Staphylococcal nuclease substrate specificity

Staphylococcal nuclease is a phosphodiesterase which can cleave either DNA or RNA to produce 3 -phosphomononucleosides (3-16, 20,25). The rates of hydrolysis of these substrates are dependent on the conformation of the substrate, Ca2+ concentration, and the ionic strength and the pH of the buffer (3, 54). Denatured DNA is hydrolzed more rapidly than native DNA (3, 12, 54-56), which reflects the important effect of substrate conformation on catalysis. In native DNA the Xp-dTp and Xp-dAp bonds are preferentially attached (7, 12, 14, 15, 55). With denatured DNA the order of cleavage appears to be nearly random (14, 15, 56, 57). The Xp-dCp and Xp-dGp linkages in the helical regions of DNA, which are more extensively stabilized, are more resistant to hydrolysis. The specific order of release of various mononucleotides from native compared to denatured DNA suggests that in the hydrolysis of DNA specificity toward the constitutent bases is less important than the substrate conformation (54-57). 

Fig. 2). The staphylococcal enzyme may appear to be more akin in its mode of action to the spleen enzyme because they both hydrolyze DNA and RNA to 3 -nucleotides, whereas the venom enzyme releases 5 -nucleotides. However, their mode of action and specificity are quite different, and the structural requirements of the staphylococcal enzyme substrates are perhaps more nearly similar to those of the venom enzyme. The principal difference is that the staphylococcal enzyme cleaves the diester bond between the phosphate and the 5 -carbon of the sugar, whereas the venom enzyme cleaves on the other side of the phosphate, that is, between the phosphate and the nonspecific hydroxylic component of the diester bond. In contrast to both spleen and venom diesterases, the primary product released by staphylococcal nuclease hydrolysis is a derivative bearing a hydroxyl group (on the 5 position) rather than a phosphoryl group. Therefore, the 3 -phosphoryl product formed from polynucleotide hydrolysis is a secondary consequence of such cleavage. 

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. 

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