Enzymes staphylococcal nuclease

Figure 15.10 Jhh couplings in the enzyme staphylococcal nuclease (SNase) showing least-squares fit to a Karplus equation, http //www.biochem.wisc.edu/biochem801/ pdfs/LectureS.pdf (Reprinted from Journal of Magnetic Resonance, Vol 86, Kay, Lewis E. and Bax, Ad, New methods for the measurement of NHCaH coupling constants in nitrogen-15 labeled proteins, pages 11026, Copyright 1990 with permission from Elsevier.)...

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. 

The masters of catalysis are enzymes. Enzymes are biomolecules typically based on proteins and often associated with small organic molecules or metal ions known as cofactors. In recent years it has become clear that RNA molecules can also catalyze important reactions, and such catalytic RNA molecules are referred to as ribozymes. Our focus here, however, will be on the more well known, protein-based enzymes, which mediate the overwhelming majority of biochemical transformations. These are nature s catalysts, and they can be incredibly efficient. As just one example, the hydrolysis of a phosphoester such as that used to link nucleotides together in DNA is estimated to have a half-life of hundreds of millions of years in water at neutral pH. Yet, the enzyme staphylococcal nuclease can catalyze this hydrolysis reaction with a half-life of a few minutes. Since this is a physical organic textbook, not a biochemistry textbook, we do not look at the structures of enzymes and how they are formed. Instead, we simply focus upon the mechanisms and kinetics of enzymatic catalysis. 

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 Mutagenesis studies in which Glu43 has been replaced by Asp or Gin have shown Glu to be important for high catalytic activity.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. 

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... 

In a further study, Taniuchi et al. (1977) have shown that in the association of overlapping fragments of staphylococcal nuclease, two different species of active enzyme are formed. On the basis of the products of limited proteolysis, structures for the two species were deduced. In one case a structure is proposed in which fragment 1-126 assumes native-like structure over the sequence 1-48, and all of fragment 50-149 assumes native-like structure. In the other case the structure is one in which fragment 1-126 assumes native-like structure over the sequence 1-110, while that part of fragment 50-149 in the sequence interval 111-149 assumes native-like structure. The interest of these results is enhanced by the finding that the two active species initially form in relative concentrations substantially different from their equilibrium concentrations. Thus, both a mobile equilibrium and substantial kinetic control of the early products are evident. Taniuchi et al. did not reach a clear-cut mechanistic conclusion from their studies. 

Cotton, F. A., Hazen, E. E., Jr., and Legg, M. J. (1979). Staphylococcal nuclease Proposed mechanism of action based on structure of enzyme-thymidine 3, 5 -bisphosphate-calcium ion complex at 1.5-A resolution. Proc. Natl. Acad. Sci. U.S.A. 76, 2551-2555. 

Mildvan, A. S., and Serpersu, E. H. (1989). Genetic alteration of active site residues of staphylococcal nuclease Insights into the enzyme mechanism. In Metal Ions in Biological Systems (H. Sigel and A. Sigel, eds.), pp. 309-334. Dekker, New York. 

In addition to interest in this enzyme because of its catalytic characteristics, a considerable body of information has accumulated on staphylococcal nuclease as a protein molecule. Its relatively small size, the absence of covalent cross-linkages, and its behavior upon binding a variety of ligands have made it an ideal model substance for the study of various aspects of protein chemistry including X-ray crystallography. These investigations are reviewed in the present chapter and in Chapter 7 by Cotton and Hazen, this volume, on the three-dimensional structure (19). 

Early studies on the purification of staphylococcal nuclease led to conflicting reports on whether the enzyme could hydrolyze both DNA... 

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... 

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. 

Fig. 3. Ultraviolet spectral changes resulting from hydrolysis of nitrophenyl-pdTp-nitropheny] (32 /iM) by staphylococcal nuclease (14 nM). The spectrum at zero time is that of the mixture of enzyme and substrate before addition of Ca. The reaction is started by addition of Ca + (10 mA/) by 3 min the reaction is complete. Data from Cuatrecasas et al. (61).

The apparent usefulness of the modeling approach suggested that possible active site interactions important in understanding the mode of action of the well-characterized enzymes, ribonuclease (16) and staphylococcal nuclease (17). may be revealed. Both have been the subject of extensive crystallographic studies (18,19) with suitable inactive substrates in place. We considered the first step of hydrolytic action of ribonuclease (RNase) on the dinucleotide substrate uridylyl-(3 -5 )-adenosine(UpA). Our results (20) on the enzyme mechanism were consistent with the main features summarized by Roberts et al (21). The first step is a transphosphorylation leading to cleavage "oT the phosphodiester... 

Actual RNA has been cleaved by the artificial enzyme shown in Fig. 6.14 which is a mimic of Staphylococcal nuclease. This is molecular-cleft-type artificial enzyme, where guanidinium residues connected to a rigid backbone... 

RA. Cotton, E.E. Elazen, M.J. Legg, Staphylococcal Nuclease - Proposed Mechanism of Action Based on Structure of Enzyme-Thymidine 3, 5 -Bisphophate-Calcium ion Complex at 1.5 A Resolution , Proc. Natl. Acad. Sci. USA, 76, 2551 (1979)... 

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).]...

FRET pair that reports conformational changes triggered by enzymes, such as staphylococcal nuclease [24], a ribozyme [25], T4 Lysozyme [26]... 

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). 

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