Leakage of Fatty Acid Radicals During Lipoxygenase Catalysis
The kinetics of soybean lipoxygenase-1 catalyzed oxygenation of linoleate (LH) was studied as a function of the concentrations of LH and antioxidants 2,6-di-tert-butyl-4-methyl-phenol (BHT) and N,N,N′,N′-tetramethyl-p-phenylene-diamine (TMPD). The results revealed two distinct oxidative pathways that contribute to O2 uptake. In one of these, oxygenation of LH occurred at the active site of the enzyme and in the other it occurred in solution. The latter pathway was an autoxidation process initiated by linoleate-derived radicals released by the enzyme during turnover. Product analysis revealed that 13-hydroperoxy-(9Z,11E)-octadeca-9,11-dienoic acid (ZE13-LOOH) was the major (98%) hydroperoxide formed when LH was used at a concentration of 100 μM. At 1.8 mM LH, after consumption of 100-200 μM O2, significant amounts of three isomers of ZE13-LOOH (EE13-,EZ9-, and EE9-LOOH) also formed (total yield 35%). That these isomers result from solution autoxidation initiated by LH-derived radicals released by the enzyme during turnover was demonstrated by selectively inhibiting solution autoxidation with antioxidants. By quantifying the amount of BHT and TMPD consumed, it was established that one LH-derived radical is released by the enzyme about once every 13 turnovers.
KeywordsHigh Pressure Liquid Chromatography Fatty Acid Substrate Soybean Lipoxygenase Linoleic Acid Hydroperoxide Epoxy Alcohol
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