| Autor: |
Frankel EA; Department of Chemistry, Pennsylvania State University , University Park, Pennsylvania 16802, United States.; Center for RNA Molecular Biology, Pennsylvania State University , University Park, Pennsylvania 16802, United States., Strulson CA; Department of Chemistry, Pennsylvania State University , University Park, Pennsylvania 16802, United States.; Center for RNA Molecular Biology, Pennsylvania State University , University Park, Pennsylvania 16802, United States., Keating CD; Department of Chemistry, Pennsylvania State University , University Park, Pennsylvania 16802, United States., Bevilacqua PC; Department of Chemistry, Pennsylvania State University , University Park, Pennsylvania 16802, United States.; Center for RNA Molecular Biology, Pennsylvania State University , University Park, Pennsylvania 16802, United States.; Department of Biochemistry and Molecular Biology, Pennsylvania State University , University Park, Pennsylvania 16802, United States. |
| Abstrakt: |
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 Mg 2+ ions. We introduce global fitting for analyzing ribozyme rate-pH data parametric in Mg 2+ 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 Mg 2+ 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 Mg 2+ 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. |
