ENERGETICS OF CATALYSIS BY RIBONUCLEASES

核糖核酸酶的催化能量

基本信息

批准号：
6298202
负责人：
RONALD T RAINES
金额：
$ 0.75万
依托单位：
UNIVERSITY OF WISCONSIN-MADISON
依托单位国家：
美国
项目类别：
财政年份：
1999
资助国家：
美国
起止时间：
1999-03-01 至 2000-02-29
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6298202
关键词：
biological products biomaterials biomedical resource enzymes nuclear magnetic resonance spectroscopy proteins

项目摘要

Ribonucleases catalyze the hydrolysis of the P-O5? bond in RNA. This reaction occurs in two steps: transphosphorylation of RNA to a 2?,3?-cyclic phosphodiester intermediate and hydrolysis of this intermediate to a 3?-phosphomonoester. 31P NMR spectroscopy was used to monitor the accumulation of the 2?,3?-cyclic phosphodiester intermediate during the transphosphorylation and hydrolysis reactions catalyzed by various ribonucleases and by small molecules. The intermediate was found to accumulate during catalysis by monomeric bovine pancreatic ribonuclease A (RNase A), a dimer and a trimer of RNase A, bovine seminal ribonuclease, RNase T1, barnase, and RNase I. These enzymes, which are of widely disparate phylogenetic origin, released rather than hydrolyzed most of the intermediate formed by transphosphorylation of RNA. In contrast, the intermediate did not accumulate during catalysis by hydroxide ion or imidazole buffer. In the presence of these small molecules, hydrolysis is faster than transphosphorylation. A trapping experiment was used to assess the throughput of the reaction catalyzed by RNase A. [5,6-3H]Uridylyl-3?-5?)adenosine was incubated with RNase A in the presence of excess unlabeled uridine 2?,3?-cyclic phosphodiester, which dilutes the specific radioactivity of any released cyclic intermediate. Only 0.1% of the RNA substrate was found to be both transphos-phorylated and hydrolyzed without dissociating from the enzyme. These results suggest that ribonucleases have evolved primarily to catalyze RNA transphosphorylation and not RNA hydrolysis.

核糖核酸酶催化 P-O5? 的水解？ RNA 中的键。该反应分两步进行：RNA 转磷酸化为 2?,3?-环状磷酸二酯中间体及其水解 3′-磷酸单酯的中间体。使用 31P NMR 光谱监测 2?,3?-环磷酸二酯的积累转磷酸和水解反应期间的中间体由各种核糖核酸酶和小分子催化。这发现单体催化过程中中间体积累牛胰核糖核酸酶 A (RNase A)，二聚体和三聚体 RNase A、牛精液核糖核酸酶、RNase T1、RNA 酶和 RNase I。这些酶具有广泛不同的系统发育起源，释放而不是水解大部分形成的中间体 RNA 的转磷酸化。相比之下，中间体则没有在氢氧根离子或咪唑缓冲液的催化过程中积累。在这些小分子的存在，水解速度比转磷酸化。捕获实验用于评估 RNase A 催化反应的通量。 [5,6-3H]尿苷基-3?-5?)腺苷与RNase A在存在过量未标记的尿苷 2?,3?-环磷酸二酯，它稀释了任何释放的循环的比放射性中间的。仅 0.1% 的 RNA 底物被发现同时存在转磷酸化和水解而不解离酶。这些结果表明核糖核酸酶已经进化主要催化RNA转磷酸化而不是RNA水解。