Cooperative Interactions in the Hammerhead Ribozyme Drive pKa 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 pKa 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 plc 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 pKa 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-pi 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.