Prostacyclin is a potent mediator of vasodilation and anti-platelet aggregation. It is synthesized from prostaglandin H(2) by prostacyclin synthase (PGIS), a member of Family 8 in the cytochrome P450 superfamily. Unlike most P450s, which require exogenous reducing equivalents and an oxygen molecule for mono-oxygenation, PGIS catalyzes an isomerization with an initial step of endoperoxide bond cleavage of prostaglandin H(2) (PGH(2)). The low abundance of PGIS in natural tissues necessitates heterologous expression for studies of structure/function relationships and reaction mechanism. We report here a high-yield prokaryotic system for expression of enzymatically active human PGIS. The PGIS cDNA is modified by replacing the hydrophobic amino-terminal sequence with the more hydrophilic amino-terminal sequence from P450 2C5 and by adding a four-histidine tag at the carboxyl terminus. The resulting recombinant PGIS associates with host cell membranes and was purified to electrophoretic homogeneity by nickel affinity, hydroxyapatite and CM Sepharose column chromatography. The recombinant PGIS, with a heme:protein ratio of 0.9:1, catalyzes prostacyclin formation at a K(m) of 13.3 muM PGH(2) and a V(max) of 980 per min. The dithionite-reduced PGIS binds CO with an on-rate of 5.6 x 10(5) M(-1) s(-1) and an off-rate of 15 s(-1). The ferrous-CO complex of PGIS is very short-lived and decays at a rate of 0.7 s(-1). Spectral binding assays showed that imidazole binds weakly to PGIS (K(d) approximately 0.5 mM,) but clotrimazole, a bulky and rigid imidazole derivative, binds strongly (K(d) approximately 1 microM). The transient nature of the CO complex and the weak imidazole binding seem to support an earlier proposal that PGIS active site has a limited space, but the tight binding of clotrimazole argues against this view. It appears that the heme distal pocket of PGIS is fairly adaptable to ligands of various structures. UV-visible absorption, magnetic circular dichroism and electron paramagnetic resonance spectra indicate that PGIS has a typical low-spin heme with a hydrophobic active site. PGIS catalyzes homolytic scission of the peroxide bond of a test substrate, 10-hydroperoxyoctadeca-8,12-dienoic acid, accompanied by formation of a heme intermediate with a Compound II-like optical spectrum.
Prostaglandin H synthase-1 (PGHS-1) is a bifunctional heme protein catalyzing both a peroxidase reaction, in which peroxides are converted to alcohols, and a cyclooxygenase reaction, in which arachidonic acid is converted into prostaglandin G 2 . Reaction of PGHS-1 with peroxide forms Intermediate I, which has an oxyferryl heme and a porphyrin radical. An intramolecular electron transfer from Tyr385 to Intermediate I forms Intermediate II, which contains two oxidants: an oxyferryl heme and the Tyr385 radical required for cyclooxygenase catalysis. Self-inactivation of the peroxidase begins with Intermediate II, but it has been unclear which of the two oxidants is involved. The kinetics of tyrosyl radical, oxyferryl heme, and peroxidase inactivation were examined in reactions of PGHS-1 reconstituted with heme or mangano protoporphyrin IX with a lipid hydroperoxide, 15-hydroperoxyeicosatetraenoic acid (15-HPETE), and ethyl hydrogen peroxide (EtOOH). Tyrosyl radical formation was significantly faster with 15-HPETE than with EtOOH and roughly paralleled oxyferryl heme formation at low peroxide levels. However, the oxyferryl heme intensity decayed much more rapidly than the tyrosyl radical intensity at high peroxide levels. The rates of reactions for PGHS-1 reconstituted with MnPPIX were approximately an order of magnitude slower, and the initial species formed displayed a wide singlet (WS) radical, rather than the wide doublet radical observed with PGHS-1 reconstituted with heme. Inactivation of the peroxidase activity during the reaction of PGHS-1 with EtOOH or 15-HPETE correlated with oxyferryl heme decay, but not with changes in tyrosyl radical intensity or EPR line shape, indicating that the oxyferryl heme, and not the tyrosyl radical, is responsible for the self-destructive peroxidase side reactions. Computer modeling to a minimal mechanism was consistent with oxyferryl heme being the source of peroxidase inactivation.Prostagladin H synthases (PGHSs) catalyze the first two steps in biosynthesis of prostaglandins, the cyclooxygenase reaction that converts arachidonic acid (AA) 1 to prostaglandin (PG)G 2 and the peroxidase reaction that reduces PGG 2 to PGH 2 (1). The cyclooxygenase reaction is dependent upon formation of the Tyr385 radical during the peroxidase reaction (2). A simplified mechanistic model is shown in Figure 1. According to † This work was supported by U.S. Public Health Service Grants GM44911 (to A.-L.T.), GM52170 (to R.J.K.), and GM55807 (to G.P.) and Postdoctoral Fellowship DK61929 (to C.E.R.), and Welch Foundation Grant C636 (to G.P. Reaction with peroxide drives the redox events that lead to strong feedback amplification of cyclooxygenase catalysis, and it also drives the irreversible self-inactivation of both peroxidase and cyclooxygenase activities that imposes an upper limit on prostanoid synthesis (7,8).Cyclooxygenase inactivation induced by peroxide occurs on the same time scale as peroxidase inactivation, although the former seems more sensitive to the peroxide structure (9,1...
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