The Binding of Nucleotides and Calcium to the Extracellular Nuclease of Staphylococcus aureus

Abstract Staphylococcal nuclease binds various nucleotides in a 1:1 molar ratio. This binding is completely dependent on the presence of Ca++ and is prevented by high urea concentrations and by various chemical modifications of the protein. Dissociation constants have been determined for a number of nucleotide-enzyme complexes. There are good correlations between the affinities of binding and the strengths of inhibition of polynucleotide hydrolysis by the various nucleotides. The dissociation constants vary, just as enzyme activity varies, with pH and Ca++ concentration. The nucleotides studied all bind to the same region of the enzyme, which appears to be the same as the catalytically active site and is probably responsible for hydrolysis of deoxyribo- and ribonucleic acid. It is suggested that the structural unit of the substrate responsible for specific enzyme recognition and binding is a 3',5'-diphosphonucleoside region. Ca++ is bound weakly to the enzyme in a 1:1 molar ratio if monophosphonucleoside derivatives are present, and in a 2:1 ratio if deoxythymidine 3',5'-diphosphate is present. No Ca++ binding occurs in the absence of nucleotide or substrate. Nucleotides (or substrates) and Ca++ are bound to nuclease in a unique complex which forms only when all three components are present concurrently.