The catalytic mechanism of peptide bond formation on the ribosome is not known. The crystal structure of 50S ribosomal subunits shows that the catalytic center consists of RNA only and suggests potential catalytic residues. Here we report rapid kinetics of the peptidyl transferase reaction with puromycin at rates up to 50 s(-1). The rate-pH profile of the reaction reveals that protonation of a single ribosomal residue (pK(a) = 7.5), in addition to protonation of the nucleophilic amino group, strongly inhibits the reaction (>100-fold). The A2451U mutation within the peptidyl transferase center has about the same inhibitory effect. These results suggest a contribution to overall catalysis of general acid-base and/or conformational catalysis involving an ionizing group at the active site.

Biochemical and crystallographic evidence suggests that 23S ribosomal RNA (rRNA) is the catalyst of peptide bond formation. To explore the mechanism of this reaction, we screened for nucleotides in Escherichia coli 23S rRNA that may have a perturbed pKa (where Ka is the acid constant) based on the pH dependence of dimethylsulfate modification. A single universally conserved A (number 2451) within the central loop of domain V has a near neutral pKa of 7.6 +/- 0.2, which is about the same as that reported for the peptidyl transferase reaction. In vivo mutational analysis of this nucleotide indicates that it has an essential role in ribosomal function. These results are consistent with a mechanism wherein the nucleotide base of A2451 serves as a general acid base during peptide bond formation.

Urea destabilizes helical and folded conformations of nucleic acids and proteins, as well as protein-nucleic acid complexes. To understand these effects, extend previous characterizations of interactions of urea with protein functional groups, and thereby develop urea as a probe of conformational changes in protein and nucleic acid processes, we obtain chemical potential derivatives (μ23 = dμ2/dm3) quantifying interactions of urea (component 3) with nucleic acid bases, base analogs, nucleosides and nucleotide monophosphates (component 2) using osmometry and hexanol-water distribution assays. Dissection of these μ23 yields interaction potentials quantifying interactions of urea with unit surface areas of nucleic acid functional groups (heterocyclic aromatic ring, ring methyl, carbonyl and phosphate O, amino N, sugar (C,O)); urea interacts favorably with all these groups, relative to interactions with water. Interactions of urea with heterocyclic aromatic rings and attached methyl groups (as on thymine) are particularly favorable, as previously observed for urea-homocyclic aromatic ring interactions. Urea m-values determined for double helix formation by DNA dodecamers near 25°C are in the range 0.72 to 0.85 kcal mol−1 m−1 and exhibit little systematic dependence on nucleobase composition (17–42% GC). Interpretation of these results using the urea interaction potentials indicates that extensive (60–90%) stacking of nucleobases in the separated strands in the transition region is required to explain the m-value. Results for RNA and DNA dodecamers obtained at higher temperatures, and literature data, are consistent with this conclusion. This demonstrates the utility of urea as a quantitative probe of changes in surface area (ΔASA) in nucleic acid processes.