title: On the mechanism of prostacyclin and thromboxane A2 biosynthesis.
abstract: The present research describes studies which address the mechanism of prostacyclin (PGI2) and thromboxane A2 (TXA2) biosynthesis. In addition to prostaglandin H1 (PGH1), PGG2, PGH2, and PGH3, also 8-iso-PGH2, 13(S)-hydroxy-PGH2, and 15-keto-PGH2 were applied to determine the substrate specificities and kinetics of prostacyclin and thromboxane synthase in more detail. Human platelet thromboxane synthase converted PGH1, 8-iso-PGH2, 13(S)-hydroxy-PGH2 and 15-keto-PGH2 into the corresponding heptadecanoic acid (C17) plus malondialdehyde, whereas the thromboxane derivative was formed only from PGG2, PGH2, and PGH3 together with the corresponding C17 metabolite and malondialdehyde in a 1:1:1 ratio. In contrast, PGG2, PGH2, 13(S)-hydroxy-PGH2, 15-keto-PGH2 and PGH3 were almost completely isomerized to the corresponding prostacyclin derivative by bovine aortic prostacyclin synthase, whereas PGH1 and 8-iso-PGH2 only produced the corresponding C17 hydroxy acid plus malondialdehyde. Isotope-labeling experiments with [5,6,8,9,11,12,14,15-2H]PGH2 revealed complete retention of label and no isotope effect in the course of thromboxane biosynthesis, but the loss of one 2H atom at C-6 with an isotope effect of 1.20 during PGI2 formation. Prostacyclin and thromboxane synthase bind both 9,11-epoxymethano-PGF2 alpha and 11,9-epoxymethano-PGF2 alpha at the heme iron, but according to their difference spectra in opposite ways with respect to the 9- and 11-position. In agreement with published model studies, a cage radical mechanism is proposed for both enzymes according to which the initial radical process is terminated through oxidation of carbon-centered radicals by the iron-sulfur catalytic site, followed by ionic rearrangement to PGI2 or TXA2. Various Fe(III) model compounds as well as liver microsomes or cytochrome P-450CAM can also form small amounts of PGI2 and TXA2, but mainly yield 12(S)-hydroxy-5,8,10-heptadecatrienoic acid plus malondialdehyde probably by a radical fragmentation pathway.
authors.affiliation: Faculty of Biology, University of Konstanz, Federal Republic of Germany.
authors.forename: M
authors.initials: M
authors.lastname: Hecker
authors.forename: V
authors.initials: V
authors.lastname: Ullrich
mesh_terms: D000818:Animals; D001011:Aorta / Q000201:enzymology; D016718:Arachidonic Acid; D001095:Arachidonic Acids / Q000378:metabolism; D001792:Blood Platelets / Q000201:enzymology; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D011464:Epoprostenol / Q000096:biosynthesis*; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D019746:Intramolecular Oxidoreductases; D007535:Isomerases / Q000378:metabolism*; D008861:Microsomes / Q000201:enzymology*; D015394:Molecular Structure; D011453:Prostaglandins / Q000378:metabolism; D013379:Substrate Specificity; D013929:Thromboxane B2 / Q000096:biosynthesis*; D013930:Thromboxane-A Synthase / Q000097:blood*
chemical_list: D001095:Arachidonic Acids; D011453:Prostaglandins; D016718:Arachidonic Acid; D013929:Thromboxane B2; D003577:Cytochrome P-450 Enzyme System; D011464:Epoprostenol; D007535:Isomerases; D019746:Intramolecular Oxidoreductases; C022461:prostacyclin synthetase; D013930:Thromboxane-A Synthase