Chloroperoxidase can utilize chloride, bromide, and iodide ions and catalyze the formation of a carbon-halogen bond in the presence of suitable acceptor molecules. Suitable halogen acceptor molecules are β-keto acids, cyclic β-diketones, and substituted phenols. An optical assay has been developed to measure the halogenating activity of chloroperoxidase. The assay is based on the conversion of monochlorodimedon to the corresponding gem-dihalo derivative. The optical assay is based on the loss of absorbance at 278 mµ associated with the formation of the dihalo derivative from the monohalo compound. In the optical assay, crystalline chloroperoxidase preparations catalyze the formation of the dichlorodimedon from monochlorodimedon, chloride ion, and hydrogen peroxide at a rate of 67,000 moles of product formed per min per mole of enzyme. When bromide ion replaces chloride ion in the optical assay, the rate of formation of monobromo-monochlorodimedon is approximately twice the rate of formation of dichlorodimedon. The chloroperoxidase-catalyzed halogenation of tyrosine can be followed by measuring the decrease in tyrosine fluorescence associated with the formation of the mono- and dihalogenated tyrosines. At low concentrations of halogen anion (0.33 mm) the relative activity among the halogen anions for the halogenation of tyrosine is approximately 5.1:4.8:1 for iodide, bromide, and chloride, respectively. At higher concentrations of halogen anion, 3.3 mm, the ratio of activity between bromide and chloride falls to 3:1. At 3.3 mm iodide concentration, the enzymatic formation of elemental iodine masks the tyrosine fluorescences measurements so that rate comparisons cannot be made in this instance. Crystalline chloroperoxidase preparations catalyze the chlorination of tyrosine at a rate of approximately 7500 moles of product formed per min per mole of enzyme. Fluoride is not a substrate for chloroperoxidase but is an inhibitor of the halogenation reaction. The results indicate that fluoride ion can compete for both the hydrogen peroxide- and the halogen anion-binding sites on chloroperoxidase. Chloroperoxidase shares with other peroxidases the ability to catalyze the oxidation of iodide to iodine. The rate of iodine formation is about 1.5 times the rate of chlorination of monochlorodimedon when the reactions are compared, with equivalent amounts of enzyme and under saturating conditions for all substrates.
This research was originally published in the Journal of Biological Chemistry. Hager, L.P., Morris, D.R., Brown, F.S., and Eberwein, H. Chloroperoxidase: II. UTILIZATION OF HALOGEN ANIONS. Journal of Biological Chemistry. 1966.241:1749-1777. © the American Society for Biochemistry and Molecular Biology
Lowell P. Hager, David R. Morris, Frederick S. Brown and Horst Eberwein. 1966. "Chloroperoxidase: II. UTILIZATION OF HALOGEN ANIONS." Journal of Biological Chemistry 241:1769-1777.