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.
|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|
|Clapp, C H; Grandizio, A M; McAskill, S A et al. (1995) Action of soybean lipoxygenase 1 on 12-iodo-cis-9-octadecenoic acid and 12-bromo-cis-9-octadecenoic acid. Biochemistry 34:264-72|
|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|
|Wiseman, J S; Skoog, M T; Clapp, C H (1988) Activity of soybean lipoxygenase in the absence of lipid hydroperoxide. Biochemistry 27:8810-3|