Investigation of the pKa 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.

Automated summary

This study used molecular dynamics to calculate the pK(a) of the nucleophilic O2' in the hairpin ribozyme at 18.5+/-0.8, higher than the reference compound, and identified the importance of monovalent ions and the exocyclic amine in impacting the pK(a) of the O2'. The study suggests that the pK(a) of the O2' is elevated in the ground state and decreases during the reaction process due to positioning and metal ion interactions.