Rapid Kinetics of Pistol Ribozyme: Insights into Limits to RNA Catalysis.

Pistol ribozyme (Psr) is a distinct class of small endonucleolytic ribozymes, which are important experimental systems for defining fundamental principles of RNA catalysis and designing valuable tools in biotechnology. High-resolution structures of Psr, extensive structure-function studies, and computation support a mechanism involving one or more catalytic guanosine nucleobases acting as a general base and divalent metal ion-bound water acting as an acid to catalyze RNA 2'--transphosphorylation. Yet, for a wide range of pH and metal ion concentrations, the rate of Psr catalysis is too fast to measure manually and the reaction steps that limit catalysis are not well understood.

Syntheses and structures of two benzoyl amides: 2-chloro-4-eth-oxy-3,5-dimeth-oxy--(3-oxo-cyclo-hex-1-en-1-yl)benzamide and 2-chloro--(5,5-dimethyl-3-oxo-cyclo-hex-1-en-1-yl)-4-eth-oxy-3,5-di-meth-oxy-benzamide.

The first title benzoyl amide, CHClNO (), crystallizes in the monoclinic space group 2/ with = 4 and the second, CHClNO (), also crystallizes in 2/ with = 8 (' = 2), thus there are two independent mol-ecules in the asymmetric unit. In , the phenyl ring makes a dihedral angle of 50.8 (3)° with the amide moiety with the C=O group on the same side of the mol-ecule as the C-Cl group.

Isotope effect analyses provide evidence for an altered transition state for RNA 2'-O-transphosphorylation catalyzed by Zn(2+).

Solvent D2O and (18)O kinetic isotope effects on RNA 2'-O-transphosphorylation catalyzed by Zn(2+) demonstrate an altered transition state relative to specific base catalysis. A recent model from DFT calculations involving inner sphere coordination to the non-bridging and leaving group oxygens is consistent with the data.