Co-oxidation of NADH and NADPH by a mammalian 15-lipoxygenase: inhibition of lipoxygenase activity at near-physiological NADH concentrations.
The purified 15-lipoxygenase from rabbit reticulocytes is capable of oxidizing NADH in the presence of linoleic acid and oxygen. This co-oxidation proceeds at a rate that amounts to approx. 7% of linoleic acid oxygenation rates. Although NADH inhibits the lipoxygenase reaction with linoleic acid as substrate (46% inhibition at 0.2 mM NADH), the reaction specificity of the enzyme was not altered since (13S)-hydroperoxy-(9Z,11E)-octadecadienoic acid was identified as the major reaction product. NADH oxidation was inhibited by NAD+ (uncompetitive with respect to linoleate and mixed/competitive with respect to NADH), and NADPH or NMNH could substitute for NADH with slightly different apparent Km values. NADH oxidation was enhanced at lower oxygen tension, but was completely prevented under anaerobic conditions. Computer-assisted modelling of 15-lipoxygenase/NADH interaction and sequence alignments of mammalian lipoxygenases with NADH-dependent enzymes suggested that there is no specific binding of the coenzyme at the putative fatty acid-binding site of lipoxygenases. These results suggest that NAD(P)H might be oxidized by a radical intermediate formed during the dioxygenase cycle of the lipoxygenase reaction but that NADH oxidation might not proceed at the active site of the enzyme. The mechanism and possible biological consequences of 15-lipoxygenase-catalysed NAD(P)H oxidation are discussed.
O'donnell
V B
VB
Institute of Biochemistry and Molecular Biology, University of Bern, Bühlstrasse 28, CH-3012 Bern, Switzerland.
Kühn
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D000818:Animals; D001093:Arachidonate 15-Lipoxygenase / Q000378:metabolism*; D001665:Binding Sites; D003198:Computer Simulation; D005231:Fatty Acids, Unsaturated / Q000378:metabolism; D005609:Free Radicals / Q000378:metabolism; D007700:Kinetics; D019787:Linoleic Acid / Q000378:metabolism; D008054:Lipid Peroxides / Q000737:chemistry / Q000378:metabolism; D016859:Lipoxygenase Inhibitors / Q000494:pharmacology; D009243:NAD / Q000378:metabolism* / Q000494:pharmacology; D009249:NADP / Q000378:metabolism*; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D011817:Rabbits; D012156:Reticulocytes / Q000201:enzymology; D013481:Superoxides / Q000378:metabolism
D005231:Fatty Acids, Unsaturated; D005609:Free Radicals; D008054:Lipid Peroxides; D016859:Lipoxygenase Inhibitors; D009243:NAD; D013481:Superoxides; D009249:NADP; D019787:Linoleic Acid; D001093:Arachidonate 15-Lipoxygenase; D010100:Oxygen