Direct raman measurement of an elevated base pKa in the active site of a small ribozyme in a precatalytic conformation

Catalytic RNA mols. can achieve rate acceleration by shifting base pKa values toward neutrality. Prior evidence has suggested that base A38 of the hairpin ribozyme plays an important role in phosphoryl transfer, possibly functioning as a general acid, or by orienting a specific water mol. for proton transfer. To address the role of A38, Raman spectroscopy was used to measure directly the pKa of the N1-imino moiety in the context of hairpin ribozyme crystals representative of a precatalytic conformation. The results revealed that the pKa of A38 is shifted to 5.46 ± 0.05 relative to 3.68 ± 0.06 derived from a ref. soln. of the nucleotide AMP. The elevated pKa correlates well with the first titrn. point of the macroscopic pH-rate profile of the hairpin ribozyme in soln. and strongly supports A38 as a general acid catalyst in bond scission. The results confirm that A38 is protonated before the transition state, which would promote phosphorane development. Overall, the results establish a cogent structure-function paradigm that expands our understanding of how RNA structure can enhance nucleobase reactivity to catalyze biol. reactions.