department of Biochemistry, University of Iowa, Iowa City 52242.
authors.forename: F
authors.initials: F
authors.lastname: Fan
authors.forename: J A
authors.initials: JA
authors.lastname: Lorenzen
authors.forename: B V
authors.initials: BV
authors.lastname: Plapp
mesh_terms: D000426:Alcohol Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001224:Aspartic Acid; D001483:Base Sequence; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism*; D015345:Oligonucleotide Probes; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity
chemical_list: D015345:Oligonucleotide Probes; D009243:NAD; D005182:Flavin-Adenine Dinucleotide; D001224:Aspartic Acid; D000426:Alcohol Dehydrogenase