Phosphodiesterases staphylococcal nuclease

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). 

Apart from important similarities in the endo- and exonucleolytic properties of staphylococcal nuclease and other well-studied phosphodiesterases (67), those from snake venom and spleen, the basic structural substrate elements for these enzymes appear to be quite different... 

Staphylococcal nuclease (SNase) is a phosphodiesterase that cleaves DNA and RNA at the 5 position of the phosphodiester bond to form 3 -phosphate monoester and a free 5 -hydroxyl group through the reaction displayed above (Tucker et al., 1978). The... 

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). 

In contrast to the lack of detailed structural information for the 3, 5 -cyclic nucleotide phosphodiesterase, staphylococcal (or micrococcal) nuclease (SNase), an extracellular nuclease produced by Staphylococcus aureus, is well characterized. SNase is an extraordinarily efficient catalyst for the endo- and exonucleo-lytic hydrolysis of single-stranded DNA and RNA, with the rate acceleration for DNA being approximately 10 relative to the uncatalyzed rate the final products of the reaction are 3 -mononucleotides. The sequence of 149 amino acids that constitute the enzyme has been determined both by classical degradation procedures and by sequence analysis of the cloned gene. A highly refined 1.65-A X-ray structure determined in the presence of Ca and the competitive inhibitor thymidine 3, 5 -bisphosphate (pdTp) was recently completed (87) this structure differs only slightly from a less refined 1.5-A structure that was reported in 1979... 

The guanidinium unit plays a crucial role in the activity of some enzymes such as staphylococcal nuclease [23] and has been used as an activating and/or anchoring group in the design of supramolecular catalysts [24-28]. Artificial phosphodiesterases 27 30 were developed by introducing from two to four guanidinium units at the upper rim of a cone calix[4]arene scaffold [26]. 

Staphylococcal nuclease (SNase), also commonly known as micrococcal nuclease, is a Ca -dependent phosphodiesterase which cleaves both DNA and RNA to yield 3 -phosphomononucleotide and 3 -phosphooligonucleotide end products (Scheme 3.1). 

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. 

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