pKa of adenine 2451 in the ribosomal peptidyl transferase center remains elusive

The universally conserved A2451 of 23S rRNA has been proposed to participat e directly in the catalysis of peptide bond formation in the ribosomal pept idyl transferase center. An unusually high, near neutral, pKa of A2451 is a prerequisite for its action as a general acid-base catalyst. Increased rea ctivity of A2451 to dimethylsulfate (DMS) at pH 8.5 compared to pH 6.5 was taken as evidence that the pKa of this nucleotide falls within this pH rang e. Structural data suggested that the interaction between A2451 and G2447 i n the ribosome is responsible for A2451 pKa perturbation. In contrast to ex pectation, our studies did not show pH dependence of A2451 dimethylsulfate modification in ribosomes of Thermus aquaticus and Mycobacterium smegmatis. Other rRNA regions, however, showed major alterations in DMS reactivity at pH 8.5 compared to pH 6.5, suggesting that conformational rearrangements i n the structure of the large ribosomal subunit may occur upon the pH shift. The G2447U mutant of M. smegmatis was viable, indicating that the G2447-A2 451 interaction is not critical for the ribosome function. We concluded tha t the proposed unusual pKa of A2451, if existing, may not be crucial for th e ribosome activity and that the previously reported pH-dependent alteratio ns in the DMS modification of A2451 do not necessarily reveal an unusual pK a of this nucleotide.