The N-terminal membrane anchor domain of the membrane-bound prostacyclin synthase involved in the substrate presentation of the coupling reaction with cyclooxygenase.
To mimic the native conditions, the cyclooxygenase (COX)/prostaglandin I(2) synthase (PGIS) coupling reaction system was used to determine the coordination of PGIS with COX for the biosynthesis of prostacyclin (PGI(2)) using arachidonic acid (AA) as a substrate in a membrane-bound environment. The membrane-bound PGIS exhibited a faster isomerization of PGH(2) produced by COX to PGI(2) than the detergent-solubilized PGIS. To determine whether the N-terminal domain of PGIS responds to the facilitation of PGH(2) movement (presentation) from COX to the active site of PGIS, the first 20 residues of PGIS (Delta20-PGIS) were deleted and expressed in COS-7 cells. Delta20-PGIS retained membrane-bound properties and exhibited a slower substrate presentation property. Furthermore, a chimeric molecule (PGIS/TXAS(8-27)) with the replacement of the first 20 residues of PGIS by the corresponding membrane anchor region (residues 8-27) of thromboxane A(2) synthase was created to evaluate the mechanism influencing the biosynthesis of PGI(2) in coordination with COX. The chimera revealed a multiple fold delay in the PGH(2) presentation in low range concentrations of AA (0.3-3muM) at 30s reactions. However, the delay could be recovered by a longer incubation time in high range concentrations of AA (>10muM), but not in low range concentrations of AA. These results demonstrated that the N-terminal domain of PGIS plays a role in the facilitation of the substrate presentation to the PGIS active site in low concentrations of AA, which may be a physiological condition. The TXAS N-terminal domain could not replace the function of the corresponding domain of PGIS, indicating that the facilitation of the substrate presentation is specific.
Ruan
Ke-He
KH
Department of Internal Medicine, The University of Texas Health Science Center, Houston, TX 77030, USA. kruan@uth.tmc.edu
Deng
Hui
H
Wu
Jiaxin
J
So
Shui-Ping
SP
D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism; D001665:Binding Sites; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism; D002522:Chlorocebus aethiops; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D011464:Epoprostenol / Q000235:genetics / Q000378:metabolism*; D019746:Intramolecular Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D011451:Prostaglandin-Endoperoxide Synthases / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary / Q000235:genetics / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D013930:Thromboxane-A Synthase / Q000235:genetics / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; D016718:Arachidonic Acid; D003577:Cytochrome P-450 Enzyme System; D011464:Epoprostenol; D011451:Prostaglandin-Endoperoxide Synthases; D019746:Intramolecular Oxidoreductases; C022461:prostacyclin synthetase; D013930:Thromboxane-A Synthase