Role of lipid hydroperoxides in the activation of 15-lipoxygenase.
We have used stopped-flow rapid reaction methods, employing both fluorescence and absorbance monitoring, together with HPLC analysis of the products to study the activation of soybean 15-lipoxygenase by 13(S)-hydroperoxy-9, 11(E,Z)-octadecadienoic acid (13-HPOD). When lipoxygenase is mixed with an equimolar concentration of 13-HPOD, the enzyme undergoes a rapid change in fluorescence. The rate of the change of fluorescence is dependent on the concentration of the 13-HPOD (k = 6.7 x 10(6) M-1 s-1) and is accompanied by activation of the enzyme. The fluorescence change is not accompanied by any change in the UV absorbance of the 13-HPOD, suggesting no loss of the conjugated diene during enzyme activation, and HPLC analysis of the products of the reaction confirms that the 13-HPOD can be recovered unchanged following this reaction. In the presence of an inhibitor (BWA4C, a hydroxamate inhibitor) that reduces the active-site iron, the 13-HPOD and the inhibitor are destroyed in a peroxidase-like reaction. On the basis of these observations we propose that 13-HPOD binds to the enzyme and facilitates activation of the enzyme, possibly through the formation of a protein radical, and that the 13-HPOD is not changed chemically in this process.
Jones
G D
GD
Department of Biological and Chemical Sciences, University of Essex, Colchester, United Kingdom.
Russell
L
L
Darley-Usmar
V M
VM
Stone
D
D
Wilson
M T
MT
D001093:Arachidonate 15-Lipoxygenase / Q000302:isolation & purification / Q000378:metabolism*; D044482:Benzeneacetamides; D001881:Borates / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D004789:Enzyme Activation; D006877:Hydroxamic Acids / Q000494:pharmacology; D007700:Kinetics; D019787:Linoleic Acid; D008041:Linoleic Acids / Q000378:metabolism; D015227:Lipid Peroxidation; D008054:Lipid Peroxides / Q000378:metabolism*; D016859:Lipoxygenase Inhibitors / Q000494:pharmacology; D008956:Models, Chemical; D013025:Glycine max / Q000201:enzymology; D013050:Spectrometry, Fluorescence
D044482:Benzeneacetamides; D001881:Borates; D006877:Hydroxamic Acids; D008041:Linoleic Acids; D008054:Lipid Peroxides; D016859:Lipoxygenase Inhibitors; C056614:N-(3-phenoxycinnamyl)acetohydroxamic acid; C038649:13-hydroperoxy-9,11-octadecadienoic acid; C010634:sodium borate; D019787:Linoleic Acid; D001093:Arachidonate 15-Lipoxygenase