Inhibition of potato lipoxygenase by linoleyl hydroxamic acid: kinetic and EPR spectral evidence for a two-step reaction.
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.
Butovich
Igor A
IA
Department of Pharmaceutical Sciences, College of Pharmacy, Medical University of South Carolina, Charleston, SC 29425, U.S.A. butovich@musc.edu
Reddy
C Channa
CC
D004578:Electron Spin Resonance Spectroscopy; D004789:Enzyme Activation; D008041:Linoleic Acids / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D008054:Lipid Peroxides / Q000378:metabolism; D008084:Lipoxygenase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D016859:Lipoxygenase Inhibitors / Q000378:metabolism / Q000494:pharmacology*; D015394:Molecular Structure; D011198:Solanum tuberosum / Q000201:enzymology*; D013696:Temperature
D008041:Linoleic Acids; D008054:Lipid Peroxides; D016859:Lipoxygenase Inhibitors; C089594:linoleic acid hydroxamate; D008084:Lipoxygenase