International Journal of Biological Macromolecules 49 (2011) 1078–1082 Contents lists available at SciVerse ScienceDirect International Journal of Biological Macromolecules journa l h o me pag e: www.elsevier.com/locate/ijbiomac Suicide inactivation of peroxidase from Chamaerops excelsa palm tree leaves Nazaret Hidalgo Cuadrado a , Galina G. Zhadan b , Manuel G. Roig a,∗ , Valery L. Shnyrov b a Departamento de Química Física, Facultad de Ciencias Químicas, Universidad de Salamanca, 37008 Salamanca, Spain b Departamento de Bioquímica y Biología Molecular, Universidad de Salamanca, 37007, Spain a r t i c l e i n f o Article history: Received 27 July 2011 Received in revised form 31 August 2011 Accepted 1 September 2011 Available online 8 September 2011 Keywords: Chamaerops excelsa peroxidase Hydrogen peroxide Mechanism-based inactivation Suicide inactivation Ageing a b s t r a c t The concentration and time-dependences and the mechanism of the inactivation of Chamaerops excelsa peroxidase (CEP) by hydrogen peroxide were studied kinetically with four co-substrates (2,2 ′ -azino- bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), guaiacol, o-dianisidine and o-phenylenediamine). The turnover number (r) of H 2 O 2 required to complete the inactivation of the enzyme varied for the different substrates, the enzyme most resistant to inactivation (r = 4844) with ABTS being the most useful substrate for biotechnological applications, opening a new avenue of enquiry with this peroxidase. © 2011 Elsevier B.V. All rights reserved. 1. Introduction Peroxidases (EC 1.11.1.7; donor: hydrogen peroxide oxidorre- ductase) form a group of enzymes that utilize H 2 O 2 to oxidize a second (reducing) substrate. These enzymes share a simi- lar catalytic cycle in which H 2 O 2 reacts with the resting ferric enzyme to form the intermediate Compound I, which carries two oxidizing equivalents. Compound I is subsequently reduced by reactions with two reducing substrate molecules. The first of these reduction steps generates the intermediate, Compound II, which is then further reduced back to the ferric native enzyme. Palm peroxidase reacts with a large variety of reducing sub- strates, using H 2 O 2 as an oxidizing agent [1–3]. In the absence of reducing substrates, an excess of H 2 O 2 leads to inactivation of the enzyme, in this case the H 2 O 2 acting as a suicide sub- strate of peroxidase and being irreversibly bound to its active site [4,5]. However, it has been suggested by several authors [6,7] that the HRP inactivation by hydrogen peroxide is due to the formation of one or several non-active enzyme prod- ucts likely through the formation of Compound III (peroxyl-FeIII porphyrin). The oxidative inactivation of peroxidases is mechanism- based. The molecular mechanism underlying this hydrogen ∗ Corresponding author. Tel.: +34 923 294 487; fax: +34 923 294 579. E-mail address: mgr@usal.es (M.G. Roig). peroxide-mediated inactivation is extraordinarily complex because a multitude of reactions can occur subsequent to the reaction of the haem iron with the hydroperoxide. Despite the peculiarities among the different peroxidases, a common inacti- vation mechanism comprising several stages can be proposed. In the absence of substrate, or when exposed to high concentrations of hydrogen peroxide, peroxidases show the kinetic behavior of suicide inactivation, in which hydrogen peroxide is the sui- cide substrate that converts Compound II into a highly reactive peroxy-iron(III) porphyrin free-radical named Compound III [8]. Compound III is not part of the peroxidase cycle, but is produced under excessive exposure of protonated Compound II to oxida- tive species in a reaction partially mediated by free superoxide radical [9]. Despite representing different structural groups, kinetic mod- els for the hydrogen peroxide-mediated inactivation of horseradish peroxidase (HRP) [5], ascorbate peroxidase (APX) [10], peroxidase from the Royal Palm Tree (RPTP) [11], microperoxidase-11 [12] and CEP are similar in that they are time-dependent and show satura- tion kinetics. From the stoichiometry of the inactivation, it has been concluded that for APX only 2.5 molecules of hydrogen peroxide are required per active site to generate the inactivation form [10], in contrast to the 265 molecules required for HRP [5]. This difference is due to the low catalytic activity of HRP, which is absent in APX [13]. For APX peroxidase, inactivation is correlated with enzyme bleaching, suggesting haem destruction [10]. Another factor in this difference is the glycosylation of the enzyme, which appears to be significant in protecting the enzyme from inactivation [5]. 0141-8130/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ijbiomac.2011.09.001