A cationic peroxidase was isolated and characterized from the latex of the perennial Mediterranean plant Euphorbia characias. The purified enzyme contained one heme prosthetic group identified as ferric iron-protoporphyrin IX. In addition, the purified peroxidase contained 1 mol of endogenous calcium per mol of enzyme; removal of this calcium ion resulted in almost complete loss of the enzyme activity. However, when excess Ca2+ was added to the native enzyme the catalytic efficiency was enhanced by 3 orders of magnitude. The mechanism of activation was studied using a wide range of spectroscopic and analytic techniques. Analysis of the steady state by stopped-flow measurements suggests that the main effect of calcium ions is to favor the oxidation of the ferric enzyme by hydrogen peroxide to form compound I, whereas the other steps of the catalytic cycle seem to be affected to a lesser extent. UV/vis absorption spectra and CD measurements show that the heme iron is pentacoordinated high-spin in native enzyme and remains so after the binding of Ca2+. Only minor changes in the secondary or tertiary structure of the protein could be detected by fluorescence or CD measurements in the presence of Ca2+ ions, except for a significant perturbation of the Fe3+ inner sphere geometry, as detected by EPR measurements. We propose that Ca2+ binding to a low affinity site induces a reorientation of the distal histidine changing the almost inactive form of Euphorbia peroxidase to a high activity form. This is the first example of a peroxidase that responds as an on/off switch to variations in the external Ca2+ level.
Medda, R., Padiglia, A., Longu, S., Bellelli, A., Arcovito, A., Cavallo, S., et al. (2003). Critical role of Ca2+ ions in the reaction mechanism of Euphorbia characias peroxidase. BIOCHEMISTRY, 42(29), 8909-8918 [10.1021/bi034609z].
|Tipologia:||Articolo su rivista|
|Citazione:||Medda, R., Padiglia, A., Longu, S., Bellelli, A., Arcovito, A., Cavallo, S., et al. (2003). Critical role of Ca2+ ions in the reaction mechanism of Euphorbia characias peroxidase. BIOCHEMISTRY, 42(29), 8909-8918 [10.1021/bi034609z].|
|IF:||Con Impact Factor ISI|
|Settore Scientifico Disciplinare:||Settore BIO/10|
|Revisione (peer review):||Sì, ma tipo non specificato|
|Digital Object Identifier (DOI):||http://dx.doi.org/10.1021/bi034609z|
|Stato di pubblicazione:||Pubblicato|
|Data di pubblicazione:||2003|
|Titolo:||Critical role of Ca2+ ions in the reaction mechanism of Euphorbia characias peroxidase|
|Autori:||Medda, R; Padiglia, A; Longu, S; Bellelli, A; Arcovito, A; Cavallo, S; Pedersen, JZ; Floris, G|
|Appare nelle tipologie:||01 - Articolo su rivista|