An Uncharged Amine in the Transition State of the Ribosomal Peptidyl Transfer Reaction

Abstract

The ribosome has an active site comprised of RNA that catalyzes peptide bond formation. To understand how RNA promotes this reaction requires a detailed understanding of the chemical transition state. Here, we report the Brønsted coefficient of the α-amino nucleophile (β nuc) using a series of puromycin derivatives. 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.

The transition state for peptide bond formation reveals the ribosome as a water trap

“…The six-membered transition state reported here has a higher barrier, but this is most likely because the 2′-and 3′-oxygen atoms could not be adequately solvated and EVB simulations (15,17) show that the auxiliary water also stabilizes a six-membered TS. Regardless of the proton shuttle mechanism, the transition states all have an essentially uncharged amine, which is in agreement with Brønstedt plots for this reaction (25), and they are all late. It thus takes more energy to break the C − O bond than for the amine to approach the ester carbonyl carbon.…”

“…Strobel and coworkers measured 15 N kinetic isotope effects (KIEs) for the reaction with an A-site substrate analog, a CC-puromycin derivative, on the 50S ribosomal subunit and suggested an early TS based on these experiments (24). Subsequently reported Brønstedt plots (25) showed a very weak dependence on nucleophile pK a , compatible with both early and late transition states, and were interpreted in terms of an essentially neutral nucleophile in the TS. Computer simulations employing the empirical valence bond (EVB) method as well as quantum chemical ab initio calculations on a small model system (15)(16)(17), on the other hand, indicated a late TS that is mostly associated with elongation of the ester C − O3 0 bond.…”

“…As mentioned above, Brønstedt plots suggest an essentially uncharged amine in the TS (25), and it is still not clear whether there is an accumulation of negative charge on the ester carbonyl oxygen. That is, can we speak of an "oxyanion" in this reaction?…”

Modulation of the Rate of Peptidyl Transfer on the Ribosome by the Nature of Substrates

“…Rapid peptide bond formation requires fulllength tRNA in both A and P sites, and the reaction rate is influenced by the length of the tRNA fragments when model substrates are used (8, 10 -14). The reaction rate is also influenced by the nature of the amino acid side chain of the A-site substrate (13,(15)(16)(17), but is independent of the nucleophilicity of the attacking amino group in model substrates (18). Moreover, the length of the peptidyl chain and the nature of the C-terminal amino acid of the peptidyl-tRNA in the P site seem to have an effect (10,12,13,19).…”

How RNA acts as a nuclease: some mechanistic comparisons in the nucleolytic ribozymes

“…Ribozymes are catalytic RNA molecules, some of which catalyse extremely important cellular reactions, notably processing tRNA and mRNA (1) and condense amino acids to form polypeptides (2)(3)(4)(5). Ribozymes are very widespread; for example ribonuclease P (6, 7) processes precursor tRNA in all domains of life.…”