INHIBITION OF SOYBEAN LIPOXYGENASE-1 BY A DIARYL-N-HYDROXYUREA BY REDUCTION OF THE FERRIC ENZYME

It has been proposed that catechols and other antioxidants inhibit lipoxygenase activity by reducing the active Fe3+ form of the enzyme [Kemal et al. (1987) Biochemistry 26, 7064-7072]. In this model, reductively inactivated lipoxygenase can be reactivated by reaction with the hydroperoxide product in a pseudoperoxidase reaction. The contribution of enzyme reduction in the inhibition of the activity of soybean lipoxygenase-1 by the reducing inhibitor N-(4-chlorophenyl)-N-hydroxy-N'-(3-chlorophenyl urea (CPHU) has been evaluated quantitatively. The inhibition by CPHU of the oxygenation of linoleic acid to 13-hydroperoxy-9,11-octadecadienoic acid (13-HpODE) was accompanied by an initial lag phase which could be eliminated by the presence of exogenous 13-HpODE at the initiation of the reaction. In addition, both 13-HpODE and CPHU were found to be consumed during the lipoxygenase reaction, indicating occurrence of both oxygenase and pseudoperoxidase reactions. When analyzed individually, both the oxygenase reaction at different linoleic acid and O-2 concentrations and the pseudoperoxidase reaction at different 13-HpODE and CPHU concentrations were found to follow ping-pong kinetics. A rate equation for the lipoxygenase-catalyzed reaction in the presence of reducing agent was derived considering that the inhibition of the oxygenase reaction is the combined result of 13-HpODE consumption and formation of inactive Fe2+ enzyme due to occurrence of the pseudoperoxidase reaction. By comparing the experimental data with those predicted by the rate equation, it is concluded that the inactivation of the enzyme by reduction can quantitatively account for the inhibition caused by CPHU.