Alterations in intracellular deoxyribonucleotide levels of mutationally altered ribonucleotide reductases in Escherichia coli
Alterations in intracellular deoxyribonucleotide levels of mutationally altered ribonucleotide reductases in Escherichia coli
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大肠杆菌中突变核糖核苷酸还原酶的胞内脱氧核糖核苷酸水平的变化
DOI：
10.1128/jb.164.3.1194-1199.1985
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发表时间：
1985
影响因子：
3.2
通讯作者：
B. Sjöberg
中科院分区：
文献类型：
--
作者：
A. Platz;M. Karlsson;S. Hahne;S. Eriksson;B. Sjöberg
编码大肠杆菌核糖核苷酸还原酶的四个重组质粒克隆（pPS305、pPS308、pPS317 和 pPS319）已在体内和体外进行了表征。每个克隆都携带不同的错义突变，影响 B1 亚基。对脱氧核糖核苷三磷酸池进行测量。携带野生型质粒 pPS2 的细胞过量产生核糖核苷酸还原酶 10 至 20 倍。由于酶水平升高，脱氧核糖核苷酸池大约高出三倍。所有四个突变克隆都显示出受干扰的脱氧核糖核苷酸库。体外研究涉及亲和介质上的色谱法、酶活性的测量和各种底物和效应分子的变构调节，以及在 dTTP 存在下的直接光亲和标记。克隆pPS305和pPS308被证明编码催化缺陷酶，而克隆pPS317和pPS319被证明编码变构改变的酶。因此，所表征的错义突变可以定位于涉及底物特异性调节的区域或蛋白 B1 的活性位点。在含有变构缺陷克隆 pPS317 和 pPS319 的细胞中发现的脱氧核糖核苷酸库的改变清楚地证明了大肠杆菌细胞中蛋白质 B1 的变构控制的体内意义。
Four recombinant plasmid clones (pPS305, pPS308, pPS317, and pPS319) coding for Escherichia coli ribonucleotide reductase have been characterized in vivo and in vitro. Each clone carried a different missense mutation affecting the B1 subunit. Measurements were made of deoxyribonucleoside triphosphate pools. Cells carrying the wild-type plasmid, pPS2, overproduced ribonucleotide reductase 10 to 20 times. As a consequence of this elevated enzyme level, the deoxyribonucleotide pools were approximately three times higher. All four mutant clones showed disturbed deoxyribonucleotide pools. The in vitro studies involved chromatography on affinity media, measurements of enzyme activity and allosteric regulation with a variety of substrates and effector molecules, and direct photoaffinity labeling in the presence of dTTP. Clones pPS305 and pPS308 were shown to code for catalytically defective enzymes, whereas clones pPS317 and pPS319 were shown to code for allosterically altered enzymes. The characterized missense mutations can thus be localized to areas involved in regulation of the substrate specificity or to the active site of protein B1. The alteration of the deoxyribonucleotide pools found in cells containing the allosterically defective clones pPS317 and pPS319 clearly demonstrated in vivo significance for the allosteric control of protein B1 in E. coli cells.