12-Iodo-cis-9-octadecenoic acid (12-IODE) is a time-dependent, irreversible inactivator of soybean lipoxygenase 1. The rate of inactivation is independent of 12-IODE concentration above 20 microM and is half-maximal at about 4 microM. Inactivation by 12-IODE requires lipid hydroperoxide, which must be present even after the initial oxidation of the iron in the enzyme from ferrous to ferric. Inactivation by 12-IODE is also dependent on O2. These findings suggest that 12-IODE is converted by the enzyme into a more reactive species, which is responsible for inactivation. No inactivation has been detected with 12-iodooctadecanoic acid, 12-bromo-cis-9-octadecenoic acid, 12-iodo-trans-9-octadecenoic acid, or a mixture of stereoisomers of 9,11-octadecadienoic acid.

The ferric form of soybean lipoxygenase catalyzes an elimination reaction on 12-iodo-cis-9-octadecenoic acid (12-IODE) to produce iodide ions and 9,11-octadecadienoic acid (9, 11-ODA). If excess 13(S)-hydroperoxy-cis-9,trans-11-octadecadienoic acid (13-HPOD) is present, the reaction proceeds until about one-half of the racemic 12-IODE is consumed; in the absence of excess 13-HPOD, the reaction stops after about three turnovers. Ferric lipoxygenase also catalyzes the conversion of 12-bromo-cis-9-octadecenoic acid (12-BrODE) to 9,11-ODA at a rate that is less than 25% of that observed with 12-IODE. These elimination reactions cannot be detected with ferrous lipoxygenase or with lipoxygenase that has been inactivated by 5,8,11,14-eicosatetraynoic acid. In the case of 12-IODE, elimination is accompanied by a loss of enzymatic activity; at pH 9.0, about 10 iodide ions are produced per molecule of enzyme inactivated. No inactivation can be detected with 12-BrODE. Ascorbate and hydroxylamine, which can act as free-radical traps, block the inactivation by 12-IODE but do not inhibit the elimination reaction. When the enzyme is inactivated by [1-14C]-12-IODE at pH 9.0, the amount of radioactivity that is covalently bound to the protein is less than 30% of that expected for 1:1 incorporation.(ABSTRACT TRUNCATED AT 250 WORDS)

Previous work has demonstrated that the ferric form of soybean lipoxygenase-1 will catalyze an elimination reaction on 12-iodo-cis-9-octadecenoic acid (12-IODE) to produce 9, 11-octadecadienoic acid and iodide ion. Elimination is accompanied by irreversible inactivation of the enzyme on 1 out of 10 turnovers. In the present work, 11,11-dideuterio-12-IODE (D(2)-12-IODE) was synthesized and used to demonstrate that both the elimination reaction and inactivation of the enzyme exhibit very large kinetic isotope effects. The rates with the deuterated compound are so low that the isotope effects are difficult to quantify, but they appear to be comparable to the isotope effects previously observed for the normal reaction catalyzed by lipoxygenase and much larger than can be explained by zero-point energy considerations. ESR spectroscopy was used to demonstrate that 12-IODE can reduce ferric lipoxygenase to the ferrous form, and a large isotope effect on this process was observed with D(2)-12-IODE. It is proposed that the pathway leading to reduction and inactivation by 12-IODE is initiated by homolytic cleavage of the C(11)-H bond. Elimination could be initiated either by homolytic or by heterolytic cleavage of this bond. The results suggest that very large isotope effects may be a general feature of C-H bond cleavages catalyzed by this enzyme.

Soybean lipoxygenase-1 is inactivated by micromolar concentrations of the following hydrophobic thiols: 1-octanethiol, 12(S)-mercapto-9(Z)-octadecenoic acid (S-12-HSODE), 12(R)-mercapto-9(Z)-octadecenoic acid (R-12-HSODE), and 12-mercaptooctadecanoic acid (12-HSODA). In each case, inactivation is time-dependent and not reversed by dilution or dialysis. Inactivation requires 13-hydroperoxy-9(Z),11(E)-octadecadienoic acid (13-HPOD), which suggests that it is specific for the ferric form of the enzyme. Lipoxygenase catalyzes an oxygenation reaction on each of the aforementioned thiols, as judged by the consumption of O(2). These reactions also require 13-HPOD. 1-Octanethiol is converted to 1-octanesulfonic acid, which was identified by GC/MS of its methyl ester. The rates of oxygen uptake for R- and S-12-HODE are about 5- and 2.5-fold higher than the rate with 1-octanethiol. The stoichiometries of inactivation imply that inactivation occurs on approximately 1 in 18 turnovers for 12-HSODA, 1 in 48 turnovers for 1-octanethiol, 1 in 63 turnovers for S-12-HSODE, and 1 in 240 turnovers for R-12-HSODE. These data imply that close resemblance to lipoxygenase substrates is not a crucial requirement for either oxidation or inactivation. Under the conditions of our experiments, inactivation was not observed with several more polar thiols: mercaptoethanol, dithiothreitol, L-cysteine, glutathione, N-acetylcysteamine, and captopril. The results imply that hydrophobic thiols irreversibly inactivate soybean lipoxygenase by a mechanism that involves oxidation at sulfur.