Suicide inactivation of porcine leukocyte 12-lipoxygenase associated with its incorporation of 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid derivative.
Two isozymes of arachidonate 12-lipoxygenase, platelet-type and leukocyte-type, which were distinguished by their substrate specificities and primary structures, were investigated with reference to 'suicide' inactivation. Upon reaction with arachidonic acid the leukocyte-type enzyme was inactivated rapidly during the catalysis, whereas the platelet-type enzyme did not show such a rapid inactivation. The two 12-lipoxygenase isozymes were incubated with various hydroperoxy and hydroxy products from arachidonic acid. (15S)-Hydroperoxy-5,8,11,13-eicosatetraenoic acid (15-HPETE) was found to be a unique substrate of the leukocyte-type 12-lipoxygenase as follows. (1) 15-HPETE was an active substrate for porcine leukocyte 12-lipoxygenase, and converted anaerobically to a 14,15-epoxy compound (14,15-leukotriene A4). (2) A rapid inactivation of the enzyme was observed within 2 min upon aerobic and anaerobic incubations with 15-HPETE. (3) 15-HPETE was rapidly incorporated into the enzyme in a nearly equimolar amount under both aerobic and anaerobic conditions. (4) Several findings suggested a covalent binding of 15-HPETE or its derivative to the enzyme. (5) Such a rapid and stoichiometric incorporation of 15-HPETE was not observed with the platelet-type 12-lipoxygenase. On the basis of these findings we presumed that 15-HPETE was transformed to 14,15-leukotriene A4, which was covalently bound to the leukocyte-type 12-lipoxygenase leading to the suicide inactivation of the enzyme.
Kishimoto
K
K
Department of Biochemistry, Tokushima University, School of Medicine, Japan.
Nakamura
M
M
Suzuki
H
H
Yoshimoto
T
T
Yamamoto
S
S
Takao
T
T
Shimonishi
Y
Y
Tanabe
T
T
D000818:Animals; D001092:Arachidonate 12-Lipoxygenase / Q000737:chemistry; D016718:Arachidonic Acid / Q000378:metabolism; D007962:Leukocytes / Q000201:enzymology*; D015289:Leukotrienes / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D008054:Lipid Peroxides / Q000737:chemistry / Q000494:pharmacology*; D016859:Lipoxygenase Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors; D013552:Swine
D015289:Leukotrienes; D008054:Lipid Peroxides; D016859:Lipoxygenase Inhibitors; D009097:Multienzyme Complexes; D016718:Arachidonic Acid; C021201:12-HPETE; C025086:15-hydroperoxy-5,8,11,13-eicosatetraenoic acid; D001092:Arachidonate 12-Lipoxygenase