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Inhibition of lipoxygenase from S. Tuberosum (L.) by linoleyl hydroxamic acid: kinetic and EPR spectral evidence for a two-step reaction.
Butovich I.A., Reddy C.C.Biochem. J. 365: 865-871 (2002)
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 0.5 K showed signals of a high-spin (pseudo) axial Fe3+ with g-values of around 6.3 0.1 and a shoulder at g=5.9 0.1, and a 'rhombic' Fe3+ signal with g=4.35 0.05. When the enzyme was treated with 2-x 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 1/4 of the 'native' ptLOX iron was in ferrous state. Also, the positional isomer, 9(S)-HPODE, caused similar spectral changes. Therefore, the ptLOX catalytic center appears to accommodate both the positional isomers of linoleic acid hydroperoxides in such a way that ensures proper alignment of their hydroperoxyl groups with the iron center of the enzyme. Treatment of Fe3+-ptLOX form with 3-x molar excess of linoleyl hydroxamic acid (LHA) completely quenched the g=6.3 signal. Concurrently, a dramatic increase in the signal with g=4.35 was detected, which is attributed to a newly formed LHA-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 which may be responsible for the inhibitory activity of LHA, at least at the initial stages of enzyme inhibition. A prolonged 15-min incubation of the complex at 23 1 oC 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 corresponding kinetic scheme of the inhibition is proposed.