Probing general acid catalysis in the hammerhead ribozyme.

Recent crystallographic and computational studies have provided fresh insights into the catalytic mechanism of the RNA-cleaving hammerhead ribozyme. Based on these findings, specific ribozyme functional groups have been hypothesized to act directly as the general acid and base catalysts, although the catalytic role of divalent metal cations (M(2+)) remains uncertain. We now report a functional characterization of the general acid catalysis mechanism and the role of an M(2+) cofactor therein, for the S. mansoni hammerhead (an "extended" hammerhead ribozyme). We have compared hammerhead cleavage of substrates with natural (ribo-phosphodiester) versus bridging-5'-phosphorothioate scissile linkages, in the contexts of active site mutations and M(2+) substitution. Cleavage of the natural substrate is inhibited by modification of the G8 2'-OH ribozyme residue and depends strongly upon the presence and identity of an M(2+) cofactor; in contrast, cleavage of the bridging-phosphorothioate substrate is conspicuously insensitive to any of these factors. These results imply that (1) both an M(2+) cofactor and the G8 2'-OH play crucial roles in hammerhead general acid catalysis and (2) the M(2+) cofactor does not contribute to general acid catalysis via Lewis acid stabilization of the leaving group. General acid pK(a) perturbation was also demonstrated for both M(2+) substitution and G8 2'-OH modification, which suggests transition state M(2+) coordination of the G8 2'-OH, to lower its pK(a) and improve its ability to transfer a proton to the leaving group. We also report a simple method for synthesizing radiolabeled bridging-5'-phosphorothioate substrates.