Nuclease enzyme

Composition and Methods of Manufacture. The vaccine consists of a mixture of purified capsular polysaccharides from 23 pneumococcal types that are responsible for over 90% of the serious pneumococcal disease in the world (47,48). Each of the polysaccharide types is produced separately and treated to remove impurities. The latter is commonly achieved by alcohol fractionation, centrifugation, treatment with cationic detergents, proteolytic enzymes, nucleases or activated charcoal, diaffltration, and lyophilization (49,50). The vaccine contains 25 micrograms of each of the types of polysaccharide and a preservative such as phenol or thimerosal. 

To isolate a functional macromolecular component from bacterial cells, you must accomplish three things. First, you must efficiently disrupt the bacterial cell wall and cell-membrane system to facilitate extraction of desired components. Second, you must work under conditions that either inhibit or destroy the many degradative enzymes (nucleases, proteases) released during cell disruption. Finally, you must employ a fractionation procedure that separates the desired macromolecule from other cellular components in satisfactory yield and purity. 

The nuclease which degrades oligo-isoadenylate copurified with the iso-A activated ribonuclease P over several steps, but was separated on hydroxylapatite. The iso-A degrading enzyme (nuclease D of Figure 1) splits (2 -5 ) P-A- to yield 5 -AMP, and more slowly (3 -5 )-A-pA. It clearly differs from known ribonucleases that are inhibited by 2 -5 dinucleotides (22). 

Pepsin, rennin Proteases Glucose oxidase Penicillin amidase Galactosidase Steroidal modifying enzymes Nucleases Urease... 

Another class of promising new generation nucleases includes artificial and semiartificial nucleases. Chemical nucleases are typical artificial nucleases that make use of redox-active compounds (e.g., phenanthroline-copper and ferrous-EDTA) as the cutter and an attached oligonucleotide as the site-specific recognition module. Although the importance of chemical nucleases and the potential for their applications are bound to grow, this chapter focuses on some of the classical enzyme nucleases that are most widely used in recombinant DNA technology. 

Limited tryptic digestion of SNase in the presence of pdTp and Ca produces an enzymatically active (8% of the native enzyme) nuclease derivative, called nuclease-T, which contains two noncovalently bound peptide fragments nuclease-T-(6-48) and nuclease-T-(49-149) (35,36). Nuclease-T refers to the complex formed on reconstitution of the isolated inactive nuclease-T fragments. 

Staphylococcal nuclease was initially isolated as an extracellular enzyme (nuclease A) of S. aureus (or Micrococcus pyogenes) (1,49). The extremely high affinity of the nuclease to phosphocellulose at pH 5.8 has provided the basis of convenient purification methods. SNase can also be efficiently purified by the use, as an affinity matrix, of 3 -(4-aminophenylphosphoryl)deoxythymidine 5 -P linked to the CNBr-activated Sepharose (50). 

En me Mechanism. Staphylococcal nuclease (SNase) accelerates the hydrolysis of phosphodiester bonds in nucleic acids (qv) some 10 -fold over the uncatalyzed rate (r93 and references therein). Mutagenesis studies in which Glu43 has been replaced by Asp or Gin have shown Glu to be important for high catalytic activity. The enzyme mechanism is thought to involve base catalysis in which Glu43 acts as a general base and activates a water molecule that attacks the phosphodiester backbone of DNA. To study this mechanistic possibiUty further, Glu was replaced by two unnatural amino acids. 

Figure 2.15 The Greek key motif is found in antiparallel p sheets when four adjacent p strands are arranged in the pattern shown as a topology diagram in (a). The motif occurs in many p sheets and is exemplified here by the enzyme Staphylococcus nuclease (b). The four p strands that form this motif are colored red and blue.

Fike most enzymes (see Chapter 14), nucleases exhibit selectivity or specificity for the nature of the substance on which they act. That is, some nucleases act only on DNA (DNases), while others are specific for RNA (the RNases). Still... 

Staphylococcal nuclease (SNase) is a single-peptide chain enzyme consisting of 149 amino acid residues. It catalyzes the hydrolysis of both DNA and RNA at the 5 position of the phosphodiester bond, yielding a free 5 -hydroxyl group and a 3 -phosphate monoester... 

When 10 (R configuration about phosphorus) was treated with ribo-nuclease in aqueous methanol, nucleophilic attack by methanol in the enzyme-catalyzed process led to formation of a methyl ester, 11, which has been shown by X-ray analysis to be the isomer with the R configuration about the phosphorus (60) ... 

Hydrolysis of RNA by alkali or pancreatic RNase leads initially to fragments which terminate in 2, 3 -cyclic phosphodiesters. Micrococcal nuclease, on the other hand, gives rise to fragments terminating in 3 -phos-phomonoester groups which facilitate their isolation, and this enzymic hydrolysis has been used to prepare 3 -ribodinucleotides. ... 

A second explanation of the ability of oxidative stress to cause DNA damage is that the stress tri ers a series of metabolic events within the cell that lead to activation of nuclease enzymes, which cleave the DNA backbone. Oxidative stress causes rises in intracellular free Ca, which can fiagment DNA by activating Ca -dependent endonucleases (Orrenius etal., 1989 Farber, 1990 Ueda and Shah, 1992) in a mechanism with some of the features of apoptosis (see Wyllie, 1980). An example of apoptosis is the killing of immature thymocytes by glucocorticoid hormones, which activate a cell-destructive process that apparently involves DNA fragmentation by a Ca -dependent nuclease. 

Fig. 8. Schematic for the micrococcal nuclease attack to (a) breathing ds-DNA, (b) dista-mycin-bound DNA, and (c) Zn2+-cyclen derivative-bound DNA. Arrows and dashed arrows, respectively, indicate successful and failed enzyme hydrolysis. Reproduced with permission from Ref. (37). Copyright 1999, American Chemical Society.

Pyridyl functionalized tris(pyrazolyl)borate ligands show some interesting properties including the formation of polynuclear zinc complexes.23,1 Some of these contain extensive H bonding and have potential as models for multinuclear zinc enzymes such as phospholipase C or PI nuclease.235 A bis-ligand complex of the hydrotris(5-methyl-3-(3-pyridyl)pyrazolyl)borate ligand (23) shows octahedral coordination of all six pyrazole nitrogen donors despite the steric bulk. 

As already mentioned, Lippard and co-workers have also studied the -hydroxy dizinc unit found in metallohydrolases in a model system [Zn2L(/x-OI I)(/x-02PPh2)]2+, L = 2,7-bis [2-(2-pyridylethyl)-aminomethyl]-l,8-naphthyridine) 454 Hydrolysis of phosphodiesters and beta-lactams was studied and related to the PI nuclease and beta-lactamase enzymes. The dinuclear complex... 

---