Kinetics of leukotriene A4 synthesis by 5-lipoxygenase from rat polymorphonuclear leukocytes.
When arachidonic acid is added to lysates of rat polymorphonuclear leukocytes, it is oxidized to (5S)-hydroperoxy-6(E),8(Z),11(Z),14(Z)-eicosatetraenoic acid (5-HPETE). The 5-HPETE then partitions between reduction to the 5-hydroxyeicosanoid and conversion to leukotriene A4 (LTA4). Both steps in the formation of LTA4 are catalyzed by the enzyme 5-lipoxygenase. When [3H]arachidonic acid and unlabeled 5-HPETE were incubated together with 5-lipoxygenase, approximately 20% of the arachidonic acid oxidized at low enzyme concentrations was converted to LTA4 without reduction of the specific radioactivity of the LTA4 by the unlabeled 5-HPETE. A significant fraction of the [3H]-5-HPETE intermediate that is formed from arachidonic acid must therefore be converted directly to LTA4 without dissociation of the intermediate from the enzyme. This result predicts that even in the presence of high levels of peroxidase activity, which will trap any free 5-HPETE by reduction, the minimum efficiency of conversion of 5-HPETE to LTA4 will be approximately 20%, and this prediction was confirmed. 5-HPETE was found to be a competitive substrate relative to arachidonic acid, so that it is likely that the two substrates share a common active site.
Wiseman
J S
JS
Merrell Dow Research Institute, Cincinnati, Ohio 45215.
Skoog
M T
MT
Nichols
J S
JS
Harrison
B L
BL
D000818:Animals; D001094:Arachidonate 5-Lipoxygenase / Q000378:metabolism*; D001091:Arachidonate Lipoxygenases / Q000378:metabolism*; D016718:Arachidonic Acid; D001095:Arachidonic Acids / Q000096:biosynthesis* / Q000378:metabolism; D007700:Kinetics; D017572:Leukotriene A4; D015289:Leukotrienes; D009504:Neutrophils / Q000201:enzymology*; D051381:Rats; D014316:Tritium
D001095:Arachidonic Acids; D017572:Leukotriene A4; D015289:Leukotrienes; D014316:Tritium; D016718:Arachidonic Acid; C025348:arachidonic acid 5-hydroperoxide; D001091:Arachidonate Lipoxygenases; D001094:Arachidonate 5-Lipoxygenase