Publications

• Thromboxane synthase as a cytochrome P450 enzyme.

  Ullrich V., Haurand M.

  Adv Prostaglandin Thromboxane Leukot Res 11:105-110(1983) [PubMed] [EuropePMC]

• Thromboxane A synthase-independent production of 12-hydroxyheptadecatrienoic acid, a BLT2 ligand.

  Matsunobu T., Okuno T., Yokoyama C., Yokomizo T.

  12(S)-hydroxyheptadeca-5Z,8E,10E-trienoic acid (12-HHT) has long been considered a by-product of thromboxane A₂ (TxA₂) biosynthesis with no biological activity. Recently, we reported 12-HHT to be an endogenous ligand for BLT2, a low-affinity leukotriene B4 receptor. To delineate the biosynthetic p ... >> More

  12(S)-hydroxyheptadeca-5Z,8E,10E-trienoic acid (12-HHT) has long been considered a by-product of thromboxane A₂ (TxA₂) biosynthesis with no biological activity. Recently, we reported 12-HHT to be an endogenous ligand for BLT2, a low-affinity leukotriene B4 receptor. To delineate the biosynthetic pathway of 12-HHT, we established a method that enables us to quantify various eicosanoids and 12-HHT using LC-MS/MS analysis. During blood coagulation, 12-HHT levels increased in a time-dependent manner and were relatively higher than those of TxB₂, a stable metabolite of TxA₂. TxB₂ production was almost completely inhibited by treatment with ozagrel, an inhibitor of TxA synthase (TxAS), while 12-HHT production was inhibited by 80-90%. Ozagrel-treated blood also exhibited accumulation of PGD₂ and PGE₂, possibly resulting from the shunting of PGH₂ into synthetic pathways for these prostaglandins. In TxAS-deficient mice, TxB₂ production during blood coagulation was completely lost, but 12-HHT production was reduced by 80-85%. HEK293 cells transiently expressing TxAS together with cyclooxygenase (COX)-1 or COX-2 produced both TxB₂ and 12-HHT from arachidonic acid, while HEK293 cells expressing only COX-1 or COX-2 produced significant amounts of 12-HHT but no TxB₂. These results clearly demonstrate that 12-HHT is produced by both TxAS-dependent and TxAS-independent pathways in vitro and in vivo. << Less

  J. Lipid Res. 54:2979-2987(2013) [PubMed] [EuropePMC]

• Thromboxane synthase catalyses hydroxylations of prostaglandin H2 analogs in the presence of iodosylbenzene.

  Hecker M., Baader W.J., Weber P., Ullrich V.

  Human platelet microsomes supplemented with iodosylbenzene converted the stable prostaglandin H2 analog 15(S)-hydroxy-11 alpha,9 alpha-epoxymethano-5(Z),13(E)-prostadienoic acid (U46619) into three metabolites (17.5% yield) which were not formed in the presence of specific thromboxane synthase inh ... >> More

  Human platelet microsomes supplemented with iodosylbenzene converted the stable prostaglandin H2 analog 15(S)-hydroxy-11 alpha,9 alpha-epoxymethano-5(Z),13(E)-prostadienoic acid (U46619) into three metabolites (17.5% yield) which were not formed in the presence of specific thromboxane synthase inhibitors. The same three products were also formed among others by incubation of U46619 with liver microsomes from phenobarbital-pretreated rats with NADPH/O2 or with iodosylbenzene. The NADPH-supported metabolism of U46619 was suppressed in the presence of carbon monoxide. Combined gas chromatography/negative-ion chemical ionization mass spectrometry analysis revealed for all three compounds the incorporation of one oxygen atom which according to the electron impact fragmentation pattern had to be introduced either at the 9-methylene group or at the cyclopentane ring. The identification of these metabolites as 9 beta,15(S)-dihydroxy-11 alpha,9 alpha-epoxymethano-5(Z),13(E)-prostadienoic acid and the R and S isomer of 15(S)-hydroxy-11 alpha,9 alpha-(C-hydroxy-epoxymethano)-5(Z),13(E)-prostadienoic acid is only tentative since no reference compounds were available, but clearly thromboxane synthase was acting as an oxene transferase in this reaction. In contrast to U46619, its 9,11-epoxymethano isomer U44069 was found to be only a poor substrate for the oxene transferase activity of thromboxane synthase (1% yield) which indicates a preference for the 9-methylene group of U46619 which is orientated close to the heme iron of thromboxane synthase as evidenced by spectroscopic studies. Low-level chemiluminescence detected following incubation of iodosylbenzene with partly purified thromboxane synthase is in agreement with the formation of an activated (FeO)3+ oxygen species. In summary, these results point to a common role of the thiolate ligand in the oxygen activation mechanism by thromboxane and prostacyclin synthase and liver cytochrome P-450 monooxygenases. << Less

  Eur J Biochem 169:563-569(1987) [PubMed] [EuropePMC]

• Substrate binding is the rate-limiting step in thromboxane synthase catalysis.

  Wang L.H., Tsai A.L., Hsu P.Y.

  Thromboxane synthase (TXAS) is a "non-classical" cytochrome P450. Without any need for an external electron donor, or for a reductase or molecular oxygen, it uses prostaglandin H2 (PGH2) to catalyze either an isomerization reaction to form thromboxane A2 (TXA2) or a fragmentation reaction to form ... >> More

  Thromboxane synthase (TXAS) is a "non-classical" cytochrome P450. Without any need for an external electron donor, or for a reductase or molecular oxygen, it uses prostaglandin H2 (PGH2) to catalyze either an isomerization reaction to form thromboxane A2 (TXA2) or a fragmentation reaction to form 12-l-hydroxy-5,8,10-heptadecatrienoic acid and malondialdehyde (MDA) at a ratio of 1:1:1 (TXA2:heptadecatrienoic acid:MDA). We report here kinetics of TXAS with heme ligands in binding study and with PGH2 in enzymatic study. We determined that 1) binding of U44069, an oxygen-based ligand, is a two-step process; U44069 first binds TXAS, then ligates the heme-iron with a maximal rate constant of 105-130 s(-1); 2) binding of cyanide, a carbon-based ligand, is a one-step process with k(on) of 2.4 M(-1) s(-1) and k(off) of 0.112 s(-1); and 3) both imidazole and clotrimazole (nitrogen-based ligands) bind TXAS in a two-step process; an initial binding to the heme-iron with on-rate constants of 8.4 x 10(4) M(-1) s(-1) and 1.5 x 10(5) M(-1) s(-1) for imidazole and clotrimazole, respectively, followed by a slow conformational change with off-rate constants of 8.8 s(-1) and 0.53 s(-1), respectively. The results of our binding study indicate that the TXAS active site is hydrophobic and spacious. In addition, steady-state kinetic study revealed that TXAS consumed PGH2 at a rate of 3,800 min(-1) and that the k(cat)/K(m) for PGH2 consumption was 3 x 10(6) M(-1) s(-1). Based on these data, TXAS appears to be a very efficient catalyst. Surprisingly, rapid-scan stopped-flow experiments revealed marginal absorbance changes upon mixing TXAS with PGH2, indicating minimal accumulation of any heme-derived intermediates. Freeze-quench EPR measurements for the same reaction showed minimal change of heme redox state. Further kinetic analysis using a combination of rapid-mixing chemical quench and computer simulation showed that the kinetic parameters of TXAS-catalyzed reaction are: PGH2 bound TXAS at a rate of 1.2-2.0 x 10(7) M(-1) s(-1); the rate of catalytic conversion of PGH2 to TXA2 or MDA was at least 15,000 s(-1) and the lower limit of the rates for products release was 4,000-6,000 s(-1). Given that the cellular PGH2 concentration is quite low, we concluded that under physiological conditions, the substrate-binding step is the rate-limiting step of the TXAS-catalyzed reaction, in sharp contrast with "classical" P450 enzymes. << Less

  J. Biol. Chem. 276:14737-14743(2001) [PubMed] [EuropePMC]

• Expression of human thromboxane synthase using a baculovirus system.

  Yokoyama C., Miyata A., Suzuki K., Nishikawa Y., Yoshimoto T., Yamamoto S., Nuesing R., Ullrich V., Tanabe T.

  Human thromboxane (TX) synthase (EC 5.3.99.5) was produced by the baculovirus expression system using cDNA encoding human TX synthase [(1991) Biochem. Biophys. Res. Commun. 78, 1479-1484]. A recombinant baculovirus TXS7 was expressed in Spodoptera frugiperda Sf9 insect cells. The expressed protein ... >> More

  Human thromboxane (TX) synthase (EC 5.3.99.5) was produced by the baculovirus expression system using cDNA encoding human TX synthase [(1991) Biochem. Biophys. Res. Commun. 78, 1479-1484]. A recombinant baculovirus TXS7 was expressed in Spodoptera frugiperda Sf9 insect cells. The expressed protein was recognized by monoclonal antibody, Kon 7 raised against human TX synthase [(1990) Blood 76, 80-85]. The recombinant TX synthase catalyzed the conversion of prostaglandin (PG) H2 to TXA2 and 12-hydroxy-heptadecatrienoic acid (HHT). Both conversions of PGH2 to TXA2 and HHT by the expressed TX synthase were completely inhibited by a specific TX synthase inhibitor, OKY-046 (5 microM). << Less

  FEBS Lett. 318:91-94(1993) [PubMed] [EuropePMC]