N-(4-chlorophenyl)-N-hydroxy-N'-(3-chlorophenyl)urea, a general reducing agent for 5-, 12-, and 15-lipoxygenases and a substrate for their pseudoperoxidase activities.
Lipoxygenases contain a nonheme iron that undergoes oxidation and reduction during the catalytic cycle. The conversion from the Fe3+ enzyme form to the Fe2+ form can be achieved using reducing inhibitors, a reaction that can be reversed with lipid hydroperoxides. The present study describes the properties of N-(4-chlorophenyl)-N-hydroxy-N'-(3-chlorophenyl)urea (CPHU), which functions as a reducing agent for various lipoxygenases and stimulates the degradation of lipid hydroperoxide catalyzed by these enzymes (pseudoperoxidase activity). CPHU was a substrate for the pseudoperoxidase reaction of purified soybean lipoxygenase-1 with apparent Km values for CPHU and 13-hydroperoxy-9,11-octadecadienoic acid (13-HpODE) of 14 and 15 microM, respectively. CPHU was converted during the pseudoperoxidase reaction to a mixture of products that can be resolved by reverse-phase high pressure liquid chromatography. By comparison with the chemical reaction of CPHU and potassium nitrosodisulfonate, the major enzymatic reaction product was tentatively identified as a one-electron oxidation product of CPHU. At low concentrations (50 microM), dithiothreitol completely protected against the degradation of hydroxyurea without inhibiting the pseudoperoxidase reaction. Under these conditions, the rate of the pseudoperoxidase reaction with CPHU as a substrate can be quantitated by the change in absorbance at 234 nm owing to the consumption of 13-HpODE. In addition to soybean lipoxygenase-1, CPHU was found to be a substrate for the pseudoperoxidase activities of purified recombinant human 5-lipoxygenase and porcine leukocyte 12-lipoxygenase. The results are consistent with CPHU reacting with lipoxygenase by a one-electron oxidation to generate the ferrous enzyme form and the nitroxide radical, which could be reduced back to CPHU by DTT.(ABSTRACT TRUNCATED AT 250 WORDS)
Falgueyret
J P
JP
Department of Biochemistry, Merck Frosst Centre for Therapeutic Research, Kirkland, Que., Canada.
Desmarais
S
S
Roy
P J
PJ
Riendeau
D
D
D000818:Animals; D001092:Arachidonate 12-Lipoxygenase / Q000378:metabolism; D001093:Arachidonate 15-Lipoxygenase / Q000378:metabolism; D001094:Arachidonate 5-Lipoxygenase / Q000378:metabolism; D044482:Benzeneacetamides; D002224:Carbanilides / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D006877:Hydroxamic Acids / Q000494:pharmacology; D006918:Hydroxyurea / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D007700:Kinetics; D008084:Lipoxygenase / Q000378:metabolism*; D016859:Lipoxygenase Inhibitors / Q000494:pharmacology; D010084:Oxidation-Reduction; D010544:Peroxidases / Q000187:drug effects / Q000378:metabolism*; D010944:Plants / Q000201:enzymology; D013552:Swine
D044482:Benzeneacetamides; D002224:Carbanilides; D006877:Hydroxamic Acids; D016859:Lipoxygenase Inhibitors; C056614:N-(3-phenoxycinnamyl)acetohydroxamic acid; C076493:N-(4-chlorophenyl)-N-hydroxy-N'-(3-chlorophenyl)urea; D006861:Hydrogen Peroxide; D010544:Peroxidases; D008084:Lipoxygenase; D001092:Arachidonate 12-Lipoxygenase; D001093:Arachidonate 15-Lipoxygenase; D001094:Arachidonate 5-Lipoxygenase; C063449:zileuton; D006918:Hydroxyurea