Investigation of the pK_a of the Nucleophilic O2′ of the Hairpin Ribozyme

Abstract

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.

Isotope Labels Combined with Solution NMR Spectroscopy Make Visible the Invisible Conformations of Small-to-Large RNAs

“…As with purine C8 labeling, adenine C2 can be readily labeled. Labeling C2 is attractive because its chemical shift can monitor protonation at adenine N1, which cannot be achieved with 15 N NMR experiments due to severe line broadening. − Unlike the environments of single-stranded RNA, those in structured RNAs can shift the p K a values of protonated adenosine or cytidines significantly toward neutrality, serving both catalytic and structural functions in RNA enzymes. − The 13 C isotope can be incorporated at the purine C2 site starting with 5-aminoimidazole-4-carboxamide (AICA) and ethylsodium 13 C-xanthate to form [2- 13 C]-hypoxanthine, [2- 13 C]-adenine, or [2- 13 C]-guanine …”

The role of Na+ in catalysis by the 8–17 DNAzyme

“…Recently, it has been suggested that Na + may increase the acidity of the 2′-OH group, helping with deprotonation prior or during the nucleophilic attack in the mechanism of the hairpin ribozyme. 34 In the mechanism of the hammerhead ribozyme, Mg 2+ activates the general base G12 promoting γ-catalysis. 21,35,36 Also, it has been suggested that Mg 2+ binding increases the acidity of the 2′-OH group of G8 promoting its character as a general acid (δ-catalysis) under near-physiological conditions, 20,[37][38][39] although other pathways can be viable under different conditions for this ribozyme.…”