Inhibition of potato lipoxygenase by linoleyl hydroxamic acid: kinetic and EPR spectral evidence for a two-step reaction
| Autor: | C. Channa Reddy, Igor A. Butovich |
|---|---|
| Rok vydání: | 2002 |
| Předmět: |
Lipid Peroxides
Reaction mechanism Stereochemistry Linoleic acid Lipoxygenase Biochemistry Medicinal chemistry Redox law.invention Ferrous Catalysis chemistry.chemical_compound law Structural isomer Lipoxygenase Inhibitors Electron paramagnetic resonance Molecular Biology Solanum tuberosum Molecular Structure biology Electron Spin Resonance Spectroscopy Temperature Cell Biology Enzyme Activation Linoleic Acids chemistry biology.protein Research Article |
| Zdroj: | Biochemical Journal. 365:865-871 |
| ISSN: | 1470-8728 0264-6021 |
| Popis: | 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 Fe(3+) with a g-value of approx. 6.3+/-0.1 with a shoulder at g=5.9+/-0.1, and a rhombic Fe(3+) 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 Fe(3+)-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-Fe(3+)-ptLOX complex. The spectral characteristics of the complex are similar to those of a 4-nitrocatechol-Fe(3+)-ptLOX complex. From these observations, we conclude that LHA did not reduce Fe(3+) to Fe(2+), but rather formed a LHA-Fe(3+)-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 15 min incubation of the complex at 23+/-1 degrees C led to the partial quenching of the g=4.35 signal. The quenching is attributed to the reduction of Fe(3+)-ptLOX by LHA, with concomitant formation of its oxidation product(s). A kinetic scheme for the inhibition is proposed. |
| Databáze: | OpenAIRE |
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