The structure of the staphylococcal nuclease (EC 3.1.4.7)—thymidine 3′,5′-bisphosphate—Ca 2+ (enzyme—inhibitor) complex has been extended to 1.5-Å resolution by using much additional data and a phase refinement scheme based on an electron-density map modification procedure. By correlating this structure with the known properties of the enzyme, a mechanism of action is proposed that involves nucleophilic attack on phosphorus by a water molecule, which is bound to Glu-43, in line with the 5′-CH 2 O(H) leaving group. The carboxylate of Glu-43 promotes this attack by acting as a general base for the abstraction of a proton from the attacking water molecule. Nucleophilic attack is further facilitated by polarization of the phosphodiester by an ionic interaction between a Ca 2+ ion and a phosphate oxygen atom and by four hydrogen bonds to phosphate oxygen atoms from guanidinium ions of Arg-35 and Arg-87. These interactions may also catalyze the reaction by lowering the energy of a trigonal bipyramidal transition state. The hydrolysis of nucleic acid substrate proceeds by cleavage of the 5′—P—O bond to yield a free 5′-hydroxyl group and a terminal, 3′-phosphate monoester group. In the inhibitor complex the only general acid group found in a position to donate a proton to the leaving 5′-oxygen is the guanidinium ion of Arg-87. Alternative proton donors, presently lacking direct structural support, could be the phenolic hydroxyl group of Tyr-113 or a water molecule. The precision and rigidity of the location of the reactants at the active site and the probable dual binding and catalytic roles of the guanidinium ions of Arg-35 and Arg-87 are especially noteworthy.

Cotton, F. A., Hazen, E. E., & Legg, M. J. (1979). Staphylococcal nuclease: Proposed mechanism of action based on structure of enzyme—thymidine 3′,5′-bisphosphate—calcium ion complex at 1.5-Å resolution. Proceedings of the National Academy of Sciences, 76(6), 2551–2555.