A second catalytic metal ion in a group I ribozyme
A second catalytic metal ion in a group I ribozyme
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作者：
Weinstein, LB;Jones, BCNM;Cech, TR
尽管只有一部分蛋白酶的催化功能依赖于金属离子的存在，但所有天然存在的RNA酶都需要金属离子来稳定其结构和催化能力（1）。在来自嗜热四膜虫的自我剪接I组内含子中（2），几种二价金属可以起到结构作用，但只有Mg 2+和Mn 2+促进剪接位点切割和外显子连接（3，4）。一项对类似于鸟苷切割5 '-剪接位点的核酶反应的研究发现了第一个在催化中具有决定性作用的金属离子。用硫取代离去基团的3 '-氧导致金属特异性开关，表明离去基团和金属离子之间的相互作用（5）。在这里，我们使用3 '-（硫代葡萄糖酰基）-（3' -> 5 '）-尿苷（6），IspU，作为模拟外显子连接的反应中的底物。活性需要添加亲硫金属离子（Cd 2+或Mn 2+），这为在剪接步骤中通过金属离子稳定离去基团提供了证据。基于微观可逆性原理，该金属离子在剪接的第一步中激活鸟苷的亲核3 '-羟基，支持双金属离子活性位点模型（7）。
Although only a subset of protein enzymes depend on the presence of a metal ion for their catalytic function, all naturally occurring RNA enzymes require metal ions to stabilize their structure and for catalytic competence(1). In the self-splicing group I intron from Tetrahymena thermophila(2), several divalent metals can serve structural roles, but only Mg2+ and Mn2+ promote splice-site cleavage and exon ligation(3,4). A study of a ribozyme reaction analogous to 5'-splice-site cleavage by guanosine uncovered the first metal ion with a definitive role in catalysis. Substitution of the 3'-oxygen of the leaving group with sulphur resulted in a metal-specificity switch, indicating an interaction between the leaving group and the metal ion(5). Here we use 3'-(thioinosylyl)-(3' --> 5')-uridine(6), IspU, as a substrate in a reaction that emulates exon ligation. Activity requires the addition of a thiophilic metal ion (Cd2+ or Mn2+), providing evidence for stabilization of the leaving group by a metal ion in that step of splicing. Based on the principle of microscopic reversibility, this metal ion activates the nucleophilic 3'-hydroxyl of guanosine in the first step of splicing, supporting the model of a two-metal-ion active site(7).