Role of tyrosine-80 in the stability of kanamycin nucleotidyltransferase analyzed by site-directed mutagenesis.
A thermostable mutant of kanamycin nucleotidyltransferase (KNTase) with a single amino acid replacement of Asp at position 80 by Tyr has been isolated by a novel screening method in a previous study [Matsumura, M. & Aiba, S. (1985) J. Biol. Chem. 260, 15298-15303]. To elucidate the role of Tyr80 in stabilizing the enzyme, the KNTase gene was modified by site-directed mutagenesis so that the codon for Asp80 of the wild type was replaced by that for Ser, Thr, Ala, Val, Leu, Phe and Trp, respectively. The eight mutant KNTases including Tyr80 were all purified, as well as the wild-type enzyme. The heat-inactivation rate constants were determined at 58 degrees C and the half-life values were found to be correlated with the hydrophobicity of the amino acid residues replaced at the unique position. The Gibbs energy change of unfolding in water of KNTase assessed from urea denaturation (25 degrees C, pH 7.0) was also found to be correlated with hydrophobicity. The results suggest that different amino acids at position 80 of KNTase contribute to the stability of the protein by hydrophobic interactions. In the case of tyrosine at position 80 the unusually high stability of the enzyme compared to the Phe80 enzyme suggests that the hydroxyl group also contributes to the conformational stability.
Matsumura
M
M
Department of Fermentation Technology, Faculty of Engineering, Osaka University, Japan.
Yahanda
S
S
Yasumura
S
S
Yutani
K
K
Aiba
S
S
D001665:Binding Sites; D004247:DNA / Q000032:analysis; D004735:Energy Transfer; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D006900:Hydroxylation; D009154:Mutation; D009713:Nucleotidyltransferases / Q000235:genetics* / Q000502:physiology; D011487:Protein Conformation; D011489:Protein Denaturation; D014443:Tyrosine / Q000302:isolation & purification / Q000502:physiology*; D014508:Urea
D014443:Tyrosine; D014508:Urea; D004247:DNA; D009713:Nucleotidyltransferases; C024251:kanamycin nucleotidyltransferase