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

Abstract

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 catalysis(4), 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 nucleophilic 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.