ACTION OF SOYBEAN LIPOXYGENASE-1 ON 12-IODO-CIS-9-OCTADECENOIC ACID AND 12-BROMO-CIS-9-OCTADECENOIC 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-C-14]-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:I incorporation. Considerable radioactivity from [1-C-14]-12-IODE becomes noncovalently associated with the protein; most of this radioactivity can be removed by extraction with ethyl acetate, and thin-layer chromatography of the ethyl acetate extracts indicates that they contain a complex mixture of radioactive substances that are more polar than 12-IODE and 9,11-ODA. These materials may be formed by a free-radical pathway, since the noncovalent incorporation of radioactivity is greatly reduced by the presence of ascorbate. The noncovalent incorporation of these materials does not appear to be the cause of inactivation, since extraction of most of the radioactivity with ethyl acetate does not restore catalytic activity, and since noncovalent incorporation occurs to about the same extent at pH 7.6 as at pH 9.0, even though inactivation is much less at pH 7.6. The results suggest that the main pathway for inactivation of lipoxygenase by 12-IODE involves the enzymatic conversion of 12-IODE to a radical (or some other reactive species that can be trapped by ascorbate and hydroxylamine), which irreversibly modifies the enzyme without covalent attachment of the carbon skeleton of the inactivator to the protein.