Staphylococcal nuclease: proposed mechanism of action based on structure of enzyme-thymidine 3',5'-bisphosphate-calcium ion complex at 1.5-A resolution.

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-A 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.

Staphylococcal nuclease reviewed: a prototypic study in contemporary enzymology. IV. The nuclease as a model for protein folding.

This is the last in a series of four articles in which the chemical, enzymological and crystallographic work on Ribonucleate (deoxyribonucleate)-3'-nucleotidohydrolase, EC 3.1.4.4 (staphylococcal nuclease, micrococcal nuclease) will be reviewed and correlated. This article discusses the use of the nuclease as a model system for the study of the mechanisms and energetics of the folding-unfolding reaction in proteins and for the study of the interrelationships between amino acid sequence and three-dimensional structure.

Staphylococcal nuclease reviewed: a prototypic study in contemporary enzymology. II. Solution studies of the nucleotide binding site and the effects of nucleotide binding.

This is the second of a series of four articles in which the chemical, enzymological and crystallographic work on Ribonucleate (deoxyribonucleate)-3'-nucleotidohydrolase, EC 3.1.4.4. (staphylococcal nuclease, micrococcal nuclease) will be reviewed and correlated. This article discusses studies in solution delineating the extent of the binding site of the enzyme and identifying some of the particular amino acid residues that form this site. In addition, the effects of the very potent inhibitory combination of thymidine-3',5'-diphosphate and Ca2+ on the conformation of the enzyme and its physical, chemical and enzymological properties will be reviewed.

Staphylococcal nuclease reviewed: a prototypic study in contemporary enzymology. III. Correlation of the three-dimensional structure with the mechanisms of enzymatic action.

This is the third in a series of four articles in which the chemical, enzymological and crystallographic work on Ribonucleate (deoxribonucleate)-3'-nucleotidohydrolase, EC 3.1.4.4 (staphylococcal nuclease, micrococcal nuclease) will be reviewed and correlated. This article describes the structure of the nuclease and of a nuclease-inhibitor complex as determined by x-ray crystallography. The crystal structures are correlated with some of the known chemical and enzymological properties of the enzyme, and the three areas combined to propose a mechanism of action.

Staphylococcal nuclease reviewed: a prototypic study in contemporary enzymology. I. Isolation; physical and enzymatic properties.

This is the first of a series of four articles in which the chemical, enzymological, and crystallographic work on Ribonucleate (deoxyribonucleate)-3'-nucleotidohydrolase, EC 3.1.4.7, (Staphylococcal nuclease, Micrococcal nuclease) will be reviewed and correlated. This article discusses the purification of the enzyme and its general physical and enzymological properties. Subsequent articles will deal with specific studies of the nucleotide binding site, crystallographic studies of a nuclease-inhibitor complex, use of the nuclease as a model for protein folding and possible mechanisms for the action of the enzyme.

The role of arginine residues at enzyme active sites. The interaction between guanidinium ions and p-nitro-phenyl phosphate and its effect on the rate of hydrolysis of the ester.

In alcoholic solutions a relatively strong complex forms among two guanidinium ions and one p-nitrophenylphosphate dianion. The effect of this complex formation on the hydrolysis of the ester is to lower the rate by a factor of 4 in solutions containing 1 M guanidine hydrochloride when compared with solutions of the same total ionic strength containing no guanidinium ion. It is therefore suggested that, for the enzymatically catalyzed hydrolysis of phosphate compounds going via the formation of a metaphosphate intermediate, the role of any arginine residues at the active site is primarily one of binding and positioning the substrate.

Tumor-inhibiting properties of the neutral P-O-ethyl ester of adenosine 3':5'-monophosphate in correlation with its crystal and molecular structure.

The P-O-ethyl ester of cAMP has been synthesized, its inhibition of solid and ascites tumors studied, and its pattern of urinary excretion followed. Et-cAMP is more effective than cAMP against solid sarcoma 180 in mice and against Ehrlich ascites carcinoma cells in tissue culture. The urinary excretion pattern of injected E-t-cAMP suggests that about two-thirds of the injected dose (13 mumol per animal) is retained in the rat rather than being promptly excreted. Liver slice studies of the effect on glycogenolysis suggest that the Et-cAMP is converted to cAMP intracellularly. The compound crystallizes in space group P21 with one molecule per asymmetric unit. The base ring has the anti conformation. The ethyl group is endo to the base ring and is axial in the flattened chair-conformer six-membered ring formed by the 3'-5' O-P-O cyclization. In most other respects the structure of the compound is closely similar to the known structures of other cyclic nucleotides.

The extracellular nuclease of Staphylococcus aureus: structures of the native enzyme and an enzyme-inhibitor complex at 4 A resolution.

Independent 4 A electron density maps calculated for the extracellular nuclease of Staphylococcus aureus (based on data from three heavy-atom derivatives) and for a nuclease-thymidine-3',5'-diphosphate-calcium ion complex (based on a single isomorphous derivative) show about 60 per cent of the chain resolved, including 3(1/2) turns of helix. The pyrimidine ring of the inhibitor fits into a pocket in the enzyme and appears to be parallel to the ring of a tyrosyl residue. Conformational changes can be observed between the nuclease and the nuclease-inhibitor complex, but the two structures seem to be identical over most of the molecule.