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 react ion on 12-iodo-cis-9-octadecenoic acid (12-IODE) to produce iodide ion s and 9,11-octadecadienoic acid (9,11-ODA). If excess 13 (S)-hydropero xy-cis-9,trans-11-octadecadienoic acid (13-HPOD) is present, the react ion 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-br omo-cis-9-octadecenoic acid (12-BrODE) to 9,11-ODA at a rate that is l ess than 25% of that observed with 12-IODE. These elimination reaction s cannot be detected with ferrous lipoxygenase or with lipoxygenase th at has been inactivated by 5,8,11,14-eicosatetraynoic acid. In the cas e of 12-IODE, elimination is accompanied by a loss of enzymatic activi ty; at pH 9.0, about 10 iodide ions are produced per molecule of enzym e inactivated. No inactivation can be detected with 12-BrODE. Ascorbat e and hydroxylamine, which can act as free-radical traps, block the in activation by 12-IODE but do not inhibit the elimination reaction. Whe n 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 prote in; 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 substance s that are more polar than 12-IODE and 9,11-ODA. These materials may b e formed by a free-radical pathway, since the noncovalent incorporatio n of radioactivity is greatly reduced by the presence of ascorbate. Th e noncovalent incorporation of these materials does not appear to be t he cause of inactivation, since extraction of most of the radioactivit y with ethyl acetate does not restore catalytic activity, and since no ncovalent incorporation occurs to about the same extent at pH 7.6 as a t 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 som e other reactive species that can be trapped by ascorbate and hydroxyl amine), which irreversibly modifies the enzyme without covalent attach ment of the carbon skeleton of the inactivator to the protein.