Staphylococcal nuclease is produced by Staphylococcus aureus and catalyzes the hydrolysis of DNA and RNA to yield 3'-mononucleotides. The nuclease is a structurally very well characterized enzyme that is particularly amenable to genetic and NMR studies of its structure and chemical mechanism of action: the enzyme contains only 149 amino acids in a linear sequence established by both amino acid sequence analysis of the protein and DNA sequence analysis of the cloned gene; the three dimensional structure of the enzyme has been determined to 1.5 A resolution; and the gene for the enzyme has been expressed at high levels in Escherichia coli by the use of several expression plasmids. We have previously investigated several aspects of the chemical mechanism by which the enzyme catalyzes the hydrolysis of DNA and RNA. In this proposal we describe a comprehensive application of primer directed site specific mutagenesis to further investigate the role of a number of amino acids present in the active site of the enzyme. In particular, we plan to probe the role of the carboxylate group present at residue 43 (glutamate in the wild type enzyme), the amino acids presumed to effect binding of substrate to the enzyme (lysines, arginines, and tyrosines that interact with the anionic phosphates and other residues that form the base binding site), and the amino acids presumed to bind the essential Ca2+ required for catalysis. The properties of the mutant enzymes generated in this study will be analyzed kinetically and structurally by both X-ray crystallography and by NMR spectroscopy. While the X-ray crystallography will be carried out elsewhere, the NMR studies will be conducted in our laboratory and will develop methodology for the simplification of the NMR spectral properties of the nuclease so that proton chemical shifts, proton-proton coupling constants, and proton-proton nuclear Overhauser effects can be used to deduce information about the conformations of wild type and mutant forms of the nuclease.