Reaction mechanisms of 15-hydroperoxyeicosatetraenoic acid catalyzed by human prostacyclin and thromboxane synthases.
Prostacyclin synthase (PGIS) and thromboxane synthase (TXAS) are atypical cytochrome P450s. They do not require NADPH or dioxygen for isomerization of prostaglandin H(2) (PGH(2)) to produce prostacyclin (PGI(2)) and thromboxane A(2) (TXA(2)). PGI(2) and TXA(2) have opposing actions on platelet aggregation and blood vessel tone. In this report, we use a lipid hydroperoxide, 15-hydroperoxyeicosatetraenoic acid (15-HPETE), to explore the active site characteristics of PGIS and TXAS. The two enzymes transformed 15-HPETE not only into 13-hydroxy-14,15-epoxy-5,8,11-eicosatrienoic acid (13-OH-14,15-EET), like many microsomal P450s, but also to 15-ketoeicosatetraenoic acid (15-KETE) and 15-hydroxyeicosatetraenoic acid (15-HETE). 13-OH-14,15-EET and 15-KETE result from homolytic cleavage of the O-O bond, whereas 15-HETE results from heterolytic cleavage, a common peroxidase pathway. About 80% of 15-HPETE was homolytically cleaved by PGIS and 60% was homolytically cleaved by TXAS. The V(max) of homolytic cleavage is 3.5-fold faster than heterolytic cleavage for PGIS-catalyzed reactions (1100 min(-1)vs. 320 min(-1)) and 1.4-fold faster for TXAS (170 min(-1)vs. 120 min(-1)). Similar K(M) values for homolytic and heterolytic cleavages were found for PGIS ( approximately 60 microM 15-HPETE) and TXAS ( approximately 80 microM 15-HPETE), making PGIS a more efficient catalyst for the 15-HPETE reaction.
Yeh
Hui-Chun
HC
Division of Hematology, Department of Internal Medicine, University of Texas Health Science Center, 6431 Fannin, Houston, TX 77030, USA.
Tsai
Ah-Lim
AL
Wang
Lee-Ho
LH
D002384:Catalysis; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D019746:Intramolecular Oxidoreductases / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015289:Leukotrienes / Q000378:metabolism*; D008054:Lipid Peroxides / Q000378:metabolism*; D013930:Thromboxane-A Synthase / Q000378:metabolism*
D015289:Leukotrienes; D008054:Lipid Peroxides; C025086:15-hydroperoxy-5,8,11,13-eicosatetraenoic acid; D003577:Cytochrome P-450 Enzyme System; D019746:Intramolecular Oxidoreductases; C022461:prostacyclin synthetase; D013930:Thromboxane-A Synthase