title: Inactivation of 1-aminocyclopropane-1-carboxylate oxidase involves oxidative modifications.
abstract: 1-Aminocyclopropane-1-carboxylate (ACC) oxidase catalyzes the final step in the biosynthesis of the plant signaling molecule ethylene. It is a member of the ferrous iron dependent family of oxidases and dioxygenases and is unusual in that it displays a very short half-life under catalytic conditions, typically less than 20 min, and a requirement for CO2 as an activator. The rates of inactivation of purified, recombinant ACC oxidase from tomato under various combinations of substrates and cofactors were measured. Inactivation was relatively slow in the presence of buffer alone (t1/2 > 1 h), but fast in the presence of ferrous iron and ascorbate (t1/2 approximately 10 min). The rate of iron/ascorbate-mediated inactivation was increased by the addition of ACC, unaffected by the addition of CO2 at saturation (supplied as bicarbonate) but decreased by the addition of catalase or ACC + CO2 at saturation (supplied as bicarbonate). Iron/ascorbate-mediated inactivation was accompanied by partial proteolysis as observed by SDS-PAGE analysis. The fragmentation pattern was altered when ACC was also included, suggesting that ACC can bind to ACC oxidase in the absence of bicarbonate. N-terminal sequencing of fragments resulted in identification of an internal cleavage site which we propose is proximate to active-site bound iron. Thus, ACC oxidase inactivates via relatively slow partial unfolding of the catalytically active conformation, oxidative damage mediated via hydrogen peroxide which is catalase protectable and oxidative damage to the active site which results in partial proteolysis and is not catalase protectable.
authors.affiliation: Oxford Centre for Molecular Sciences and the Dyson Perrins Laboratory, U.K.
authors.forename: J N
authors.initials: JN
authors.lastname: Barlow
authors.forename: Z
authors.initials: Z
authors.lastname: Zhang
authors.forename: P
authors.initials: P
authors.lastname: John
authors.forename: J E
authors.initials: JE
authors.lastname: Baldwin
authors.forename: C J
authors.initials: CJ
authors.lastname: Schofield
mesh_terms: D000594:Amino Acid Oxidoreductases / Q000037:antagonists & inhibitors*; D000693:Anaerobiosis; D001205:Ascorbic Acid / Q000378:metabolism; D001639:Bicarbonates / Q000378:metabolism; D001665:Binding Sites; D002374:Catalase / Q000378:metabolism; D004229:Dithiothreitol / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006861:Hydrogen Peroxide / Q000378:metabolism; D007501:Iron / Q000378:metabolism; D018551:Solanum lycopersicum; D013058:Mass Spectrometry; D008956:Models, Chemical; D010084:Oxidation-Reduction; D011487:Protein Conformation
chemical_list: D001639:Bicarbonates; D006861:Hydrogen Peroxide; D007501:Iron; D002374:Catalase; D000594:Amino Acid Oxidoreductases; C059460:1-aminocyclopropane-1-carboxylic acid oxidase; D001205:Ascorbic Acid; D004229:Dithiothreitol