Adenine protonation in domain B of the hairpin ribozyme

Protein enzymes often use ionizable side chains, such as histidine, for gen eral acid-base catalysis because the imidazole pK(a) is near neutral pH. RN A enzymes, on the other hand, are comprised of nucleotides which do not hav e apparent pK(a) values near neutral pH. Nevertheless, it has been recently shown that cytidine and adenine protonation can play an important role in both nucleic acid structure and catalysis. We have employed heteronuclear N MR methods to determine the pK(a) values and time scales of chemical exchan ges associated with adenine protonation within the catalytically essential B domain of the hairpin ribozyme. The large, adenine-rich internal loop of the B domain allows us to determine adenine pK(a) values for a variety of n on-Watson-Crick base pairs. We find that adenines within the internal loop have pK(a) values ranging from 4.8 to 5.8, significantly higher than the fr ee mononucleotide pK(a) of 3.5. Adenine protonation results in potential ch arge stabilization, hydrogen bond formation, and stacking interactions that are expected to stabilize the internal loop structure at low pH. Fast prot on exchange times of 10-50 mus were determined for the well-resolved adenin es. These results suggest that shifted pK(a) values may be a common feature of adenines in non-Watson-Crick base pairs, and identify two adenines whic h may participate in hairpin ribozyme active site chemistry.