Reinstein J, Schlichting I, Wittinghofer A
Abteilung Biophysik, Max-Planck-Institut für medizinische Forschung, Heidelberg, West Germany.
Biochemistry. 1990 Aug 14;29(32):7451-9. doi: 10.1021/bi00484a014.
Amino acids in the phosphate binding loop of adenylate kinase of Escherichia coli were mutated by site-directed mutagenesis. The mutant proteins with a Pro-9----Gly (P9G) and with a Lys-13----Gln (K13Q) exchange were overexpressed and purified. They were characterized by steady-state kinetics, fluorescence binding, and structural studies, together with the phosphate binding loop mutants P9L and G10V prepared earlier [Reinstein, J., Brune, M., & Wittinghofer, A. (1988) Biochemistry 27, 4712-4720]. The results obtained show that all these mutations change the structure of the protein as evidenced by NMR spectroscopy and temperature-stability studies. All the mutant proteins have increased dissociation constants for substrates and inhibitors, but their catalytic activity, except for K13Q, is not reduced. The results obtained with K13Q suggest that this lysine residue, which is conserved in all guanine and many adenine nucleotide proteins, might have an important role in catalysis.
通过定点诱变对大肠杆菌腺苷酸激酶磷酸结合环中的氨基酸进行了突变。将脯氨酸-9突变为甘氨酸（P9G）以及赖氨酸-13突变为谷氨酰胺（K13Q）的突变蛋白进行了过表达和纯化。通过稳态动力学、荧光结合和结构研究对它们进行了表征，同时还研究了之前制备的磷酸结合环突变体P9L和G10V [莱因斯坦，J.，布鲁内，M.，& 维廷霍费尔，A.（1988年）《生物化学》27，4712 - 4720]。所获得的结果表明，如核磁共振光谱和温度稳定性研究所证明的，所有这些突变都改变了蛋白质的结构。所有突变蛋白对底物和抑制剂的解离常数都增加了，但除了K13Q外，它们的催化活性并未降低。K13Q的结果表明，这个在所有鸟嘌呤和许多腺嘌呤核苷酸蛋白中都保守的赖氨酸残基，可能在催化中起重要作用。
