MECHANISM OF THE STAPHYLOCOCCAL NUCLEASE REACTION

这个项目旨在了解金属的机制

This project is directed toward understanding the mechanism of the metal ion (Ca 2+) assisted hydrolysis of DNA catalyzed by Staphylococcal nuclease (SNase). SNase is particularly amenable to the study of structure/function relationships in catalysis since the protein is small (149 amino acids), the structure has been determined to high resolution by x-ray crystallography, and the NMR resonances (1H, 13C, and 15N) are almost totally assigned; furthermore, a variety of kinetic tools is available to study the mechanism of the hydrolysis reaction. We have already deleted a conformationally flexible Omega-loop in the active site of SNase (to produce delta SNase) and devised a metabolic screen for activity variants of SNase. We will use casette based random mutagenesis of the gene for delta SNase to 1) determine the geometric requirements for general base catalysis by a carboxylate group (Glu and Asp) at residue 43; 2) determine the geometric requirements for general base catalysis by an imidazolium group (His) at residue 43; 3) determine whether metals ions other than Ca 2+ can promote the hydrolysis of DNA; and 4) determine whether the heterocyclic base specificity can be altered to favor G and/or C rather than A and T. In the case of Specific Aims 1 and 2, random mutagenesis will be used to alter the conformation of the Beta-turn produced by deletion of the Omega-loop; in the case of Specific Aims 3 and 4, site-directed mutagenesis and random mutagenesis will be used together to alter the identity and positions of functional groups that bind the essential metal ion and base so that the specificity of the enzyme can be altered. All four Specific Aims will involve both significant biophysical (x-ray and NMR) characterization of the structures of mutant enzymes in addition to detailed kinetic and mechanistic studies of the reactions catalyzed by the mutant enzymes.