Different substrate-dependent transition states in the active site of the ribosome

The active site of the ribosome, the peptidyl transferase center, catalyzes two reactions, peptide bond formation between peptidyl-tRNA and aminoacyl-tRNA as well as the release factor (RF)-dependent hydrolysis of peptidyl-tRNA. The 2′-OH group of A76 of the P-site tRNA substrate seems to play a key role (Brunelle et al., 2008), possibly by taking part in a concerted proton shuttle mechanism for the protonation of the leaving group, in analogy with current models of peptide bond formation (Trobro and Aqvist, 2009; Wallin and Aqvist, 2010). Here we report the kinetic solvent isotope effects and the proton inventories of the two reactions. The transition state of the RF2-dependent hydrolysis reaction is characterized by the rate-limiting formation of a single strong hydrogen bond that involves the hydrolytic water molecule. This finding argues against a concerted proton shuttle in the transition state of the hydrolysis reaction. In comparison, the proton inventory for peptide bond formation indicates the rate-limiting formation of three hydrogen bonds with about equal contributions, consistent with a concerted eight-membered proton shuttle in the transition state. Thus, the ribosome supports different rate-limiting transition states for the two reactions that take place in the peptidyl transferase center.