Reinstein J, Brune M, Wittinghofer A
Max-Planck-Institut für medizinische Forschung, Abteilung Biophysik, Heidelberg, West Germany.
Biochemistry. 1988 Jun 28;27(13):4712-20. doi: 10.1021/bi00413a020.
The adk gene of Escherichia coli has been used to overexpress the adenylate kinase protein in two ways: (1) by cloning the adk gene with its own promoter into pEMBL plasmids, which have an increased copy number, and (2) by deleting the adk promoter and cloning the gene behind the regulatable tac promoter. Adenylate kinase comprises up to 40% of the soluble cellular extracts from E. coli strains containing these plasmids. Mutations have been introduced into the gene by site-directed mutagenesis to exchange amino acids in the nucleotide binding loop, which is highly conserved in many mononucleotide binding proteins. The mutation of Lys13----Gln is nearly inactive, whereas the Pro9----Leu and the Gly10----Val mutant proteins have an increased Km for both substrates and a Vmax that is similar to wild type. Proton NMR measurements of the proteins show that a major structural change seems to have taken place for the Pro9----Leu and Gly10----Val mutants. The results are discussed in the light of the kinetic mechanism for adenylate kinase and the three-dimensional structure of the protein.
大肠杆菌的adk基因已被用于通过两种方式过表达腺苷酸激酶蛋白：（1）将带有自身启动子的adk基因克隆到拷贝数增加的pEMBL质粒中；（2）删除adk启动子并将该基因克隆到可调控的tac启动子之后。在含有这些质粒的大肠杆菌菌株的可溶性细胞提取物中，腺苷酸激酶占比高达40%。已通过定点诱变将突变引入该基因，以替换核苷酸结合环中的氨基酸，该环在许多单核苷酸结合蛋白中高度保守。Lys13突变为Gln的突变体几乎无活性，而Pro9突变为Leu和Gly10突变为Val的突变蛋白对两种底物的Km值增加，Vmax与野生型相似。对这些蛋白的质子核磁共振测量表明，Pro9突变为Leu和Gly10突变为Val的突变体似乎发生了主要的结构变化。根据腺苷酸激酶的动力学机制和该蛋白的三维结构对结果进行了讨论。
