title: An inserted loop region of stromal ascorbate peroxidase is involved in its hydrogen peroxide-mediated inactivation.
abstract: Ascorbate peroxidase isoforms localized in the stroma and thylakoid of higher plant chloroplasts are rapidly inactivated by hydrogen peroxide if the second substrate, ascorbate, is depleted. However, cytosolic and microbody-localized isoforms from higher plants as well as ascorbate peroxidase B, an ascorbate peroxidase of a red alga Galdieria partita, are relatively tolerant. We constructed various chimeric ascorbate peroxidases in which regions of ascorbate peroxidase B, from sites internal to the C-terminal end, were exchanged with corresponding regions of the stromal ascorbate peroxidase of spinach. Analysis of these showed that a region between residues 245 and 287 was involved in the inactivation by hydrogen peroxide. A 16-residue amino acid sequence (249-264) found in this region of the stromal ascorbate peroxidase was not found in other ascorbate peroxidase isoforms. A chimeric ascorbate peroxidase B with this sequence inserted was inactivated by hydrogen peroxide within a few minutes. The sequence forms a loop that binds noncovalently to heme in cytosolic ascorbate peroxidase of pea but does not bind to it in stromal ascorbate peroxidase of tobacco, and binds to cations in both ascorbate peroxidases. The higher susceptibility of the stromal ascorbate peroxidase may be due to a distorted interaction of the loop with the cation and/or the heme.
authors.affiliation: Research Institute of Innovative Technology for the Earth (RITE), Soraku-gun, Kyoto, Japan.
authors.forename: Sakihito
authors.initials: S
authors.lastname: Kitajima
authors.forename: Ken-ichi
authors.initials: K
authors.lastname: Tomizawa
authors.forename: Shigeru
authors.initials: S
authors.lastname: Shigeoka
authors.forename: Akiho
authors.initials: A
authors.lastname: Yokota
mesh_terms: D000595:Amino Acid Sequence; D060387:Ascorbate Peroxidases; D001665:Binding Sites; D002736:Chloroplasts / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D003600:Cytosol / Q000201:enzymology / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000378:metabolism / Q000494:pharmacology*; D008969:Molecular Sequence Data; D010544:Peroxidases / Q000737:chemistry* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000461:Rhodophyta / Q000166:cytology / Q000201:enzymology; D016415:Sequence Alignment; D018724:Spinacia oleracea / Q000166:cytology / Q000201:enzymology; D014026:Nicotiana / Q000166:cytology / Q000201:enzymology
chemical_list: D011993:Recombinant Fusion Proteins; D006861:Hydrogen Peroxide; D010544:Peroxidases; D060387:Ascorbate Peroxidases