Suicide inactivation of D-amino acid oxidase by 1-chloro-1-nitroethane.

Given that the oxidation of nitroethane by D-amino acid oxidase proceeds through a transient carbinolamine adduct at the N5 position of the active-site FAD cofactor (Porter, D. J. T., Voet, J. G., and Bright, H. J. (1973) J. Biol. Chem. 248, 4400-4416), it follows that 1-chloro-1-nitroethane should generate a stable amide at N5 and thereby function as a suicide inactivator of this enzyme. This hypothesis was validated as follows. 1-Chloro-1-nitroethane, as the nitronate ion (pKa = 7.0), inactivated D-amino acid oxidase completely with a Km value of 2 mM and a maximum rate of 0.02 s-1. The chloro and nitro groups were quantitatively recovered as free Cl- and NO2(-) after the enzyme was inactivated by 1.5 flavin equivalents of 1-chloro-1-nitroethane. Inactivation did not require O2 and was accompanied by bleaching of the flavin under both anaerobic and aerobic conditions. The modified coenzyme of the inactivated enzyme was identified as N5-acetyl-1,5-dihydro FAD. The enzyme catalyzes the oxidation of 1-chloro-1-nitroethane to acetate approximately 0.5 times as rapidly as the enzyme catalyzes suicide inactivation. The transient intermediate which is common to both the inactivation and oxidation pathways must be N5-(1-X-1-hydroxyethyl)-1,5-dihydro FAD, where X = nitro or chloro.