Site-directed mutagenesis of Gly-15 and Gly-20 in the glycine-rich region of adenylate kinase.
Glycine-15 or Glycine-20 in the glycine-rich region of chicken adenylate kinase was replaced by Ala via site-directed mutagenesis. The two mutant enzymes showed lower enzymatic activities under the standard assay conditions. Kinetic analyses of the mutant enzymes revealed that they have markedly lower affinities for AMP and ATP, the Vmax values being comparable to that of the wild-type enzyme. These properties are similar to those of the (Pro-17----Leu, Gly or Val) enzymes (Reinstein, J., Brune, M., & Wittinghofer, A. (1988) Biochemistry 27, 4712-4720; Tagaya, M., Yagami, T., Noumi, T., Futai, M., Kishi, F., Nakazawa, A., & Fukui, T. (1989) J. Biol. Chem. in press), providing evidence that the glycine-rich region in adenylate kinase is important for the binding of both substrate nucleotides. The substrate specificity and the susceptibilities as to thermal denaturation and proteolysis were also affected by the mutations.
Yoneya
T
T
Institute of Scientific and Industrial Research, Osaka University.
Tagaya
M
M
Kishi
F
F
Nakazawa
A
A
Fukui
T
T
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000263:Adenylate Kinase / Q000032:analysis*; D000818:Animals; D002645:Chickens; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005998:Glycine / Q000032:analysis*; D008968:Molecular Conformation; D009154:Mutation; D010770:Phosphotransferases / Q000032:analysis*; D011489:Protein Denaturation; D013379:Substrate Specificity
D010770:Phosphotransferases; D000263:Adenylate Kinase; D000251:Adenosine Triphosphatases; D005998:Glycine