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 I2 synthase (PGIS) coupling reaction system was used to determine the coordination of PGIS with COX for the biosynthesis of prostacyclin (PGI2) using arachidonic acid (AA) as a substrate in a membrane-bound environment. The membrane-bound PGIS exhibited a faster isomerization of PGH2 produced by COX to PGI2 than the detergent-solubilized PGIS. To determine whether the N-terminal domain of PGIS responds to the facilitation of PGH2 movement (presentation) from COX to the active site of PGIS, the first 20 residues of PGIS (Δ20-PGIS) were deleted and expressed in COS-7 cells. Δ20-PGIS retained membrane-bound properties and exhibited a slower substrate presentation property. Furthermore, a chimeric molecule (PGIS/TXAS8-27) with the replacement of the first 20 residues of PGIS by the corresponding membrane anchor region (residues 8-27) of thromboxane A2 synthase was created to evaluate the mechanism influencing the biosynthesis of PGI2 in coordination with COX. The chimera revealed a multiple fold delay in the PGH2 presentation in low range concentrations of AA (0.3-3 μM) at 30 s reactions. However, the delay could be recovered by a longer incubation time in high range concentrations of AA (>10 μM), 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.