| Abstract
| - There is mounting evidence that suggests that general acid/base catalysis is operative in thehairpin ribozyme, with analogy to the protein enzyme RNaseA. Nevertheless, the extent of general basecatalysis as well as the identity of the specific chemical groups responsible remains the subject of somecontroversy. An affinity label has previously been used to alkylate histidine 12 (His12), the active generalbase in RNaseA. To date, no such experiment has been applied to a ribozyme. We have synthesized theanalogous affinity label for the hairpin ribozyme with an electrophilic 2‘-bromoacetamide group in lieu ofthe 2‘-hydroxyl (2‘OH) at the substrate cleavage site and show that guanosine 8 (G8) of the hairpin ribozymeis specifically alkylated, most likely at the N1 position. This evidence strongly implicates N1 of G8 in activesite chemistry. By direct analogy to RNase A, these findings could be consistent with the hypothesis thatdeprotonated G8 residue functions as a general base in the hairpin ribozyme. Other mechanistic possibilitiesfor N1 of G8 such as indirect general base catalysis mediated by a water molecule or transition statestabilization could also be consistent with our findings.
|
