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

核糖核酸酶催化水解的P-O 5？ RNA中的键。 该反应分两步发生：RNA转磷酸化为 两个？三个？环状磷酸二酯中间体及其水解方法 中间到3？-磷酸单酯 使用31 P NMR光谱 为了监测2？的积累，三个？环状磷酸二酯 转磷酸化和水解反应过程中的中间体 由各种核糖核酸酶和小分子催化。 的 发现中间体在催化过程中通过单体 牛胰腺核糖核酸酶A（RNase A）， RNase A、牛精液核糖核酸酶、RNase T1、芽孢杆菌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的中间体形成的 RNA转磷酸化。 相比之下，中间体没有 通过氢氧根离子或咪唑缓冲剂在催化过程中积累。 在 在这些小分子的存在下，水解速度比 转磷酸作用 捕获实验用于评估 RNase A催化的反应的通量。 [5，6 - 3 H]尿苷基-3？-五？）腺苷与核糖核酸酶A孵育， 存在过量的未标记尿苷2？，三个？环状磷酸二酯， 它稀释了任何释放的环状放射性物质的放射性 中间体 只有0.1%的RNA底物被发现是两种 转磷酸化和水解，而不从 酵素 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。

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.