Staphylococcal nuclease: Proposed mechanism of action based on structure of enzyme—thymidine 3′,5′-bisphosphate—calcium ion complex at 1.5-Å resolution

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Abstract
The structure of the staphylococcal [Staphylococcus aureus] nuclease (EC 3.1.4.7)-thymidine 3'',5''-bisphosphate-Ca2+ (enzyme-inhibitor) complex was extended to 1.5 .ANG. 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 Ph by a water molecule, which is bound to Glu-43, in line with the 5''-CH2O(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 faciliatated by polarization of the phosphodiester by an ionic interaction between a Ca2+ ion and a phosphate O atom and by 4 H bonds to phosphate O 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''.sbd.P.sbd.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''-O 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.

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