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. 1979 Jun;76(6):2551–2555. doi: 10.1073/pnas.76.6.2551

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

F Albert Cotton 1, Edward E Hazen Jr 1, Margaret J Legg 1
PMCID: PMC383645  PMID: 288045

Abstract

The structure of the staphylococcal nuclease (EC 3.1.4.7)—thymidine 3′,5′-bisphosphate—Ca2+ (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′-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 facilitated by polarization of the phosphodiester by an ionic interaction between a Ca2+ 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.

Keywords: micrococcal nuclease, arginine residues, x-ray crystallography

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Selected References

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