Yoneya T, Tagaya M, Kishi F, Nakazawa A, Fukui T
Institute of Scientific and Industrial Research, Osaka University.
J Biochem. 1989 Feb;105(2):158-60. doi: 10.1093/oxfordjournals.jbchem.a122631.
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.
通过定点诱变，将鸡腺苷酸激酶富含甘氨酸区域中的甘氨酸 -15 或甘氨酸 -20 替换为丙氨酸。在标准测定条件下，这两种突变酶表现出较低的酶活性。对突变酶的动力学分析表明，它们对 AMP 和 ATP 的亲和力显著降低，Vmax 值与野生型酶相当。这些特性与（Pro -17→Leu、Gly 或 Val）酶的特性相似（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），这证明腺苷酸激酶中富含甘氨酸的区域对于两种底物核苷酸的结合很重要。突变也影响了底物特异性以及对热变性和蛋白水解的敏感性。
