Pseudoperoxidase activity of 5-lipoxygenase stimulated by potent benzofuranol and N-hydroxyurea inhibitors of the lipoxygenase reaction.
The purified 5-lipoxygenase from porcine leukocytes was found to catalyse the degradation of lipid hydroperoxides in the presence of potent inhibitors of the lipoxygenase reaction. Derivatives of diphenyl-N-hydroxyureas, 4-hydroxybenzofurans and 5-hydroxydihydrobenzofurans all stimulated the 5-lipoxygenase-mediated destruction of 13-hydroperoxyoctadecadienoic acid (13-HPOD). The reaction was dependent on inhibitor and hydroperoxide concentrations (1-10 microM) and could not be detected using heat-inactivated enzyme, when ATP and Ca2+ were omitted or when the hydroperoxide was replaced by the corresponding alcohol. The stability of the inhibitors during this pseudoperoxidase reaction was investigated by measuring the recoveries of 5-hydroxy-2-phenethyl-6-(3-phenoxypropyl)-2,3-dihydrobenzofuran and N-(4-chlorophenyl)-N-hydroxy-N'-(3-chlorophenyl)urea from the reaction mixtures using reverse-phase h.p.l.c. By using an equimolar concentration of 13-HPOD and inhibitor (10 microM) and under conditions where 50% of the 13-HPOD was consumed, the concentration of the benzofuranol decreased by 30%, whereas the N-hydroxyurea derivative could be completely recovered from the reaction mixture. A stimulation of the pseudoperoxidase reaction could be detected only with very effective inhibitors of leukotriene B4 biosynthesis by human leucocytes [IC50 (concn. causing 50% inhibition) less than 100 nM], but not with closely related structural analogues of lower potency or other inhibitors such as nordihydroguaiaretic acid, quercetin or the hydroxamate A-64077. These results demonstrate that 5-lipoxygenase possesses a pseudoperoxidase activity and indicate that potent inhibitors in both N-hydroxyurea and benzofuranol series can function as reducing agents for the enzyme.
Riendeau
D
D
Department of Biochemistry, Merck Frosst Centre for Therapeutic Research, Pointe Claire-Dorval, Québec, Canada.
Falgueyret
J P
JP
Guay
J
J
Ueda
N
N
Yamamoto
S
S
D000818:Animals; D001094:Arachidonate 5-Lipoxygenase / Q000097:blood*; D001572:Benzofurans / Q000494:pharmacology*; D006918:Hydroxyurea / Q000031:analogs & derivatives* / Q000494:pharmacology*; D007700:Kinetics; D007962:Leukocytes / Q000201:enzymology*; D010544:Peroxidases; D013329:Structure-Activity Relationship; D013552:Swine
D001572:Benzofurans; D010544:Peroxidases; D001094:Arachidonate 5-Lipoxygenase; D006918:Hydroxyurea