Staphylococcal nuclease inhibitors

Fig. 29. An assortment of/3 barrels, viewed down the barrel axis (a) staphylococcal nuclease, 5-stranded (b) soybean trypsin inhibitor, 6-stranded (c) chymotrypsin, 6-stranded (d) immunoglobulin (McPC603 CH1) constant domain, 7-stranded (e) Cu,Zn superoxide dismutase, 8-stranded (f) triosephosphate isomerase, 8-stranded (g) im-... 

Figure 12-29 Drawing showing the hydrogen-bonding interactions between the guanidinium ions of arginines 35 and 87 of the micrococcal (staphylococcal) nuclease with the 5 -phosphate of the inhibitor thymidine 3, 5 -diphosphate in the complex of E + I + Ca2+. A possible mechanism is illustrated. A hydroxyl ion bound to Ca2+ carries out an in-line attack on the phosphorus. See Libson et al.S26...

Several routes exist for the synthesis of amides of halo acids. Cuatrecasas et al. (1969) used two different bromoacetylating reagents in the synthesis of water soluble inhibitors of staphylococcal nuclease. The N-hydroxysuccinimide ester of bromoacetate was one of the reagents used. It is particularly useful for the synthesis of radioactive derivatives since C-bromoacetic acid is commercially available. Synthesis of the hydroxysuccininimide ester is accomplished by dissolving 87 mg (630 /imoles) of bromoacetic acid and 86 mg of N-hydroxysuccinimide in 3 ml of dioxane. To this solution, 132 mg (700 /imoles) of dicyclohexylcarbodiimide is added. Urea precipitates immediately and after 1 hr, is removed. The solution of bromoacetyl N-hydroxysuccinimide ester is brought to 5 ml. It can then be used without any further purification. 

Figure 1. Stereo drawing of the a-carbon backbone of the refined stmcture of staphylococcal nuclease. The protein backbone is drawn with dark bonds and light atoms the inhibitor pdTp is drawn with light bonds and dark atoms. Also shown is the Ca " " ion, depicted as a large sphere immediately below the PdTp molecule. The protein s N-terminus appears at the upper right in this view the C-terminus is at the lower left (from ref. 1).

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).

The metallobiochemistry of staphylococcal nuclease has been extensively investigated. It was shown early that the tripositive lanthanide ions, Ln, are potent competitive inhibitors of the enzyme, binding to it with K s of about 9/uM and acting with inhibitory constants of l-2 iM. The binding of Ln + ions enhances the binding of pdTp. Ln " ions, but not Ca, stabilize the enzyme toward tryptic proteolysis. The paramagnetism of Gd " was exploited in H and P resonance relaxation studies on pdTp bound in the ternary complex to determine that the structure in solution was consistent with the observed x-ray structure. Some differences were observed but these were of uncertain significance. 

Figure 7 Correlation between predicted and experimental pK s in nine globular proteins hen egg white lysozyme, ribonuclease A, turkey ovomucid third domain, bovine pancreatic trypsin inhibitor, B1 and B2 immuno obulin G binding domains of protein G, a-chymotrypsin, ribonuclease Tj, lysozyme T4, and staphylococcal nuclease.

Staphylococcal nuclease with substrate analogues Stromelysin domain with N-TIMP-2 inhibitor Stromelysin with nonpeptide inhibitors Thioredoxin with NFkP peptide Topoisomerase-I domain with DNA Trp repressor with DNA Trypsin with proteinase inhibitors Urbs 1 with DNA... 

Fig. 3.13. Proportionality of the loss of accessible surface area to the molecular weight of proteins (from Janin, 1976). Different proteins insulin, rubredoxin, pancreatic trypsin inhibitor, HIPIP, calcium binding protein, ribonuclease S, lysozyme, staphylococcal nuclease, papain, chymotrypsin, concanavalin A, subtilisin, thermolysin, carboxypeptidase A.

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... 

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