title: Inactivation of cholinesterase by ascorbic acid in the presence of cupric ions: a possible mechanism for the inactivation of an enzyme by the metal-catalyzed oxidation system.
abstract: The mechanism of inactivation of cholinesterase (EC 3.1.1.8) by the Cu2+ -ascorbic acid (AsA) system was investigated. Incubation of the enzyme with the Cu2+ -AsA system under aerobic conditions resulted in an irreversible loss of enzyme activity. At low concentrations of Cu2+, the extent of inactivation showed the same dependence in accordance with the extent of oxidation of AsA. Saturation kinetics were observed with respect to the concentration of AsA. No change in the dissociation constant of the enzyme-AsA complex was observed at various concentrations of Cu2+. Catalase at a low concentration partially protected the enzyme from the inactivation, but did not affect the oxidation of AsA. In addition, catalase at a high concentration completely protected both the enzyme from inactivation and the AsA from oxidation. Both thiourea and thiocyanate completely protected the enzyme from the inactivation, while AsA was partially oxidized only in the initial phase. Our proposed mechanism for the inactivation of an enzyme by the Cu2+ -AsA system is as follows. A ternary complex involving the enzyme, Cu2+ and AsA is formed. This is followed by a redox reaction within the complex which generates a superoxide (.O2-) and hydrogen peroxide (H2O2). The H2O2 then reacts with .O2- in a Haber-Weiss reaction producing the hydroxyl radical (.OH). Another role of H2O2 is the conversion of the reduced Cu+ within the complex to Cu2+. Thus, repeated cycles of the redox reaction between the Cu2+ and AsA take place at the same locus, producing multiple .OH, which causes its complete inactivation.
authors.affiliation: Kyoto Pharmaceutical University, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Kanazawa
authors.forename: S
authors.initials: S
authors.lastname: Fujimoto
authors.forename: A
authors.initials: A
authors.lastname: Ohara
mesh_terms: D000818:Animals; D001205:Ascorbic Acid / Q000494:pharmacology*; D002800:Cholinesterase Inhibitors / Q000494:pharmacology*; D003300:Copper / Q000494:pharmacology*; D016166:Free Radical Scavengers / Q000494:pharmacology; D006736:Horses; D017665:Hydroxyl Radical; D010084:Oxidation-Reduction
chemical_list: D002800:Cholinesterase Inhibitors; D016166:Free Radical Scavengers; D017665:Hydroxyl Radical; D003300:Copper; D001205:Ascorbic Acid