Staphylococcal nuclease specificity

Studies of proteolytic fragments of staphylococcal nuclease (Tan-iuchi and Anfinsen, 1969) and RNase A (Taniuchi, 1970) seemed to support this view. Taniuchi (1970), in summary remarks, said Thus, the minimum information of the specific folding of a protein requiring almost the entire amino acid sequence is observed with both staph-yloccocal nuclease and bovine pancreatic ribonuclease. ... 

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. 

Abstract. Walter Kauzmann stated in a review of protein thermodynamics that volume and enthalpy changes are equally fundamental properties of the unfolding process, and no model can be considered acceptable unless it accounts for the entire thermodynamic behaviour (Nature 325 763-764, 1987). While the thermodynamic basis for pressure effects has been known for some time, the molecular mechanisms have remained rather mysterious. We, and others in the rather small field of pressure effects on protein structure and stability, have attempted since that time to clarify the molecular and physical basis for the changes in volume that accompany protein conformational transitions, and hence to explain pressure effects on proteins. The combination of many years of work on a model system, staphylococcal nuclease and its large numbers of site-specific mutants, and the rather new pressure perturbation calorimetry approach has provided for the first time a fundamental qualitative understanding of AV of unfolding, the quantitative basis of which remains the goal of current work. 

Table 7-2. Specific Staphylococcal Nuclease Affinity Chromatographic Adsorbents"...

Figure 7-11. Purification ot staphylococcal nuclease by affinity adsorption chromatography on a nuclease-specific agarose column (0.8 x 5 cm). The column was equilibrated with 50mM borate buffer, pH 8.0, containing lOmAf CaClj. Approximately 50 mg of partially purified material containing about 8 mg nuclease was applied in 3.2 ml of the same buffer. After 50 ml of buffer had passed through the column, O.IM acetic acid was added to elute the enzyme. 8.2 mg nuclease and all the original activity was recovered. The flow rate was about 70 ml/hour. [From P. Cuatrecasas, M. Wilchek, and C. B. Anfinsen, Proc. Natl. Acad. Sci. US, 61 636 (1968).]...

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. 

Hybrid enzymes - Here, molecular techniques allow researchers to put together two different biomolecules to make a fusion molecule with new, useful properties. Figure 11.28 depicts a hybrid enzyme made in this fashion. In this case, an oligonucleotide of a defined sequence has been grafted onto the enzyme staphylococcal nuclease. The specific sequence in the hybrid enzyme allows it to bind to a specific complementary nucleic acid sequence (specified by the bound oligonucleotide) and cut specifically at that point. The native, unaltered enzyme has no such specificity. 

Wang JF, LeMaster DM, Markley JL (1990) Two-dimensional NMR studies of staphylococcal nuclease. 1. Sequence-specific assignments of hydrogen-1 signals and solution structure of the nuclease H124L-thymidine 3, 5 -bisphosphate-Ca2-i- ternary complex. Biochemistry 29(1) 88-101... 

Zhi Z, Liu P, Wang P, Huang Y, Zhao XS (2011) Domain-specific folding kinetics of staphylococcal nuclease observed through single-molecule FRET in a microfluidic mixer. Chem Phys Chem 12(18) 3515-3518... 

Localization of the amino acid residues of staphylococcal nuclease with which the bromoacetyl and diazonium affinity labeling reagents specifically react is summarized in Fig. 2. Reagent 1 is the most specific and attaches to Lys-48 and Lys-49 in 80% and toTyr-115 in 15% yield. Reagent II reacts with Tyr-85. Reagent III probably reacts with Lys-24... 

Deslongchamps kindly provided us with his own view of the mechanistic path by which a-chymotrypsin and other serine proteases can hydrolyze secondary amides by stereoelectronic control (Fig. 4.7). Petkov et al. (119) also arrived at a similar proposal by studying the influence of the leaving group on the reactivity of specific anilides in a-chymotrypsin-catalyzed hydrolysis. Furthermore, the stereoelectronic control theory has been applied to the mode of action of ribonuclease A, staphylococcal nuclease and lysozyme (120). 

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. 

Staphylococcal nuclease has been extensively used as a model protein for the study of enzyme mechanisms, stability, and the kinetics of (re)folding. In recombinant DNA technology, SNase is primarily used as a nonspecific endonuclease, as is DNase I which generates 5 -phosphonucleotides (Section I, this chapter). Staphylococcal nuclease has also served as the prototype model for a new generation of nucleases called hybrid nucleases, which can cleave DNA and RNA with tailored site specificities. 

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