title: Mechanistic studies of 1-aminocyclopropane-1-carboxylic acid oxidase: single turnover reaction.
abstract: The final step in the biosynthesis of the plant hormone ethylene is catalyzed by the non-heme iron-containing enzyme 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase (ACCO). ACC is oxidized at the expense of O(2) to yield ethylene, HCN, CO(2), and two waters. Continuous turnover of ACCO requires the presence of ascorbate and HCO(3)(-) (or an alternative form), but the roles played by these reagents, the order of substrate addition, and the mechanism of oxygen activation are controversial. Here these issues are addressed by development of the first functional single turnover system for ACCO. It is shown that 0.35 mol ethylene/mol Fe(II)ACCO is produced when the enzyme is combined with ACC and O(2) in the presence of HCO(3)(-) but in the absence of ascorbate. Thus, ascorbate is not required for O(2) activation or product formation. Little product is observed in the absence of HCO(3)(-), demonstrating the essential role of this reagent. By monitoring the EPR spectrum of the sample during single turnover, it is shown that the active site Fe(II) oxidizes to Fe(III) during the single turnover. This suggests that the electrons needed for catalysis can be derived from a fraction of the initial Fe(II)ACCO instead of ascorbate. Addition of ascorbate at 10% of its K(m) value significantly accelerates both iron oxidation and ethylene formation, suggesting a novel high-affinity effector role for this reagent. This role can be partially mimicked by a non-redox-active ascorbate analog. A mechanism is proposed that begins with ACC and O(2) binding, iron oxidation, and one-electron reduction to form a peroxy intermediate. Breakdown of this intermediate, perhaps by HCO(3)(-)-mediated proton transfer, is proposed to yield a high-valent iron species, which is the true oxidizing reagent for the bound ACC.
authors.affiliation: Department of Biochemistry, Molecular Biology, and Biophysics, and Center for Metals in Biocatalysis, University of Minnesota, Minneapolis, MN 55455, USA.
authors.forename: Amy M
authors.initials: AM
authors.lastname: Rocklin
authors.forename: Keisuke
authors.initials: K
authors.lastname: Kato
authors.forename: Hung-wen
authors.initials: HW
authors.lastname: Liu
authors.forename: Lawrence
authors.initials: L
authors.lastname: Que
authors.forename: John D
authors.initials: JD
authors.lastname: Lipscomb
mesh_terms: D000594:Amino Acid Oxidoreductases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D001205:Ascorbic Acid / Q000378:metabolism; D001639:Bicarbonates / Q000378:metabolism; D001881:Borates / Q000494:pharmacology; D004578:Electron Spin Resonance Spectroscopy; D004583:Electrons; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology; D005030:Ethylenes / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D007700:Kinetics; D010084:Oxidation-Reduction; D017693:Sodium Bicarbonate / Q000494:pharmacology
chemical_list: D001639:Bicarbonates; D001881:Borates; D005030:Ethylenes; D017693:Sodium Bicarbonate; D000594:Amino Acid Oxidoreductases; C059460:1-aminocyclopropane-1-carboxylic acid oxidase; D001205:Ascorbic Acid