Anthranilic acid oxidase system of Tecoma stans - II. Studies on the properties of a purified anthranilic acid oxidase system and its separation into different enzymic components

Abstract

An enzyme system which catalysed the conversion of anthranilic acid to catechol has been purified 20-fold from a cell-free leaf extract of Tecoma stans. The optimum substrate concentration was 10^-3 M and optimum temperature for the reaction was 45°. The presence of a multi-enzyme system was inferred from inhibition studies. The formation of catechol was inhibited by Mg²⁺, Zn²⁺, and Co²⁺ ions, whereas anthranilic acid disappearance was not affected to the same extent. The effect of metal chelating agents like EDTA, cyanide and pyrophosphate showed a similar trend. PCMB inhibited catechol formation but had no effect on anthranilic acid disappearance. The reaction was not inhibited by catalase, nor was it activated by peroxide-donating systems. This ruled out the possibility of peroxidative type of reaction. The overall reaction is markedly activated by NADPH and THFA. This multi-enzyme was separated into three different components, by fractionation with Alumina C_γ and calcium phosphate gels. The overall reaction catalysed by these components can be represented as anthranilic acid→3-hydroxy anthranilic acid→ο -aminophenol→catechol.