The objective of this research proposal is to provide atomic details about the transition state for ribosomal peptide bond formation. Specifically, it is aimed at determining the charge state and bond order of the leaving group for ribosomal peptide bond catalysis. Many catalytic strategies have been proposed for the peptidyl transferase (PT) reaction but a detailed knowledge of the chemical reaction mechanism remains elusive. Elucidating the chemical details of the PT transition state is essential to solving how the rRNA surrounding the active site catalyzes peptide bond formation. Charge state and bond order are two details that would limit the possible mechanistic pathways available for PT catalysis. Particularly, it is important to define these characteristics for the groups relevant to PT chemistry: the nucleophilic a-amine of the A-site amino acid and the leaving group O3'of the P-site tRNA. Recently, we reported that the nucleophile is neutral and demonstrated that the data limits the regions on a reaction trajectory where the transition state would be expected. Further refining the transition state and possibly ruling out entire mechanisms will require the measurement of the Bronsted coefficient for the leaving group. The Bronsted coefficient reports the change in charge on a particular group as the reaction proceeds from the ground state to the transition state, providing an important constraint from which bond order can be deduced. Here, we propose to measure the Bronsted coefficient for the leaving group on the ribosome. This project will require a new A-site substrate to be produced by synthetic organic chemistry (Aim 1). Chemical footprinting and ribosomal kinetic assays (Aim 2) will establish the behavior of the new substrate on the ribosome. Finally, the kinetic data will be utilized to determine the Bronsted coefficient for the leaving group (Aim 3) that will then be employed to analyze possible reaction mechanisms. This project will provide the transition state details required to determine the mechanism of ribosomal PT catalysis. Public Health Relevance: The ribosome is an established therapeutic target but a complete mechanistic understanding of the chemical reaction mechanism for peptide bond formation is currently unavailable. The goals of this research project are to provide important atomic details of the transition state for the peptide bond reaction that are critical for elucidating the reaction mechanism. A comprehensive understanding of how the ribosome makes peptides will aid the design of improved antibiotics, a pressing medical need as the number of multi-drug resistant bacteria increase.
