One mechanism by which ribozymes can accelerate biological reactions is by adopting folds that favorably perturb nucleobase ionization. Herein we used Raman crystallography to directly measure pKa values for the Ade38 N1 imino group of a hairpin ribozyme in distinct conformational states. A transition-state analogue gave a pKa value of 6.27 � 0.05, which agrees strikingly well with apparent pKa values obtained from pH-rate analyses. To identify the chemical attributes that contribute to the shifted pKa, we determined crystal structures of hairpin ribozyme variants containing single-atom substitutions at the active site and measured their respective Ade38 N1 pKa values. This approach led to the identification of a single interaction in the transition-state conformation that elevates the base pKa > 0.8 log unit relative to the precatalytic state. The agreement of the microscopic and macroscopic pKa values and the accompanying structural analysis supports a mechanism in which Ade38 N1(H+) functions as a general acid in phosphodiester bond cleavage. Overall the results quantify the contribution of a single electrostatic interaction to base ionization, which has broad relevance for understanding how RNA structure can control chemical reactivity.

Transition-state interactions that shift nucleobase ionization toward neutrality in the hairpin ribozyme

245th National Meeting (2013)

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