The effects of pH and various salts upon the activity of a series of superoxide dismutases

Abstract

The CuZn superoxide dismutases (SODs) from ox, sheep, pig and yeast were investigated by pulse radiolysis in order to evaluate the role of electrostatic interactions between O2.- and SOD proteins in the mechanism of action of the SOD enzymes. The protein net charge in this series varies, as evaluated by the protein pI values spanning over a large range of pH: 8.0 (sheep), 6.5 (pig), 5.2 (ox) and 4.6 (yeast). The amino acid sequences are largely conserved, with the three mammalian proteins being highly homologous and the yeast protein having some distinct variations in the region surrounding the active site. At pH 8.0 the activities of the SODs from various sources are similar, though the minor differences observed suggest that in the highly homologous mammalian series the most acidic protein is the most enzymically efficient one. The pH-dependences of the various activities in the pH range 7-12 are similar, and the related curves are best fitted by two pK values, which are approx. 9.2 and 11.0 for the mammalian enzymes and 9.1 and 11.4 for the yeast enzyme. The activities of the proteins at I 0.1 are decreased by approx. 20% when compared with the activity at I 0.02 at pH 8.5, whereas at pH above 10 the pH-dependence of the activity approaches that determined at I 0.02 and at pH 11.9 the activity is essentially independent of ionic strength. The dependence upon ionic strength also depends on the salt used, with perchlorate being more effective than phosphate or borate or Mops and still effective at pH above 10.5, where the effect of other salts becomes negligible. The dual and concerted dependence of the activities of different SODs on pH and salt concentration is explained with the encounter of O2.- with the active-site copper being governed by the protonation of two positively charged groups in the vicinity of the active site. The gradient between these localized charges and the rest of the protein may explain the different activities of the mammalian proteins at lower pH. On the basis of the sequence variation of the SODs examined it is not possible to definitely identify these groups. Likely candidates are conserved basic amino acid side chains in the vicinity (less than or equal to 1.2 nm) of the active site, i.e. Lys-134 and Arg-141, but co-ordination of OH- in the first copper co-ordination sphere may be an additional factor accounting for the higher pK.(ABSTRACT TRUNCATED AT 400 WORDS)

Superoxide Dismutase from the Silkworm,Bombyx mori: Sequence, Distribution, and Overexpression

“…The bmSOD was stable in a wide pH rage under the experimental condition and the stability was similar to those of SODs. [42][43][44] Successful construction of the overproduction system of rSOD makes further characterization and studies including Western blot analysis possible. Fat bodies was dissected at various times after irradiation by gamma rays at 10 Gy ( ), 50 Gy ( ), and 100 Gy ( ), and proteins were extracted (see ''Materials and Methods'').…”

Role of the electrostatic loop charged residues in Cu, Zn superoxide dismutase

“…This result indicates either that neutralization of Lysl20 allows the occurrence of new diffusion pathways of the substrate toward the active site or that eukaryotic Cu,Zn SODs, although displaying a very similar three-dimensional structure, are characterized by subtle changes that may produce a different modulation of the electrostatic attraction of the negatively charged substrate toward the active site. A common effect observed in all the natural and mutant Cu,Zn SOD eukaryotic variants studied so far is the large decrease of the catalytic rate upon increasing the ionic strength (O'Neill et al, 1988;Getzoff et al, 1992;Polticelli et al, 1995;Fisher et al, 1997). In the case of X. Zaevis SOD, as for bovine and human SODs, the net charge is negative (-6e for X .…”

The pH‐Dependent Changes of the Enzymic Activity and Spectroscopic Properties of Iron‐Substituted Manganese Superoxide Dismutase

“…The pH of the sample solution for each spectral measurement was obtained using a micro-combination pH electrode (Microelectrodes, Inc.). Curve-fitting procedures were performed to obtain the best fits of the data based on the pH-dependent activity, spectral changes and dissociation constants of azide being governed by one prototrophic equilibrium, Ka [22]. The following equations were used to obtain the best fits: (Whatman Chemical Separation) column (1 cm X 2 cm), previously equilibrated with 10 mM potassium phosphate, pH 7.8.…”