Research Project
3292577
Soybean lipoxygenase is a nonheme iron protein (MW = 94,500) that catalyzes the oxygenation of linoleic acid to 13-hydroperoxy-9,11- octadecadienoic acid (13-HPOD). The goals of this project 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. 12-Iodo-cis-9-octadecenoic acid (12-IODE) is a time-dependent, irreversible inactivator of soybean lipoxygenase. Inactivation requires both 13-HPOD (or some other lipid hydroperoxide) and 02. These observations suggest that 12-IODE is a suicide inactivator, and this idea has been strengthened by our finding that inactivation is accompanied by formation of about 9 iodide ions per molecule of enzyme inactivated. 9,11-Octadecadienoic acid is formed in quantities that are less than stoichiometric with iodide formation, which implies that other organic products are formed from 12-IODE. 02 uptake has also been detected. During the period covered by this application we will use 1-14C-12-IODE to identify the remaining organic products from the action of lipoxygenase on 12-IODE. We will also use this material to determine whether the carbon skeleton of 12-IODE becomes incorporated into the inactivated enzyme, and of so, we will carry out degradation studies on the labelled enzyme to determine how the inhibitor has become attached. In addition, we will synthesize 11,11-dideuterio- 12-IODE and use this material to test our working hypothesis by measuring the effect of deuterium substitution on the rate of iodide formation and on the rate and extent of inactivation. We have also found that 12-mercapto-cis-9-octadecenoic acid (12- HSODE) is a time-dependent, irreversible inactivator of lipoxygenase. Inactivation is accompanied by consumption of about 67 molecules of 12 HSODE per molecule of enzyme inactivated. These observations suggest that 12-HSODE is also a suicide inactivator. During the period covered by this application we will identify the products produced by the action of lipoxygenase on 12-HSODE, attempt to determine how the inactivated enzyme has been modified, and carry out other experiments to elucidate the inactivation mechanism.
