title: 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)
authors.affiliation: M.R.C. Radiobiology Unit, Chilton, Didcot, Oxon, U.K.
authors.forename: P
authors.initials: P
authors.lastname: O'Neill
authors.forename: S
authors.initials: S
authors.lastname: Davies
authors.forename: E M
authors.initials: EM
authors.lastname: Fielden
authors.forename: L
authors.initials: L
authors.lastname: Calabrese
authors.forename: C
authors.initials: C
authors.lastname: Capo
authors.forename: F
authors.initials: F
authors.lastname: Marmocchi
authors.forename: G
authors.initials: G
authors.lastname: Natoli
authors.forename: G
authors.initials: G
authors.lastname: Rotilio
mesh_terms: D000818:Animals; D002021:Buffers; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000378:metabolism*; D009025:Morpholines / Q000494:pharmacology; D009994:Osmolar Concentration; D010472:Perchlorates / Q000494:pharmacology; D010710:Phosphates / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D012492:Salts / Q000494:pharmacology*; D013045:Species Specificity; D013482:Superoxide Dismutase / Q000378:metabolism*; D013481:Superoxides / Q000378:metabolism
chemical_list: D002021:Buffers; D007527:Isoenzymes; D009025:Morpholines; D010472:Perchlorates; D010710:Phosphates; D012492:Salts; D013481:Superoxides; C008550:morpholinopropane sulfonic acid; D013482:Superoxide Dismutase