Studies of the oxidative half-reaction of anthranilate hydroxylase (deaminating) with native and modified substrates

The oxidative half-reactions of anthranilate hydroxylase (EC 1.14.12.2) were examined in the presence of anthranilate and modified substrates. C(4a)-Hydroperoxyflavin (C(4a)-FlOOH) and C(4a)-hydroxyflavin (C(4a)-FlOH) intermediates were detected in oxidative reactions with all substrates. Thus, the oxygenation reactions of the enzyme are similar to those of flavoprotein hydroxylases that convert phenolic compounds to catechols. These observations support a mechanism proposed for this enzyme (Powlowski, J. B., Dagley, S., Massey, V., and Ballou, D. P. (1987) J. Biol. Chem. 262, 69-74) involving nucleophilic attack of the substrate on C(4a)-FlOOH, and formation of an imine intermediate that is subsequently hydrolyzed. Anthranilate hydroxylase is therefore a typical flavoprotein hydroxylase with the added capacity of hydrolyzing imine intermediates. Fluorine substituents on the aromatic ring decreased the rate of conversion of C(4a)-FlOOH to C(4a)-FlOH, as predicted by this mechanism. Hydroxylation of 3-fluoro- and 3-methylanthranilates resulted in the formation of nonaromatic products that appeared to stabilize the C(4a)-FlOH. No evidence was found for a high extinction intermediate (intermediate II) (Entsch, B., Ballou, D. P., and Massey, V. (1976) J. Biol. Chem. 251, 2550-2563) under conditions where it was readily detected with other flavoprotein hydroxylases. It was shown that the spectra of the nonaromatic products (which are quinonoid forms) could not be summed with the spectra of C(4a)-hydroxyflavin to obtain that of a putative intermediate II, thus ruling out that explanation for previous observations of II.