Nuclease, staphylococcal

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 Mutagenesis studies in which Glu43 has been replaced by Asp or Gin have shown Glu to be important for high catalytic activity.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 6 Thermodynamic cycle for multi-substate free energy calculation. System A has n substates system B has m. The free energy difference between A and B is related to the substate free energy differences through Eq. (41). A numerical example is shown in the graph (from Ref. 39), where A and B are two isomers of a surface loop of staphylococcal nuclease, related by cis-trans isomerization of proline 117. The cis trans free energy calculation took into account 20 substates for each isomer only the six or seven most stable are included in the plot.

Figure 7 Experimental and theoretical inelastic neutron scattering spectrum from staphylococcal nuclease at 25 K. The experimental spectrum was obtained on the TFXA spectrometer at Oxford. The calculated spectrum was obtained from a normal mode analysis of the isolated molecule. (From Ref. 28.)...

Tucker, P.W., Hazen, E.E., Colton, F.A. Staphylococcal nuclease reviewed a prototypic study in contemporary enzymology. III. Correlation of fhe three-dimensional structure with the mechanisms of enzymatic action. Mol. Cell. Biochem. 

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... 

FIGURE 8.9. Linear free-energy relationship for the effect of metal substitution on e2 and e3 in staphylococcal nuclease (see text for details). 

Staphylococcal nuclease Phosphoglycerides, hydrolysis of ester bond in, 204... 

SNase, see Staphylococcal nuclease (SNase) S,v2 reactions, see Substitution reactions, nucleophilic (Sw2)... 

Nitrophenyl esters of thymidine are substrates for staphylococcal nuclease and ribonucleotide 5 -(5-iodoindol-3-ol) (3) and 5 -(4-methyl-coumarin-7-ol) (4) esters have been used for the histochemical demon-... 

Another advantage of PB based pKa calculations is that effects of electrolytes are readily accounted for in the PB equation. The Coulombic contribution in conjunction with salt dependence to the abnormally depressed pAVs of histidine in staphylococcal nuclease has been experimentally tested [56], Recently, the methodology used in the PB calculations (Eqs. 10-11 and 10-12) has been combined with the generalized Born (GB) implicit solvent model [94] to offer pKa predictions at a reduced computational cost [52],... 

Lee K, Fitch CA, Lecomte JT, Garcfa-Moreno EB (2002) Electrostatic effects in highly charged proteins Salt sensitivity of pKa values of histidines in staphylococcal nuclease. Biochemistry 41 5656-5667. 

Shortle has focused on the unfolded state for more than a decade, leading up to his recent demonstration using residual dipolar couplings that staphylococcal nuclease retains global structure in 8 M urea. His chapter on The Expanded Denatured State sets the stage. Dunker etal. then explore the complementary world of disordered regions within... 

In this review, NMR analysis of the denatured state of staphylococcal nuclease is briefly reviewed in nontechnical language. Most of the work has come from the author s laboratory over the past eight years. The initial experiments, which only measure local structural parameters, reported small amounts of persisting helical structure, two turns, and indirect evidence for perhaps a three-strand beta meander. When applied to the denatured state in 6 M urea, the same experiments indicated that most of these features are lost. 

Staphylococcal nuclease is a small a + ft protein of 149 amino acids that is stable to denaturation by only +5.5 kcal/mol (Anfinsen et al.,... 

Fig. 4. Scatterplots of the Nh residual dipolar couplings from three different denatured states of staphylococcal nuclease plotted against each other. (A) Wild-type, full-length nuclease in 4 M urea (y-axis) versus A131A in buffer (x-axis). (B) Wild-type, full-length nuclease denatured by acid (25 mM citrate, pH 3.0) (y-axis) versus A131A in buffer (x-axis).

Privalov et al (1989) studied the unfolded forms of several globular proteins [ribonuclease A, hen egg white lysozyme, apomyoglobin (apoMb), cytochrome c, and staphylococcal nuclease]. Unfolding was induced by 6 M Gdm-HCl at 10°C, heating to 80°C, or by low pH at 10°C with cross-links cleaved (reduction and carboxamidomethylation or removal of heme). The unfolded forms showed CD spectra (Fig. 27)... 

Hodel, A. Rice, L. M. Simonson, T. Fox, R. O. Briinger, A. T., Proline cis-trans isomerization in staphylococcal nuclease — multi-substate free-energy perturbation calculations, Prot. Sci. 1995, 4, 636-654. 

Dunn, B.M., and Affinsen, C.B. (1974) Kinetics of Woodward s reagent K hydrolysis and reaction with staphylococcal nuclease./. Biol. Chem. 249, 3717. 

Ermacora, M.R., Delfino, J.M., Cuenoud, B., Schepartz, A., and Fox, R.O. (1992) Conformation-dependent cleavage of Staphylococcal nuclease with a disulfide-linked iron chelate. PNAS 89, 6383. 

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