General acid-base catalysis is a key mechanistic strategy that has been shown to be operative in all known small RNA enzymes. Strikingly, the majority of these small ribozymes have a guanine positioned to deprotonate the 2’OH nucleophile. Although the pKa values on the free nucleobases are far removed from neutrality, A and C nucleobases have been shown to shift from ~4 upwards towards neutrality owing to coupling of their cationic protonated states to local anionic environment and/or base pairing. However, such coupling has not been demonstrated to drive population of the anionic deprotonated states of G or U from ~9 downwards towards neutrality. There is strong support in the hammerhead ribozyme that G12 acts as the general base to deprotonate the 2’OH nucleophile, however any coupling of its pKa to local environment is unclear. We probed the mechanism of the hammerhead ribozyme as a function of pH and Mg2+ concentration. We provide evidence that Mg2+ plays a role in populating the functional anionic form of the base. The potential contribution of cooperativity between Mg2+ and G– is evaluated via global fitting of rate-pH data parametric in Mg2+ concentration to an appropriate set of kinetic equations. This analysis reveals cooperativity between Mg2+ and G– that shifts the apparent pKa values downward toward neutrality. The mechanism responsible for the cooperativity in the hammerhead ribozyme may be through-space electrostatic stabilization between G– and Mg2+. These findings are potentially applicable to other small ribozymes, including the hairpin, twister, VS, and glmS ribozymes, which also have a guanine positioned near the 2’OH. The ability of a cooperative interaction with Mg2+ to provide a downward pKa shift for guanine towards neutrality provides a general mechanism by which RNA enzymes can improve catalysis. These studies also suggest that global fitting of rate-pH profiles is a powerful way to uncover pKa coupling.

Uncovering catalytic driving forces in the hammerhead ribozyme: Binding of Mg2+ promotes pKa shifting of guanine towards neutrality

251st National Meeting (2016)

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