Weinstein L B, Jones B C, Cosstick R, Cech T R
Howard Hughes Medical Institute, Department of Chemistry and Biochemistry, University of Colorado, Boulder 80309-0215, USA.
Nature. 1997 Aug 21;388(6644):805-8. doi: 10.1038/42076.
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. In the self-splicing group I intron from Tetrahymena thermophila, several divalent metals can serve structural roles, but only Mg2+ and Mn2+ promote splice-site cleavage and exon ligation. 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. Here we use 3'-(thioinosylyl)-(3'-->5')-uridine, 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.
尽管只有一部分蛋白质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并具备催化活性。在嗜热四膜虫的自我剪接I组内含子中，几种二价金属可起到结构作用，但只有Mg2+和Mn2+能促进剪接位点切割和外显子连接。一项关于类似于鸟苷对5'-剪接位点切割的核酶反应的研究，发现了第一种在催化中起明确作用的金属离子。用硫取代离去基团的3'-氧会导致金属特异性转换，这表明离去基团与金属离子之间存在相互作用。在此，我们使用3'-(硫代肌苷基)-(3'→5')-尿苷（IspU）作为模拟外显子连接反应的底物。该反应需要添加亲硫金属离子（Cd2+或Mn2+），这为剪接该步骤中金属离子对离去基团的稳定作用提供了证据。基于微观可逆性原理，这种金属离子在剪接的第一步激活鸟苷的亲核3'-羟基，支持了双金属离子活性位点模型。
