The G2447A mutation does not affect ionization of a ribosomal group taking part in peptide bond formation

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Abstract
Peptide bond formation on the ribosome is catalyzed by RNA. Kinetic studies using Escherichia coli ribosomes have shown that catalysis (>105-fold overall acceleration) is due to a large part to substrate positioning. However, peptide bond formation is inhibited ~100-fold by protonation of a ribosomal group with pKa=7.5, indicating either a contribution of general acid-base catalysis or inhibition by a pH-dependent conformational change within the active site. The function of a general base has been attributed to A2451 of 23S rRNA, and a charge relay system involving G2447 has been postulated to bring about the extensive pKa shift of A2451 implied in the model. Using a rapid kinetic assay, we found that the G2447A mutation, which has essentially no effect on cell growth, lowers the rate of peptide bond formation about 10-fold and does not affect the ionization of the ribosomal group with pKa=7.5 taking part in the reaction. This result does not support the proposed charge relay mechanism involving G2447 and the role of A2451 as general base in the catalysis of peptide bond formation.