title: Mechanism-based suicide inactivation of white Spanish broom (Cytisus multiflorus) peroxidase by excess hydrogen peroxide.
abstract: Suicide inactivation is a common mechanism observed for haem peroxidases, in which the enzyme is inactivated as a result of self-oxidation mediated by intermediate highly oxidizing enzyme forms during the catalytic cycle. The time-dependence and the inactivation mechanism of Cytisus multiflorus peroxidase (CMP) by hydrogen peroxide were studied kinetically with four co-substrates (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), ferulic acid, guaiacol and o-dianisidine). Catalytic activity decreased following the sequence ABTS>guaiacol>ferulic acid>o-dianisidine. Once the intermediate complex (compound III-H2O2) had been formed, competition was established between the catalytic pathway and the suicide inactivation pathway. One mole of CMP afforded around 3790 turnovers of H2O2 for ABTS before its complete inactivation. These results suggest that CMP follows a suicide mechanism, the enzyme not being protected in this case. The mechanism of suicide inactivation is discussed with a view to establishing a broad knowledge base for future rational protein engineering.
authors.affiliation: Centro de Investigación y Desarrollo Tecnológico del Agua (CIDTA), Universidad de Salamanca, 37007 Salamanca, Spain.
authors.forename: Patricia Pérez
authors.initials: PP
authors.lastname: Galende
authors.affiliation: Instituto de Estudios Biofuncionales, Departamento de Química-Física II, Facultad de Farmacia, Universidad Complutense de Madrid, 28040 Madrid, Spain. Electronic address: nazahidalgo@gmail.com.
authors.forename: Nazaret Hidalgo
authors.initials: NH
authors.lastname: Cuadrado
authors.affiliation: Department of Biochemistry, Microbiology and Biotechnology, Far Eastern Federal University, 690600 Vladivostok, Russia.
authors.forename: Eduard Ya
authors.initials: EY
authors.lastname: Kostetsky
authors.affiliation: Centro de Investigación y Desarrollo Tecnológico del Agua (CIDTA), Universidad de Salamanca, 37007 Salamanca, Spain; Departamento de Química Física, Universidad de Salamanca, 37008 Salamanca, Spain. Electronic address: mgr@usal.es.
authors.forename: Manuel G
authors.initials: MG
authors.lastname: Roig
authors.affiliation: Chembiotech Laboratories, Kyrewood House, Tenbury Wells, Worhestershire WR15 8SG, UK.
authors.forename: John F
authors.initials: JF
authors.lastname: Kennedy
authors.affiliation: Departamento de Bioquímica y Biología Molecular, Universidad de Salamanca, 37007 Salamanca, Spain.
authors.forename: Valery L
authors.initials: VL
authors.lastname: Shnyrov
mesh_terms: D029865:Cytisus / Q000201:enzymology*; D004789:Enzyme Activation / Q000187:drug effects; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D007700:Kinetics; D016018:Least-Squares Analysis; D013997:Time Factors
chemical_list: D006861:Hydrogen Peroxide
keywords: Cytisus multiflorus peroxidase; Haem peroxidase; Hydrogen peroxide; Mechanism-based inactivation; Suicide inactivation; Suicide substrate