Kinetic framework for ligation by an efficient RNA ligase ribozyme
Kinetic framework for ligation by an efficient RNA ligase ribozyme
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DOI：
10.1021/bi992654u
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发表时间：
2000-03-21
期刊：
影响因子：
2.9
通讯作者：
Bartel, DP
中科院分区：
文献类型：
--
作者：
Bergman, NH;Johnston, WK;Bartel, DP
先前从随机序列中分离的I类RNA连接酶核酶进行有效的RNA连接反应。它连接两个底物RNA，促进一个底物的3 '-羟基攻击另一个底物的5'-三磷酸，并释放焦磷酸。这种连接反应与RNA聚合酶催化的反应有相似之处。使用稳态动力学测量和脉冲追逐/pH跳跃实验的数据，我们已经构建了两个版本的I类连接酶，命名为207吨和210吨的最小动力学框架。对于两种连接酶以及自连接亲本核酶，化学步骤的速率常数（k（c））与pH在5.7-8.0范围内呈对数线性。在生理pH下，k（c）为100 min（-1），这一数值与报道的最快的天然核酶相似。在较高的pH值下，207 t和210 t的产品释放量均受到限制。210 t核酶具有较快的产物释放，其多重转换速率（k（cat）= 360 min（-1），pH9.0）超过了207 t和其它报道的核酶反应。210吨核酶的动力学框架描述了这种催化的局限性，并提出了如何使用设计或组合方法来改进关键步骤。
The class I RNA ligase ribozyme, isolated previously from random sequences, performs an efficient RNA ligation reaction. It ligates two substrate RNAs, promoting the attack of the 3'-hydroxyl of one substrate upon the 5'-triphosphate of the other substrate with release of pyrophosphate. This ligation reaction has similarities to the reaction catalyzed by RNA polymerases. Using data from steady-state kinetic measurements and pulse-chase/pH-jump experiments, we have constructed minimal kinetic frameworks for two versions of the class I ligase, named 207t and 210t. For both ligases, as well as for the self-ligating parent ribozyme, the rate constant for the chemical step (k(c)) is log-linear with pH in the range 5.7-8.0. At physiological pH, the k(c) is 100 min(-1), a value similar to these reported for the fastest naturally occurring ribozymes. At higher pH, product release is limiting for both 207t and 210t. The 210t ribozyme, with its faster product release, attains multiple-turnover rates (k(cat) = 360 min(-1), pH 9.0) exceeding those of 207t and other reported ribozyme reactions. The kinetic framework for the 210t ribozyme describes the limits of this catalysis and suggests how key steps can be targeted for improvement using design or combinatorial approaches.