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.

核糖核酸催化P-O5的水解？ RNA中的键。该反应分为两个步骤：RNA转磷酸化 2？，3？ - 环磷酸酯中间和水解中间至3磷脂酯。使用31p NMR光谱法监视2？，3？ - 环状磷酸二酯的积累经磷酸化和水解反应期间的中间体由各种核糖珠和小分子催化。这在单体催化过程中发现中间体积聚牛胰腺核糖核酸酶A（RNase A），二聚体和三聚体 RNase A，牛核糖核酸酶，RNase T1，Barnase和RNaseI。这些酶，具有广泛不同的系统发育起源，释放而不是水解大多数由 RNA的磷酸化。相比之下，中级没有氢氧化离子或咪唑缓冲液在催化过程中积累。在这些小分子的存在，水解比经磷酸化。使用陷阱实验来评估由RNase A催化的反应的吞吐量。 [5,6-3h] uridylyl-3？-5？）腺苷与RNase A一起在存在过多标记的尿苷2？，3？ - 环状磷酸酯，稀释任何释放的循环的特定放射性中间的。发现只有0.1％的RNA底物是跨性别型和水解而无需与酶。这些结果表明核糖核酸已经发展主要是为了催化RNA转磷酸化而不是RNA水解。