Investigation of the pK_a of the nucleophilic O2' of the hairpin ribozyme

Small ribozymes cleave their RNA phosphodiester backbone by catalyzing a transphosphorylation reaction wherein a specific O2' functions as the nucleophile. While deprotonation of this alcohol through its acidification would increase its nucleophilicity, little is known about the pK_a of this O2' in small ribozymes, in part because high pK_a's are not readily accessible experimentally. Herein, we turn to molecular dynamics to calculate the pK_a of the nucleophilic O2' in the hairpin ribozyme and to study interactions within the active site that may impact its value. We estimate the pK_a of the nucleophilic O2' in the wild-type hairpin ribozyme to be 18.5 ± 0.8, which is higher than the reference compound, and identify a correlation between proper positioning of the O2' for nucleophilic attack and elevation of its pK_a. We find that monovalent ions may play a role in depression of the O2' pK_a, while the exocyclic amine appears to be important for organizing the ribozyme active site. Overall, this study suggests that the pK_a of the O2' is raised in the ground state and lowers during the course of the reaction owing to positioning and metal ion interactions.