New indole oxygenase from the leaves of Tecoma stans L. Part I: Affinity purification and properties

Abstract

Indole oxygenase from the leaves of Tecoma stans was purified to homogeneity using 5-hydroxyindolecoupled epoxy-activated Sepharose. The purity was checked by polyacrylamide gel electrophoresis and immunoelectrophoresis. This enzyme was shown to be identical to the one purified by conventional purification method. Indole oxidase, an enzyme catalyzing the conversion of indole to anthranil, was not co-purified during affinity chromatography. A model for the orientation of the substrate in the substrate-enzyme complex was proposed from the substrate specificity, inhibition studies, and the ability of the enzyme to bind to affinity matrices with different substrate orientation. Dissociation constants for 5-hydroxyindole, 5-bromoindole and 7-methylindole were determined by kinetic and fluorescencequenching experiments and are compared. A mechanism is. proposed for the conversion of indole to anthranilic acid, with the intermediary formation of N-formylaminobenzaIdehyde and o-aminobenzaldehyde, by indole oxygenase from the trapping experiments.