Title

Investigation of the mechanism of Ca2+ catalyzed RNA phosphodiester hydrolysis.

Date of Award

5-1-2014

Document Type

Thesis

School

College of Liberal Arts

Degree Name

Bachelor in Arts

Abstract

Metal ion catalysis is used in a number of natural enzymes and ribozymes to catalyze phosphodiester cleavage of RNA. Research into metal ion catalysis has been focused on metals such as Zn2+ and other transition metals with little emphasis on alkaline earth metals, such as Ca2+ due to lower catalytic effects. To elucidate the mechanism of catalysis, an RNA model, 5-Uridine-Guanosine-3 (UpG), was subjected to varying CaCl2 concentrations at pH 11.5 at 37oC. The RP and Sp phosphorothioate analogs of UpG (UpsG) were also examined. Solvent deuterium isotope effects were also utilized to determine if the catalytic mechanism proceeded by general acid or general base catalysis. HPLC was used to isolate the stereoisomers previous to kinetic trials and to separate reactants from products in experiment samples. Catalysis by Ca2+ was observed in all models with data normalized to the percent of reactant remaining which was graphed against time to determine the rate constant at each CaCl2 concentration. The rates for each isomer and the non-thiosubstituted model were then graphed separately against the CaCl2 concentration and fit to a rectangular hyperbola to determine differences in affinity and rate enhancement. Rate enhancement with Ca2+ for UpG was found to be a 326-fold increase, for the RP isomer a 129-fold increase and for the SP isomer a 68-fold increase. Initial results seem to indicate that there is minimal loss in affinity when comparing the UpsG models, 0.90+/- 0.95 for the RP isomer, 0.21+/-0.09 for the SP isomer, 0.63+/-0.35 for the UpG model. If Ca2+ were coordinating with the non-bridging oxygens a substantial loss in affinity should be observed with the thiosubstituted models. Since there is minimal loss in affinity, there may be little to no inner sphere coordination between the Ca2+ and non-bridging atoms during hydrolysis. UpG was the chosen model for solvent deuterium isotope effect studies and the solvent deuterium isotope effect was calculated to be 0.87 in both buffered and non-buffered solutions. This indicates that the mechanism of catalysis does not proceed through general acid/base catalysis.