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Functional structures of nucleic acids and their recognition by proteins

核酸的功能结构及其蛋白质的识别

基本信息

批准号：
07458146
负责人：
SUGIMOTO Naoki
金额：
$ 3.84万
依托单位：
KONAN UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07458146/
关键词：
Ribozyme Gene cleavage Loop structure Lead ion Rare earth ion Nearest-neighbor model Gel electrophoresis Reaction mechanism RecA蛋白質オリゴペプチド DNA結合蛋白質表面プラズモン共鳴円二色性核酸反応速度構造安定性

项目摘要

A ribozyme in an RNA enzyme that can catalyze a biochemical reaction. Because a ribozyme requires a metal ions for a catalytic function, the ribozyme is considered as one of the metalloenzymes. Ribozyme reactions are very sensitive to species and concentration of metal ions, suggesting that divalent metal ions may play important roles for the chemical reaction and structural stabilization of the ribozyme.We have investigated the combined effects of metal ions on both the structural stabilization of the complex between a ribozyme (a leadzyme), CUGGGAGUCC,and a substrate, GGACCGAGCCAG,and the cleavage step in the active center of the complex has been also investigated kinetically.The results showed that the addition of Nd^<3+> in the presence of Pb^<2+> increased significantly the yield of the RNA cleavage reaction by the leadzyme, although other rare earth ions or divalent ions except Pb^<2+> did not promote the reaction. Further, kinetics for the leadzyme reaction was measured at various concentrations of Nd^<3+> and Pb^<2+>. At low concentration rations of Nd^<3+> under a constant total concentration of metal ions, Nd^<3+> increased the stablility of the complex between the leadzyme and the substrate. In contrast, at high concentration ratios of Nd^<3+>, the addition of Nd^<3+> decreased the stability of the complex. The rate constant of the cleavage step was maximized when the ratio of Nd^<3+> to Pb^<2+> was 1 : 1. These results suggest that the complex between the leadzyme and the substrate has binding sites for Nd^<3+> ion taht influence complex stability and catalyze directly the cleavage reaction. On the basis of the results, we propose a two-metal-ion mechanism in which Pb^<2+> and Nd^<3+> play the roles of base and acid catalyst, respectively.

核酶，核酶核糖核酸酶中能催化生化反应的核酶由于核酶需要金属离子的催化作用，所以核酶被认为是金属酶的一种。核酶反应对金属离子的种类和浓度非常敏感，提示二价金属离子可能在核酶的化学反应和结构稳定中起重要作用。我们研究了金属离子对核酶(引导酶)CUGGGAGUCC和底物GGACCGAGCCAG之间的络合物结构稳定的联合作用，并对络合物活性中心的切割步骤进行了动力学研究。结果表明，在Pb^&lt；2+和Gt；除Pb2+外，其他稀土离子或二价离子对铅酶的RNA切割反应没有促进作用，但能显著提高铅酶对RNA切割反应的产率。此外，还测定了不同浓度的Nd3+和Pb2+的铅酶反应动力学。在金属离子总浓度不变的情况下，低浓度的Nd3+与Gt；在金属离子总浓度不变的情况下，Nd3+增加了酶与底物之间络合物的稳定性。相反，在较高的Nd3+浓度比下，Nd3+的加入降低了络合物的稳定性。当Nd3+与Pb2+的比例为1：1时，切割步骤的速率常数最大。这些结果表明，酶与底物之间的络合物具有与Nd^&lt；3+&gt；离子的结合部位，从而影响络合物的稳定性，直接催化切割反应。在此基础上，提出了Pb2+和Nd3+分别充当碱催化剂和酸催化剂的双金属离子机理。