A two-step chemical mechanism for ribosome-catalysed peptide bond formation

摘要

核糖体的催化中心完全由RNA组成。这意味rn着，它没有在能够帮助催化的蛋白中所见的不rn同侧基，所以过去人们认为它的功能仅仅是将rn信使RNA和带电的转移RNA带到附近。但事实rn并不是这样的。对一个“肽基转移RNA”的反rn应中心内5个位置上的动态同位素效应所做的rn一项分析显示，核糖体还能通过修饰限速过渡rn状态来增强对肽键形成的催化。这一发现对于rn有关生命起源以及关于生命从RNA世界的过渡rn的研究有参考意义。%The chemical step of natural protein synthesis, peptide bond formation, is catalysed by the large subunit of the ribosome. Crystal structures have shown that the active site for peptide bond formation is composed entirely of RNA~1. Recent work has focused on how an RNA active site is able to catalyse this fundamental biological reaction at a suitable rate for protein synthesis. On the basis of the absence of important ribosomal functional groups~2 , lack of a dependence on pH~3, and the dominant contribution of entropy to catalysis4, it has been suggested that the role of the ribosome is limited to bringing the substrates into close proximity. Alternatively, the importance of the 2'-hydroxyl of the peptidyl-transfer RNA~5 and a Bronsted coefficient near zero~6 have been taken as evidence that the ribosome coordinates a proton-transfer network. Here we report the transition state of peptide bond formation, based on analysis of the kinetic isotope effect at five positions within the reaction centre of a peptidyl-transfer RNA mimic. Our results indicate that in contrast to the uncatalysed reaction, formation of the tetrahedral intermediate and proton transfer from the nudeophilic nitrogen both occur in the rate-limiting step. Unlike in previous proposals, the reaction is not fully concerted; instead, breakdown of the tetrahedral intermediate occurs in a separate fast step. This suggests that in addition to substrate positioning, the ribosome is contributing to chemical catalysis by changing the rate-limiting transition state.