Search

A study is conducted to examine substrate acquisition and the reductive and oxidative half-reactions of stable form of kynurenine 3-monooxygenase (KMO) from Pseudomonas fluorescens. The oxidative half-reaction observed in the presence of saturating benzoylalanine or m-nitrobenzoylalanine showed the accumulation of a peroxyflavin species followed by their decay to yield hydrogen peroxide without hydroxylation.

Kynurenine 3-monooxygenase (KMO) is a fiavin-dependent hydroxylase that catalyzes the conversion of L-kynurenine (L-Kyn) to 3-hydroxykynurenine (3OHKyn) in the pathway for tryptophan catabolism. KMO inhibition has been widely suggested as an early treatment for stroke and other neurological disorders that involve ischemia. We have investigated the reductive and the oxidative half- reactions of a stable form of KMO from Pseudomonas fluorescens (KMO). The binding of L-Kyn by the enzyme is relatively slow and involves at least two reversible steps. The rate constant for reduction of the flavin cofactor by NADPH increases by a factor of ∼2.5 x 103 when L-Kyn is bound. The rate of reduction of the KMOL-Kyn complex is 160 s-1, and the Kd for the NADPH complex is 200 μM with charge- transfer absorption bands for the KMORED∙ L-Kyn∙ NADP+ complex accumulating after reduction. The reduction potential of KMO is -188 mY and is unresponsive to the addition of L-Kyn or other inhibitory ligands. KMO inhibitors whose structures are reminiscent of L-Kyn such as m-nitrobenzoylalanine and benzoylalanine also stimulate reduction of flavin by NADPH and, in the presence of dioxygen, result in the stoichiometric liberation of hydrogen peroxide, diminishing the perceived therapeutic potential of inhibitors of this type. In the presence of the native substrate, the oxidative half-reaction exhibits triphasic absorbance data. A spectrum consistent with that of a peroxyfiavin species accumulates and then decays to yield the oxidized enzyme. This species then undergoes minor spectral changes that, based on flavin difference spectra defined in the presence of 30HKyn, can be correlated with product release. The oxidative half-reaction observed in the presence of saturating benzoylalanine or m-nitrobenzoylalanine also shows the accumulation of a peroxyflavin species that then decays to yield hydrogen peroxide without hydroxylation. [ABSTRACT FROM AUTHOR]