Investigation of the chemical mechanism of ribosomal peptidyl transferase: A transition state charge analysis

The ribosome is the macromolecular machine that catalyzes cellular protein synthesis. Peptidyl transfer (PT) is the reaction catalyzed during translation that produces new peptide bonds. This biologically essential reaction occurs within an active site comprised of RNA. Much is known about the peptidyl transferase reaction; however the exact nature of the chemical mechanism is still debated. This debate cannot be settled until a detailed understanding of the PT transition state is obtained. Here, I report my investigation of the charges on the nucleophile and leaving group of the PT transition state using Bronsted analysis. The transition state charge is derived from the Bronsted coefficient which reports the change in charge on an atom as the reaction proceeds from the ground state to the transition state. Using a series of puromycin derivatives we determined the Bronsted coefficient of the nucleophile (beta nuc). Both 50S subunit and 70S ribosome catalyzed reactions displayed linear free-energy relationships with slopes close to zero under conditions where chemistry is rate limiting. These results indicate that at the transition state the nucleophile is neutral in the ribosome catalyzed reaction, in contrast to the substantial positive charge reported for typical uncatalyzed aminolysis reactions. This suggests that the ribosomal transition state involves deprotonation to a degree commensurate with nitrogen-carbon bond formation. Such a transition state is significantly different from that of uncatalyzed aminolysis reactions in solution. When combined with other experimentally obtained transition state data, the betanuc value reported here makes specific predictions about the charge on the PT 03'-leaving group in the transition state. I pursued the development of a new 50S ribosomal assay to determine the Bronsted coefficient of the leaving group (betalg). The proposed assay required the design and synthesis of a new A-site substrate that we subsequently demonstrated bound exclusively to the A-site.