Part I. Energetic analysis of hydrogen bonds in model systems: Implications for enzymatic catalysis. Part II. Probing the role of metal ions in catalysis by the Tetrahymena group I ribozyme

Part I. Charge rearrangements typically occur in the course of a reaction, leading to strengthening of hydrogen bonds between enzymatic groups and substrate moieties undergoing charge accumulation. However, the corresponding hydrogen bonds from water are also strengthened in the course of a solution reaction. Enzymes, of course, need to provide rate enhancements relative to solution reactions. Thus the question arises: can the enzymatic active site provide a greater strengthening of hydrogen bonds relative to water? Model studies suggest that the strengthening of hydrogen bonds upon charge accumulation can be greater in an enzymatic active site with low effective dielectric than in aqueous solution, thus providing a rate enhancement for an enzymatic reaction. Enzymes presumably utilize multiple hydrogen bonds with substrates for catalysis. This notion is supported by studies that show that the energetic effect of two pre-positioned hydrogen bonds within a model compound is large and nearly additive. Finally, model studies suggest that the energetics of hydrogen bonds are consistent with a simple electrostatic model.;Part II. RNA enzymes require divalent metal ions for catalysis. Determining the number of metal ions in an RNA active site and delineating their catalytic roles are crucial for understanding RNA catalysis. This presents a formidable challenge, however, as catalytic metal ions are bound within a sea of metal ions that coat an RNA. We have developed a novel approach that combines metal ion specificity switch methods with in-depth mechanistic analysis. This allows metal ion sites to be distinguished from one another, and was used to provide evidence for at least three metal ions positioned within the active site of the Tetrahymena group I ribozyme. Analysis of several metal ion sites and functional groups are described: the 2'-OH of the guanosine nucleophile and its associated metal ion, the 2'-OH preceding the cleavage site of the oligonucleotide substrate, and a metal ion that interacts with the nucleotide 3' to the scissile bond. Finally, results from these analyses suggested the presence of a network of active site interactions that may play an integral role in positioning of the substrates within the active site.