Inhibition of soybean lipoxygenase 1 by N-alkylhydroxylamines

Abstract

Micromolar concentrations of N-octylhydroxylamine dramatically increase the induction period in the conversion of linoleic acid to 13(S)-hydroperoxy-cis-9,trans-11-octadecadienoic acid (13-HPOD) catalyzed by soybean lipoxygenase 1. The induction period produced by N-octylhydroxylamine is abolished by 13-HPOD but not by the corresponding hydroxy acid. Addition of a catalytic amount of lipoxygenase to a mixture of 13-HPOD and N-octylhydroxylamine results in consumption of approximately 1 mumol of 13-HPOD/mumol of N-octylhydroxylamine present. These results can be explained by a model in which 13-HPOD oxidizes the enzyme from an inactive ferrous form to an active ferric form, as proposed by previous workers, and N-octylhydroxylamine reduces the enzyme back to the ferrous form. Consistent with this model, the ESR signal at g = 6.1 characteristic of ferric lipoxygenase is rapidly abolished by N-octylhydroxylamine and can be regenerated by 13-HPOD. These results provide additional support for earlier proposals that ferric lipoxygenase is the catalytically active form and also establish a novel method of inhibiting enzymes in this class. The octyl group of N-octylhydroxylamine appears to contribute to binding near the iron, since hydroxylamine and N-methylhydroxylamine do not extend the induction period. In the n-RNHOH series, activity passes through an optimum at R = decyl.