Studies on the metabolism of aminophenols

Abstract

Certain o-aminophenols like 3-hydroxykynurenine and 3-hydroxyanthranilic acid are known to be precursors of nicotinic acid as well as of certain phenoxazinone antibiotics and insect pigments. The metabolism of 3-hydroxyanthranilic acid, which is an intermediate in tryptophan metabolism, has received only scant attention except for its conversion to pyridine carboxylic acids. This metabolite is also a potent carcinogen. With a view to investigate the alternate routes of metabolism, rat liver nuclear fraction has been chosen, and it has been found that this fraction could convert 3-hydroxyanthranilic acid to a phenoxazinone compound which was identified as cinnabarinic acid (2-amino-3H-iso-phenoxazin-3-one-1,9-dicarboxylic acid). The enzyme cinnabarinic acid synthase (3-hydroxyanthranilic acid: O? oxidoreductase) was purified 69-fold and its properties studied. The enzyme showed maximal activity at pH 7.2 and 37°C, required ions for optimum activity, and was inhibited by heavy metal ions and metal-chelating agents. The enzyme catalyzes the oxidative condensation of 2 moles of 3-hydroxyanthranilic acid to form 1 mole of cinnabarinic acid with the consumption of 7 atoms of oxygen. The mechanism of the reaction and its significance in mammals is discussed. A similar reaction was also observed in the leaves of Tecoma stans. The purified enzyme showed striking differences in its properties from the rat liver enzyme as well as from the Tecoma stans isophenoxazine synthase reported earlier. There was a requirement for Mn²? ions, optimum pH was around 8.0, and optimum temperature was 30°C. The system is highly specific for 3-hydroxyanthranilic acid and the Km value was 3.6 × 10?? M. The enzyme preparation readily oxidized reduced cytochrome C which was prepared by reduction with 3-hydroxyanthranilic acid. An enzyme capable of converting o-aminophenol to 2-amino-3H-iso-phenoxazin-3-one was isolated from the leaves of Tecoma purpurea and purified 390-fold. The enzyme isophenoxazine synthase did not show requirement for any cofactor. The stoichiometry of the reaction was established. Substrate analogues like anthranilic acid and 3-hydroxyanthranilic acid competitively inhibited the reaction. There was liberation of free sulphydryl groups on incubating the enzyme with the substrate or 3-hydroxyanthranilic acid. The conversion of anthranilic acid to catechol in Tecoma stans involves the intermediate formation of 3-hydroxyanthranilic acid and o-aminophenol. A detailed study of the terminal step, i.e., the conversion of o-aminophenol to catechol, was carried out with partially purified preparations. Stoichiometric studies indicated the formation of one mole each of catechol and ammonia from one mole of o-aminophenol. The enzyme system showed a requirement for FAD and sulphydryl groups. A possible mechanism for the reaction is suggested.