Inhibition of potato lipoxygenase by linoleyl hydroxamic acid: kinetic and EPR spectral evidence for a two-step reaction

Abstract

The reaction mechanism of an electrophoretically pure potato tuber lipoxygenase (ptLOX) was studied by EPR spectroscopy. An EPR spectrum of the ‘native’ ptLOX recorded at 4.5±0.5K showed signals of a high-spin (pseudo) axial Fe3+ with a g-value of approx. 6.3±0.1 with a shoulder at g = 5.9±0.1, and a rhombic Fe3+ signal at g = 4.35±0.05. When the enzyme was treated with a 2-fold molar excess of 13(S)-hydroperoxyoctadecadienoic acid [13(S)-HPODE], a 3-fold increase in the integral intensity of the g = 6.3 signal was observed, indicating that 25% of the native ptLOX iron was in ferrous state. The positional isomer 9(S)-HPODE caused similar spectral changes. Therefore the catalytic centre of ptLOX appears to accommodate both positional isomers of linoleic acid hydroperoxides in a manner that ensures proper alignment of their hydroperoxy groups with the iron centre of the enzyme. Treatment of the Fe3+-ptLOX form with a 3-fold molar excess of linoleyl hydroxamic acid (LHA) completely quenched the g = 6.3 signal. Concurrently, a dramatic increase in the signal at g = 4.35 was detected, which was attributed to a newly formed LHA—Fe3+-ptLOX complex. The spectral characteristics of the complex are similar to those of a 4-nitrocatechol—Fe3+-ptLOX complex. From these observations, we conclude that LHA did not reduce Fe3+ to Fe2+, but rather formed a LHA—Fe3+-ptLOX complex. Formation of such a complex may be responsible for the inhibitory activity of LHA, at least in the initial stages of enzyme inhibition. A prolonged 15min incubation of the complex at 23±1°C led to the partial quenching of the g = 4.35 signal. The quenching is attributed to the reduction of Fe3+-ptLOX by LHA, with concomitant formation of its oxidation product(s). A kinetic scheme for the inhibition is proposed.