Complex biochemical processes such as recombination, restriction, and the repair of lesions in DNA are dependent upon enzymes which cleave the internucleotide phosphodiester bonds in DNA. Despite the importance of these processes, little is known about the mechanisms of the crucial enzymes. We propose to study the mechanism of the hydrolysis reaction catalyzed by the nuclease which is produced by Staphylococcus aureus since both the amino acid sequence and a high resolution x-ray structure of the enzyme are available. At present, the mechanism of the hydrolysis reaction catalyzed by Staphylococcal nuclease is uncertain, but elucidation of the details of the process by which this enzyme catalyzes the hydrolysis of phosphate ester bonds will be not only of intrinsic importance also may provide considerable insight into the mechanisms of many other enzymes which hydrolyze DNA. With the knowledge of the amino acid residues present in the active site of the nuclease as well as recent stereochemical information we have obtained; two fundamentally different mechanisms for the hydrolysis reaction can be formulated. The first involves the active site glutamate acting as a general basic catalyst in facilitating the direct attack of water on the substrate; the second involves the active site glutamate acting as a nucleophile in attacking the substrate to form a glutamyl phosphate ester intermediate which is then hydrolyzed by the attack of water on the glutamyl carboxyl carbon. These mechanisms can be readily distinguished by isotope labeling experiments in which the origin of the oxygen incorporated into the first product released by the enzyme is ascertained; in the first mechanism the oxygen is derived from solvent whereas in the second mechanism it is derived from the glutamate carboxylate group. We also propose to perform a necessary detailed kinetic analysis of the reaction catalyzed by the nuclease, so that additional information can be obtained regarding the number and rates of the steps involved in the catalytic process.
