Interaction of the basic protrusion of Escherichia coli ribonuclease HI with its substrate
Interaction of the basic protrusion of Escherichia coli ribonuclease HI with its substrate
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DOI：
10.1006/jmbi.1996.0609
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发表时间：
1996-11-15
影响因子：
5.6
通讯作者：
Nakamura, H
中科院分区：
文献类型：
--
作者：
Iwai, S;Wakasa, M;Nakamura, H
为了确定大肠杆菌核糖核酸酶HI的活性位点和底物结合位点之间的实际距离（称为基本突出），使用具有逐渐延伸的突出端的合成寡核苷酸双链体，其中酶促切割被限制在具有2 '-O-甲基核苷的单个位点。通过动力学分析确定酶对每种底物的亲和力。结果发现，当一个核苷酸连接到9碱基对杂交双链体的DNA链的3'端时，亲和力显著增加，然后随着DNA链的进一步延伸而略微增加，而在另一个方向上的链的延伸没有引起变化。当使用其中碱性突起中的三个赖氨酸残基被改变为丙氨酸的突变酶时，没有观察到k（cat）/K-m值的增加。结果表明，为实现生产性结合，底物双链体RNA链3'端至5'端的轴必须与酶活性位点至碱性突起的方向一致。酶-底物复合物中活性位点和基本突起之间的距离比建模研究中预期的要短。为了模拟基本突起的波动，进行了动态结构细化，称为正常模式分析。(C)1996年学术出版社
In order to determine the actual distance between the active site and the substrate binding site, termed the basic protrusion, of Escherichia coli ribonuclease HI, synthetic oligonucleotide duplexes with gradually extended overhangs were used, in which the enzymatic cleavage was restricted to a single site with 2'-O-methylnucleosides. The affinity of the enzyme for each substrate was determined by kinetic analysis. It was found that the affinity increased markedly when one nucleotide was attached to the 3' end of the DNA strand of the nine-base-pair hybrid duplex and then increased slightly as the DNA strand was extended further, whereas elongation of the strand in the other direction caused no change. When a mutant enzyme, in which three lysine residues in the basic protrusion were altered to alanine, was used, no increase in the k(cat)/K-m value was observed. The results indicate that, for the productive binding, the axis from the 3' to the 5' end of the RNA strand of the substrate duplex must be oriented in agreement with the direction from the active site to the basic protrusion of the enzyme. The distance between the active site and the basic protrusion in the enzyme-substrate complex was shorter than that anticipated in modeling studies. A dynamic structure refinement, referred to as the normal mode analysis, was carried out in order to simulate the fluctuations of the basic protrusion. (C) 1996 Academic Press Limited