3-Hydroxyanthranilate-3,4-dioxygenase (HAD) is a non-
heme Fe(II) dependent
enzyme that catalyzes the oxidative ring-opening of 3-hydroxyanthranilate to 2-amino-3-carboxymuconic semialdehyde. The enzymatic product subsequently cyclizes to
quinolinate, an intermediate in the biosynthesis of
nicotinamide adenine dinucleotide.
Quinolinate has also been implicated in important
neurological disorders. Here, we describe the mechanism by which
4-chloro-3-hydroxyanthranilate inhibits the HAD catalyzed reaction. Using overexpressed and purified bacterial HAD, we demonstrate that
4-chloro-3-hydroxyanthranilate functions as a mechanism-based inactivating agent. The inactivation results in the consumption of 2 +/- 0.8 equiv of
oxygen and the production of
superoxide. EPR analysis of the inactivation reaction demonstrated that the inhibitor stimulated the oxidation of the active site Fe(II) to the catalytically inactive Fe(III) oxidation state. The inactivated
enzyme can be reactivated by treatment with DTT and Fe(II). High resolution ESI-FTMS analysis of the inactivated
enzyme demonstrated that the inhibitor did not form an adduct with the
enzyme and that four conserved cysteines were oxidized to two
disulfides (Cys125-Cys128 and Cys162-Cys165) during the inactivation reaction. These results are consistent with a mechanism in which the
enzyme, complexed to the inhibitor and O2, generates
superoxide which subsequently dissociates, leaving the inhibitor and the oxidized
iron center at the active site.