Catalytic importance of a protonated adenosine in the hairpin ribozyme active site

The hairpin ribozyme accelerates the rate of phosphodiester transfer reactions by at least 5 orders of magnitude. To achieve this rate enhancement, the active site forms via a substrate helix docking event that constrains the scissile phosphate linkage and positions G8 and A38 for catalysis, both of which have been implicated as sites of proton transfer in general acid-base catalysis. To investigate the functional groups required for hairpin activity, we previously reported a series of nucleotide analogue interference mapping experiments [Ryder, S. P., et al. (2001) RNA 7, 1454-1463]. The critical functional groups implicated in those studies were largely consistent with subsequent X-ray crystal structures, but the lack of A38 interference with 8-azaadenosine (n(8)A), a pK(a) perturbed nucleotide analogue, argued against functional base ionization at this site. This is inconsistent with a transition state crystal structure and other biochemical studies. To address this discrepancy, we investigated the hairpin ribozyme with an expanded set of pK(a) perturbed adenosine analogues containing fluorine. A38 was the only site that showed persistent and strong interference with low pK(a) analogues across a variety of construct/substrate pairs. This interference pattern suggests that A38 base ionization is required for catalytic activity. The lack of n(8)A interference at A38, in spite of its reduced pK(a), likely results from n(8)A stabilization of the docked state, which requires an unusual syn glycosidic base conformation at A38 for active site assembly. The fluorinated adenosine analogues are better suited to identify sites of functional ionization in systems where structural rearrangements are closely coupled to catalytic steps. All pK(a) reduced analogues, including those of the previous study, produce selective interference at A38 when substrates are stably bound and docked, consistent with the importance of base ionization at this site.