A Two-Step Chemical Mechanism for Ribosome-Catalyzed Peptide Bond Formation

The chemical step of protein synthesis, peptide bond formation, is catalyzed by the large subunit of the ribosome. Crystal structures have demonstrated 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 catalyze this fundamental biological reaction at a suitable rate for protein synthesis. Based on 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-tRNA(5) and a Bronsted coefficient near zero(6) were taken as evidence that the ribosome coordinates a proton-transfer network. Here we report the transition state of peptide bond formation based upon kinetic isotope effect analysis at five positions at the reaction center of a peptidyl-tRNA mimic. Our results indicate that in contrast to the uncatalyzed reaction, formation of the tetrahedral intermediate and proton-transfer from the nucleophilic nitrogen both occur in the rate-limiting step. Unlike 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.