title: Purification and characterization of a novel class III peroxidase isoenzyme from tea leaves.
abstract: A novel, basic (isoelectric point > 10), heme peroxidase isoenzyme (TP; relative molecular weight = 34,660 +/- 10, mean +/- SE) that can account for a significant part of the ascorbate peroxidase activity in tea (Camellia sinensis) leaves has been purified to homogeneity. The ultraviolet/visible absorption spectrum is typical of heme-containing plant peroxidases, with a Soret peak at 406 nm (epsilon = 115 mM-1 cm-1) and an A406/A280 value of 3.4. The enzyme has a high specific activity for ascorbate oxidation (151 mumol min-1 mg-1), with a pH optimum in the range of 4.5 to 5.0. Substrate-specificity studies have revealed significant differences between TP and other class III peroxidases, as well as similarities with class I ascorbate peroxidases. TP, like ascorbate peroxidase, exhibits a preference for ascorbate over guaiacol, whereas other class III isoenzymes are characterized by 2-orders-of-magnitude higher activity for guaiacol than for ascorbate. TP also forms an unstable porphyrin pi cation radical-type compound I, which is converted to compound II within approximately 2 min in the absence of added reductant. Amino acid sequence data show TP to be the first example, to our knowledge, of a class III peroxidase with a high specificity for ascorbate as an electron donor.
authors.affiliation: Nitrogen Fixation Laboratory, John Innes Centre, Norwich, United Kingdom.
authors.forename: M
authors.initials: M
authors.lastname: Kvaratskhelia
authors.forename: C
authors.initials: C
authors.lastname: Winkel
authors.forename: R N
authors.initials: RN
authors.lastname: Thorneley
mesh_terms: D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D010084:Oxidation-Reduction; D010449:Peptide Mapping; D009195:Peroxidase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D013379:Substrate Specificity; D013662:Tea / Q000201:enzymology*
chemical_list: D007527:Isoenzymes; D013662:Tea; D009195:Peroxidase