Role of the electrostatic loop of Cu,Zn superoxide dismutase in the copper uptake process.
Cu,Zn superoxide dismutases are characterized by the presence of four highly conserved charged residues (Lys120, Glu/Asp130, Glu131 and Lys134), which are placed at the edge of the active site channel and have been shown to be individually involved in the electrostatic attraction of the substrate toward the catalytically active copper ion. By genetic engineering we mutated these four residues into neutrally charged ones (Leu120, Gln130, Gln131, Thr134). The effects of these mutations on the rate of superoxide dismutation were not dramatic. In fact, at two different pH and ionic strength values, the mutant enzyme had a catalytic constant even higher with respect to the wild-type protein, showing that electrostatic interaction at these surface sites is not essential for high catalytic efficiency of the enzyme. The mutant and the wild-type enzyme showed the same degree of inhibition by CN(-), and both were not affected by I(-), showing that mutations did not alter the sensitivity of the enzyme to anions. On the other hand, reconstitution of active enzyme from either the wild-type or mutant copper-free enzymes with a copper(I)-glutathione [Cu(I)-GSH] complex showed that metal uptake by the mutant was much slower than by the wild-type enzyme. The demonstration that the 'electrostatic loop' is apparently conserved to assure optimal copper uptake by the enzyme, rather than fast dismutation, may provide further support to the idea that Cu,Zn superoxide dismutase is a bifunctional protein, acting in cellular defense against oxidative stress both as a copper buffer and as a superoxide radical scavenger.
Ciriolo
M R
MR
Department of Biomedical Sciences, University of Chieti G. D'Annunzio, Via dei Vestini 66100 Chieti, Italy. ciriolo@bio.uniroma2.it
Battistoni
A
A
Falconi
M
M
Filomeni
G
G
Rotilio
G
G
D001665:Binding Sites; D002384:Catalysis; D003300:Copper / Q000378:metabolism / Q000493:pharmacokinetics*; D004926:Escherichia coli / Q000201:enzymology; D005982:Glutathione Transferase / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007477:Ions; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D018384:Oxidative Stress; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary; D013482:Superoxide Dismutase / Q000737:chemistry* / Q000502:physiology*; D013997:Time Factors
D007477:Ions; D003300:Copper; D013482:Superoxide Dismutase; D005982:Glutathione Transferase