Determining the mechanism of peptidyl transferase in the 50S ribosomal subunit

The ribosome is the macromolecular machine responsible for all protein synthesis in cells. It catalyzes peptide bond formation between the amino-acyl tRNA and the peptidyl-tRNA. Here I report the framework for kinetic analysis of the 50S ribosomal subunit using a modified version of the fragment assay which utilizes two small synthetic substrates and 50S ribosomal subunits. The modified fragment assay can be used to the characterize the peptidyl transferase transition state. Towards this goal a heavy atom nitrogen substitution at the a-amino group of the A-site substrate was analyzed. It showed a small normal isotope effect suggesting that the transition state of the reaction lies very close to the substrate. Multiple pKa perturbed A-site substrates were used to test the pH dependence of the 50S subunit. The pH rate profile analysis is consistent with the kinetic isotope effect studies and suggests there is a ionizable functional group in the 50S ribosomal subunit. The results presented here give a better understanding of how the ribosome catalyzes peptide bond formation and the role the 50S ribosomal subunit plays in the chemical step of this reaction. These studies provide a basis for further analysis aimed at completely describing the transition state of peptide bond formation.