title: Purification and unusual kinetic properties of a tobacco anionic peroxidase.
abstract: The tobacco anionic peroxidase has been isolated from the leaves of transgenic Nicotiana sylvestris plants overproducing this enzyme. The plant expression system and the purification protocol developed allow the preparation of greater than 60 mg of homogeneous enzyme (M(r) 36 kDa, pI 3.5) from 1 kg of fresh leaves, which is an order of magnitude higher than for wild-type tobacco plants. The tobacco anionic peroxidase exhibits rather unusual catalytic properties in comparison with horseradish peroxidase (HRP C). Compound I is less active than Compound II in the tobacco enzyme. The enzyme is nearly inactive towards iodide, reflecting the peculiarities of its molecular structure. In particular, the presence of the negatively charged glutamate residue 141 at the entrance of the haeme-binding pocket seems to affect the stabilities of Compounds I, II and III, leading to a different enzyme substrate specificity than that of HRP C. Investigation of thermal stability towards a number of electron donors reveals the following 'order of stabilities': ferrocyanide > guaiacol > 2,2'-azino-bis(3-ethyl-6- benzothiazoline sulphonate) > iodide > o-dianisidine, which may indicate different binding sites and rate-limiting steps in the mechanism of the substrate oxidation.
authors.affiliation: Department of Horticulture and Crop Sciences, Ohio State University, Columbus 43210-1096, USA.
authors.forename: I G
authors.initials: IG
authors.lastname: Gazaryan
authors.forename: L M
authors.initials: LM
authors.lastname: Lagrimini
mesh_terms: D000595:Amino Acid Sequence; D000838:Anions; D002384:Catalysis; D002848:Chromatography, DEAE-Cellulose; D007700:Kinetics; D008969:Molecular Sequence Data; D010544:Peroxidases / Q000302:isolation & purification* / Q000378:metabolism*; D030821:Plants, Genetically Modified; D010947:Plants, Toxic; D013057:Spectrum Analysis; D013379:Substrate Specificity; D014026:Nicotiana / Q000201:enzymology*
chemical_list: D000838:Anions; D010544:Peroxidases