COMPARISONS:39K TRANSVERSE RELAXATION IN VITRO SOLUTIONS & E COLI K 12 CYTOPLASM

比较：39K 横向松弛体外解决方案

• 批准号: 6120992
• 负责人: M. THOMAS RECORD
• 金额: $ 0.13万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 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/6120992
• 关键词: biological products; biomaterials; biomedical resource; enzymes; nuclear magnetic resonance spectroscopy; proteins

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？结合在RNA中。 该反应分两步进行：将RNA转磷酸化为 2？，3？-环磷酸二酯中间体及其水解物 中间体为3？-磷酸单酯。使用31P核磁共振波谱 监测2？，3？-环磷酸二酯的积累 转磷酰化和水解反应的中间体 由各种核糖核酸酶和小分子催化。这个 发现中间体在单体催化过程中积累 牛胰腺核糖核酸酶A(RNaseA)、二聚体和三聚体 核糖核酸酶A、牛精液核糖核酸酶、核糖核酸酶T1、棒状核糖核酸酶和核糖核酸酶I。 这些酶有着截然不同的系统起源， 释放而不是水解大部分由 RNA的转磷酸化。相比之下，中间人则没有。 在氢氧离子或咪唑缓冲液催化过程中积累。在……里面 在这些小分子的存在下，水解率比 转磷酸化。诱捕实验被用来评估 核糖核酸酶A催化的反应通量。 腺苷与核糖核酸酶A孵育 存在过量的未标记尿苷2？，3？-环磷酸二酯， 稀释任何释放的环状化合物的特定放射性 中级的。只有0.1%的RNA底物同时存在 转磷酰化和水解而不解离 酵素。这些结果表明核糖核酸酶已经进化。 主要是催化RNA转磷酸化，而不是RNA水解。