Active site staphylococcal nuclease

Mildvan, A. S., and Serpersu, E. H. (1989). Genetic alteration of active site residues of staphylococcal nuclease Insights into the enzyme mechanism. In Metal Ions in Biological Systems (H. Sigel and A. Sigel, eds.), pp. 309-334. Dekker, New York. 

The apparent usefulness of the modeling approach suggested that possible active site interactions important in understanding the mode of action of the well-characterized enzymes, ribonuclease (16) and staphylococcal nuclease (17). may be revealed. Both have been the subject of extensive crystallographic studies (18,19) with suitable inactive substrates in place. We considered the first step of hydrolytic action of ribonuclease (RNase) on the dinucleotide substrate uridylyl-(3 -5 )-adenosine(UpA). Our results (20) on the enzyme mechanism were consistent with the main features summarized by Roberts et al (21). The first step is a transphosphorylation leading to cleavage "oT the phosphodiester... 

Staphylococcal nuclease (SNase) is one of the most powerful enzymes known in terms of its rate acceleration, with a catalytic rate that exceeds that of the non-enzymatic reaction by as much as 1016.211 This enzyme is a phosphodiesterase, and utilizes a Ca2+ ion for catalysis to hydrolyze the linkages in DNA and RNA. In addition to the metal ion, the active site has two Arg residues in a position to interact with the phosphoryl group, and a glutamate. X-ray structures212 215 of SNase have been solved for the wild-type enzyme and mutants, but the exact roles of active-site residues are still uncertain. SNase cleaves the 5 O-P nucleotide bond to yield a free 5 -hydroxyl group (Fig. 30). 

A number of bis(guanidinium) receptors (Figure 25) have been synthesized meso and J, I) by Anslyn and co workers [38]. These molecules mimic part of the active site of staphylococcal nuclease. The binding of dibenzyl phosphate was investigated in detail by H and P NMR spectroscopy. It was found that binding occurred through the formation of four hydrogen bonds between the... 

Figure 2. Schematic drawing of the active site of staphylococcal nuclease. Protein side chains are shown by light bonds, while the PdTp molecule is in dark. The Ca ion is shown as the large sphere below the inhibitor molecule. Also shown are the three inner sphere water ligands of the calcium ion and the water molecule bridging Glu-43 and the 5 -phosphate of the inhibitor (this bridging water is the putative nucleophile in the hydrolysis of phosphoesters) (from ref. 1).

Phospholipase A2 (EC 3.1.1.4) " " is a member of a class of lypolytic enzymes that hydrolyze their lipid substrates at an organized lipid-water interface. This enzyme specifically catalyses the hydrolysis of the 2-acyl ester bond of 3-5 -phyosphoglycerides. It has an absolute requirement for Ca " and binds this ion in a 1 1 molar ratio to the enzyme, with a dissociation constant of 2-4 mM. The x-ray structure of the 124-residue bovine enzyme has been determined. It has about 50% a-helical and 10% j8-sheet structure. Ca " " is bound at the active site (Figure 3) and is coordinated to backbone carbonyl atoms of Tyr-28, Gly-30, Gly-32, the two carboxylate oxygens of Asp-49 and two HjO molecules, for a total coordination number of seven. As was the case for staphylococcal nuclease, the Ca " " ligands are supplied from noncontiguous regions of the polypeptide chain. 

Fig. 12. Active site of wild-type staphylococcal nuclease with bound active site ligands Ca and thymidine 3, 5 -diphosphate. Reproduced with permission from Ref. 88.

Gobel and coworkers reported the synthesis of h(5(guanidinium) alcohols as mimics for the active site of staphylococcal nuclease.The phosphorylation of... 

Genetic Alteration of Active Site Residues of Staphylococcal Nuclease Insights into the Enzyme Mechanism... 

Serpersu, E. H. Shortle, D. Mildvan, A. S., Kenetic and magnetic resonance studies of the active-site mutants of staphylococcal nuclease Factors contributing to catalysis. Biochemistry 1987,26, 1289-1300. 

Semiartificial nucleases include (1) in concept, chimeric nucleases which are composed of heterogenous structural parts brought together by the use of recombinant DNA techniques and (2) hybrid nucleases which are derived from classical nucleases by incorporating a piece of synthetic oligonucleotide in the active site to harness a sequence specificity. An example of the hybrid nuclease is described in Section II (this chapter) on staphylococcal nuclease. 

The nuclease requires Ca + ions for activity, with no other divalent metal ion being able to support catalysis. A large number of other phospodiester-ases have been found to be dependent on divalent metal ions for activity, including the restriction endonuclease icoRI (Barton et al, 1982). Thus elucidation of the mechanism of the reaction catalyzed by staphylococcal nuclease may provide important clues to the mechanisms of the other metal-dependent phosphodiesterases. Fortunately, staphylococcal nuclease will catalyze, albeit at a low rate, the hydrolysis of a number of mononucleotide esters (Cuatrecasas et al., 1969), including thymidine 5 -(4-nitro-phenyl phosphate) this ester is hydrolyzed to thymidine and 4-nitrophenyl phosphate. We have determined the stereochemical course of the hydrolysis of thymidine 5 -(4-nitrophenyl [ 0, 0]phosphate) and interpreted the result in terms of the structure of the active site of the enzyme. 

CuATRECASAS, P., S. FucHS, and C. B. Anfinsen Tyrosyl Residues at the Active Site of Staphylococcal Nuclease. Modifications by Tetranitromethane. J. Biol. Chem. 243, 4787-4798 (1968). 

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