Cooperative Interactions in the Hammerhead Ribozyme Drive pK_a Shifting of G12 and Its Stacked Base C17

General acid-base catalysis is a key mechanistic strategy in protein and RNA enzymes. Ribozymes use hydrated metal ions, nucleobases, and organic cofactors to carry this out. In most small ribozymes, a guanosine is positioned to participate in proton transfer with the nucleophilic 2′-OH. The unshifted pK_a values for nucleobases and solvated metal ions are far from neutrality, however, and thus nonideal for general acid-base catalysis. Herein, evidence is provided for cooperative interaction in the hammerhead ribozyme among the guanine that interacts with the nucleophilic 2′-OH, G12, the -1 nucleobase C17, and Mg²⁺ ions. We introduce global fitting for analyzing ribozyme rate-pH data parametric in Mg²⁺ concentration and benchmark this method on data from the hepatitis delta virus ribozyme. We then apply global fitting to new rate-pH data for the hammerhead ribozyme using a minimal three-dimensional, four-channel cooperative model. The value for the pK_a of G12 that we obtain is channel-dependent and varies from 8.1 to 9.9, shifting closest toward neutrality in the presence of two cationic species: C17H⁺ and a Mg²⁺ ion. The value for the pK_a of the -1 nucleotide, C17, is increased a remarkable 3.5-5 pK_a units toward neutrality. Shifting of the pK_a of C17 appears to be driven by an electrostatic sandwich of C17 between carbonyl groups of the 5′-neighboring U and of G12 and involves cation-π interactions. Rate-pH profiles reveal that the major reactive channel under biological Mg²⁺ and pH involves a cationic C17 rather than a second metal ion. Substitution of a cationic base for a metal underscores the versatility of RNA.