Peptide release on the ribosome involves substrate-Assisted base catalysis

Abstract

Termination of protein synthesis on the ribosome involves hydrolysis of the ester bond between the P-site tRNA and the nascent peptide chain. This reaction occurs in the peptidyl transferase center and is triggered by the class I release factors RF1 and RF2 in prokaryotes. Peptidyl-tRNA hydrolysis is pH-dependent, and experimental results suggest that an ionizable group with pKa > 9 is involved in the reaction. The nature of this group is, however, unknown. To resolve this problem, we conducted density functional theory calculations using a large cluster model of the peptidyl transferase center. Our calculations reveal that peptidyl-tRNA hydrolysis occurs via a base-catalyzed mechanism with a predicted activation energy of 15.8 kcal mol−1, which is in good agreement with experimental data. In this mechanism, the P-site A76 2′-OH group is deprotonated and acts as the general base by activating the nucleophilic water molecule. The energy cost of deprotonating the 2′-hydroxyl group at pH 7.5 is estimated to be about 8 kcal mol−1, on the basis of its experimental pKa in aqueous solution, and this step is predicted to be the source of the observed pH dependence. The proposed mechanism is consistent not only with experimentally derived activation energies but also with the observed kinetic solvent isotope effect.