Kinetic isotope effect analysis of the ribosomal peptidyl transferase reaction

The ribosome is the macromolecular machine responsible for protein synthesis in all cells. Here, we establish a kinetic framework for the 50S modified fragment reaction that makes it possible to measure the kinetic effects that result from isotopic substitution in either the A or P site of the ribosome. This simplified peptidyl transferase assay follows a rapid equilibrium random mechanism in which the reverse reaction is nonexistent and the forward commitment is negligible. A normal effect (1.009) is observed for N-15 substitution of the incoming nucleophile at both low and high pH. This suggests that the first irreversible step is the formation of the tetrahedral intermediate. The observation of a normal isotope effect that does not change as a function of pH suggests that the ribosome promotes peptide bond formation by a mechanism that differs in its details from an uncatalyzed aminolysis reaction in solution. This implies that the ribosome contributes chemically to catalysis of peptide bond formation.