Soybean lipoxygenase is a nonheme iron protein that catalyzes the oxygenation of lineolic acid to 13-hydroperoxy-9,11-octadecadienoic acid (13-HPOD). We have found that 12-iodo-cis-9-octadecenoic acid (12-IODE) is a time-dependent (t1/2 = 4 min at 4 Mum 12-IODE) irreversible inhibitor of this enzyme. This result is interesting, since 12-IODE is a poor alkylating agent and soybean lipoxygenase is not inhibited by high concentrations of good alkylating agents, such as iodoacetamide. Furthermore, inhibition by 12-IODE requires O2 and a low concentration of 13-HPOD, which also stimulates the normal catalytic reaction. These results suggest that 12-IODE may be a suicide inhibitor, and the goal of this project is to elucidate the inhibition mechanism. The knowledge gained from this study should be applicable to the design of specific inhibitors of a mammalian lipoxygenase involved in anaphylaxis and others that may be involved in platelet aggregation and in regulation of the activity of natural killer cells. Our working hypothesis is that 12-IODE reacts with a ferryl intermediate in the lipoxygenase-catalyzed decomposition of 13-HPOD and is thereby converted to a highly reactive iodoso compound, which reacts with an active-site nucleophile. To test this hypothesis we will carry out the following experiments. (1) We will inhibit that enzyme with 125I-12-IODE and determine if the iodine is released as 125IO-, as predicted by our hypothesis. (2) We will determine whether lipoxygenase catalyzes the O2- and 13-HPOD-dependent oxidation of the sulfur atom in 12-mercapto-cis-9-octadecenoic acid. (3) We will inhibit the enzyme with 1-14C-12-IODE and determine if radioactivity is incorporated into the protein in a way that is consistent with our hypothesis. Our proposed synthesis of 1-14C-12-IODE may provide a general method of incorporating isotopic carbon into unsaturated fatty acids that is less cumbersome than methods now in use.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Physiological Chemistry Study Section (PC)
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Bucknell University
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Clapp, Charles H; Grandizio, Anna Marie; Yang, Yingmei et al. (2002) Irreversible inactivation of soybean lipoxygenase-1 by hydrophobic thiols. Biochemistry 41:11504-11
Clapp, C H; McKown, J; Xu, H et al. (2000) The action of soybean lipoxygenase-1 on 12-iodo-cis-9-octadecenoic acid: the importance of C11-H bond breaking. Biochemistry 39:2603-11
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Rotenberg, S A; Grandizio, A M; Selzer, A T et al. (1988) Inactivation of soybean lipoxygenase 1 by 12-iodo-cis-9-octadecenoic acid. Biochemistry 27:8813-8