Differential characteristics of human 15-lipoxygenase isozymes and a novel splice variant of 15S-lipoxygenase.
The lipoxygenases (LOs) are a family of nonheme iron dioxygenases that catalyse the insertion of molecular oxygen into polyunsaturated fatty acids. Five members of this gene family have been described in man, 5-LO, 12S-LO, 12R-LO, 15-LO and 15S-LO. Using partially purified recombinant 15S-LO enzyme and cells constitutively expressing this protein, we have compared the activity, substrate specificity, kinetic characteristics and regulation of this enzyme to that previously reported for 15-LO. 15S-LO has a threefold higher Km, similar Vmax and increased specificity of oxygenation for arachidonic acid, and a similar Km but decreased Vmax for linoleic acid in comparison to 15-LO. Unlike 15-LO, 15S-LO is not suicide inactivated by the products of fatty acid oxygenation. However, in common with other LOs, 15S-LO activity is regulated through calcium-dependent association of the enzyme with the membrane fraction of cells. In addition, whilst independently cloning the recently described 15S-LO, we identified a splice variant containing an in-frame 87-bp deletion corresponding to amino acids 401-429 inclusive. Modelling of the 15S-LO and subsequent studies with partially purified recombinant protein suggest that the deleted region comprises a complete alpha-helix flanking the active site of the enzyme resulting in decreased specificity of oxygenation and affinity for fatty acid substrates. Alternative splicing of 15S-LO would therefore provide a further level of regulation of fatty acid metabolism. These results demonstrate that there are substantial differences in the enzyme characteristics and regulation of the 15-LO isozymes which may reflect differing roles for the proteins in vivo.
Kilty
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Discovery Biology, Pfizer Central Research, Sandwich, UK. Iain_Kilty@sandwich.pfizer.com
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Vickers
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D000595:Amino Acid Sequence; D001093:Arachidonate 15-Lipoxygenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005227:Fatty Acids / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D015227:Lipid Peroxidation; D016859:Lipoxygenase Inhibitors; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009928:Organ Specificity; D011487:Protein Conformation; D012326:RNA Splicing; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D004791:Enzyme Inhibitors; D005227:Fatty Acids; D007527:Isoenzymes; D016859:Lipoxygenase Inhibitors; D011993:Recombinant Fusion Proteins; D001093:Arachidonate 15-Lipoxygenase