| Abstract: | The purpose of the present research is to gain further chemical and crystallographic information concerning the structure and function of the Staphylococcal nuclease, especially in regard to mechanism of action. A description of the active site is given as seen in the nuclease-dinucleoside phosphonate-Ca²⁺ complex at 2.0A resolution. Within the structural framework of this complex and that of nuclease-pdTp-Ca²⁺, several solution studies are reported. Comparative kinetics and binding data on dinucleotide substrate analogues, in which the diester 5'-oxygen atom has been replaced with either a methylene group (phosphonates) or a sulfur atom (phosphorothioates), are discussed in regard to electronic and steric requirements. The results of the synthesis, enzyme kinetics, and esr characterization of a nuclease transition state analogue, in which the normally tetrahedral phosphate group of the nucleotide has been replaced by a tetragonal bipyramidal V0²⁺ ion, are correlated with the structural information. Phenylglyoxas modification of arginine residues, with concomitant loss of enzymatic activity is reduced in the presence of active site ligands. A low pH induced conformational change is suggested from purification, electrophoresis, and amino acid analysis studies. |
|---|