Irreversible inactivation of 5-lipoxygenase by leukotriene A4. Characterization of product inactivation with purified enzyme and intact leukocytes.
We report that leukotriene A4, the electrophilic product of 5-lipoxygenase catalysis, irreversibly inactivates the enzyme. Leukotriene A4 inhibits 5-hydroxyeicosatetraenoic acid formation by human neutrophils and differentiated granulocytic HL-60 cells in a concentration-dependent manner with IC50 values = 22.4 +/- 2.5 and 29.0 +/- 8.0 microM, respectively. Recovery of cellular enzymatic activity is negligible (< 6%) following inactivation. Leukotriene A4 inactivates cellular 5-lipoxygenase without inhibiting its translocation from the cytosol to the membrane, suggesting that it impairs catalysis without impairing formation of the complex between 5-lipoxygenase and its membrane-associated activating protein. Consistent with this, leukotriene A4 inactivates purified 5-lipoxygenase from human neutrophils, via saturable, pseudo first-order kinetics with a rate constant, ki = 0.14 min-1 and a dissociation constant, Ki = 2.1 +/- 0.7 microM. Purified 5-lipoxygenase incubated with [3H]arachidonic acid incorporated a radiolabeled species that was not removed by electrophoresis under reduced denaturing conditions. Preincubation with leukotriene A4 diminished the incorporation of radiolabeled material, consistent with irreversible modification of 5-lipoxygenase by its metastable product, leukotriene A4. This unusual product inactivation mechanism may contribute to the decline in 5-lipoxygenase activity observed during catalysis.
Lepley
R A
RA
Department of Pharmacology C236, University of Colorado Health Science Center, Denver 80262.
Fitzpatrick
F A
FA
D001094:Arachidonate 5-Lipoxygenase / Q000097:blood / Q000302:isolation & purification; D002454:Cell Differentiation; D002460:Cell Line; D006801:Humans; D006893:Hydroxyeicosatetraenoic Acids / Q000032:analysis / Q000378:metabolism; D006918:Hydroxyurea / Q000031:analogs & derivatives / Q000494:pharmacology; D007700:Kinetics; D015473:Leukemia, Promyelocytic, Acute; D007962:Leukocytes / Q000201:enzymology*; D017572:Leukotriene A4 / Q000031:analogs & derivatives / Q000494:pharmacology*; D016859:Lipoxygenase Inhibitors / Q000494:pharmacology; D009504:Neutrophils / Q000201:enzymology*; D014407:Tumor Cells, Cultured
D006893:Hydroxyeicosatetraenoic Acids; D017572:Leukotriene A4; D016859:Lipoxygenase Inhibitors; C022022:5-hydroxy-6,8,11,14-eicosatetraenoic acid; C043073:leukotriene A methyl ester; D001094:Arachidonate 5-Lipoxygenase; C063449:zileuton; D006918:Hydroxyurea