This project concerns soybean lipoxygenase, a nonheme iron protein that catalyzes the oxygenation of linoleic acid to 13-hydroperoxy-9,11-octadecadienoic acid (13-HPOD). The goals of the work are to understand the chemical properties and catalytic mechanism of this enzyme and to develop strategies that can be applied to the design of inhibitors of several physiologically and pharmaceutically interesting mammalian lipoxygenases. We have been studying the action of soybean lipoxygenase on 9-octadecenoic acid derivatives with substituents at position 12. 12-Iodo-cis-9-octadecenoic acid (12-IODE) and 12-mercapto-cis-9-octadecenoic acid (12-HSODE) are both irreversible inactivators of this enzyme. 12-BrODE does not inactivate the enzyme but is slowly converted to 9,11-octadecadienoic acid (9,11-ODE). Our working hypothesis is that ferric lipoxygenase can react with these compounds by two pathways: an elimination reaction to produce 9,11-ODE (Pathway 1) and a second pathway that is initiated by one-electron oxidation of X (Pathway 2). We propose that 12-BrODE reacts by Pathway 1, 12-HSODE reacts by Pathway 2, and 12-IODE reacts by both pathways. Pathway 2 leads to inactivation; in the case of 12-IODE there is evidence that inactivation occurs by a radical mechanism. During the period covered by this proposal we will test the hypothesis that 12-IODE reacts by both pathways by studying the action of soybean lipoxygenase on 11,11-dideuterio-12-IODE. If our hypothesis is correct, introduction of deuterium at position 11 should increase the flux through Pathway 2 due to a primary isotope effect on Pathway 1. We will also synthesize the R- and S- enantiomers of 12-BrODE to determine the stereospecificity of Pathway 1. In addition, we will use 1-14C-12-HSODE to determine whether inactivation by 12-HSODE is accompanied by covalent incorporation of the carbon skeleton of the inactivator into the protein. We will also use this labelled material to facilitate the isolation of the products from the action of lipoxygenase on 12-HSODE, and we will identify these products. Finally, the elimination reactions that we have observed provide evidence for a basic residue at the active site. We propose to synthesize and test 11-bromo-cis-9-octadecenoic acid and 11,12-oxido-9-octadecenoic acid as possible active-site directed-labelling reagents for this base.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Physiological Chemistry Study Section (PC)
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Bucknell University
Schools of Arts and Sciences
United States
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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
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