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

The active site of the ribosome, the peptidyl transferase centre, catalyses two reactions, namely, peptide bond formation between peptidyl-tRNA and aminoacyl-tRNA as well as the release-factor-dependent hydrolysis of peptidyl-tRNA. Unlike peptide bond formation, peptide release is strongly impaired by mutations of nucleotides within the active site, in particular by base exchanges at position A2602 (refs 1, 2). The 2'-OH group of A76 of the peptidyl-tRNA substrate seems to have a key role in peptide release(3). According to computational analysis(4), the 2'-OH may take part in a concerted 'proton shuttle' by which the leaving group is protonated, in analogy to similar current models of peptide bond formation(4-6). Here we report kinetic solvent isotope effects and proton inventories (reaction rates measured in buffers with increasing content of deuterated water, D(2)O) of the two reactions catalysed by the active site of the Escherichia coli ribosome. The transition state of the release factor 2 (RF2)-dependent hydrolysis reaction is characterized by the rate-limiting formation of a single strong hydrogen bond. 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(5). Thus, the ribosome supports different rate-limiting transition states for the two reactions that take place in the peptidyl transferase centre.