Meccanismo catalitico della perossidasi da Euphorbia characias

Peroxidases (EC 1.11.1.1-16) are a group of oxidoreductases utilizing hydrogen peroxide, or other peroxides, to oxidize a second reducing substrate belonging to a large array of organic and inorganic compounds. A class III peroxidase isolated from the latex of Mediterranean shrub Euphorbia characias (ELP), has peculiar features with respect to the other eme-peroxidases. In fact ELP is the first example of peroxidase that responds as an on/off switch to variation in the external Ca2+ level and represents the first peroxidase regulated by the Ca2+/calmodulin system. The catalytic mechanism of ELP was well studied and characterized. In the present thesis we investigate the catalyc pathways of ELP with hydrogen peroxide as the unique substrate and in both the presence and absence of calcium ions showing the presence oh three different pathways. Pathway I: ELP shows catalise-like activity catalyzing decomposition of hydrogen peroxide to water and oxygen; Pathway II involves the return to native enzyme with the concomitant formation of superoxyde anion; Pathway III involves an irresistible loss of enzyme activity, with hydrogen peroxide acting as a suicide substrate. Because the importance of the Ca2+ ions to influence the catalytic activity of Euphorbia peroxidase, we investigate the effects of another divalent cation (Ni2+) on the catalytic pathways of native ELP and in the presence of calcium ions. The enzyme is strongly inhibited by nickel ions an the absence of Ca2+, whereas, in the presence of both Ca2+ and Ni2+, the enzymatic activity is enhanced. The reported results are compatible with the hypothesis that ELP has Ni2+-binding sites eith opposite functional effects.