Reaction mechanisms of 15-hydroperoxyeicosatetraenoic acid catalyzed by human prostacyclin and thromboxane synthases

Abstract

Prostacyclin synthase (PGIS) and thromboxane synthase (TXAS) are atypical cytochrome P450s. They do not require NADPH or dioxygen for isomerization of prostaglandin H(2) (PGH(2)) to produce prostacyclin (PGI(2)) and thromboxane A(2) (TXA(2)). PGI(2) and TXA(2) have opposing actions on platelet aggregation and blood vessel tone. In this report, we use a lipid hydroperoxide, 15-hydroperoxyeicosatetraenoic acid (15-HPETE), to explore the active site characteristics of PGIS and TXAS. The two enzymes transformed 15-HPETE not only into 13-hydroxy-14,15-epoxy-5,8,11-eicosatrienoic acid (13-OH-14,15-EET), like many microsomal P450s, but also to 15-ketoeicosatetraenoic acid (15-KETE) and 15-hydroxyeicosatetraenoic acid (15-HETE). 13-OH-14,15-EET and 15-KETE result from homolytic cleavage of the O-O bond, whereas 15-HETE results from heterolytic cleavage, a common peroxidase pathway. About 80% of 15-HPETE was homolytically cleaved by PGIS and 60% was homolytically cleaved by TXAS. The V(max) of homolytic cleavage is 3.5-fold faster than heterolytic cleavage for PGIS-catalyzed reactions (1100 min(-1)vs. 320 min(-1)) and 1.4-fold faster for TXAS (170 min(-1)vs. 120 min(-1)). Similar K(M) values for homolytic and heterolytic cleavages were found for PGIS ( approximately 60 microM 15-HPETE) and TXAS ( approximately 80 microM 15-HPETE), making PGIS a more efficient catalyst for the 15-HPETE reaction.

Fig.1

SDS-PAGE analysis of purified hPGIS and TXAS (1 μg each) on a 10 % polyacrylamide gel. The proteins were stained by Coomassie Blue. The sizes of molecular weight markers (10 kDa markers from GibcoBRL) are indicated.

Fig.2

Chromatographic analyses of products from the reaction of 15-HPETE with PGIS or TXAS. HPLC radio-chromatograms of reaction products of (A) 0.5 μM of Mn-PGHS with 100 μM 15-HPETE for 60 s in the presence of 1 mM guaiacol, (B) 2 μM of boiled PGIS with 15-HPETE for 10 s, (C) 2 μM of PGIS with 75 μM 15-HPETE for 10 s and (D) 5 μM of TXAS with 60 μM 15-HPETE for 10 s.

Fig.3

Concentration dependence of PGIS- and TXAS-catalyzed homolytic and heterolytic cleavage of 15-HPETE. Reactions used 0.5 μM PGIS (Panel A) and 1.8 μM TXAS (Panel B) with indicated concentrations of [1-¹⁴C]-15-HPETE for 5 s and 10 s, respectively. Products were separated by HPLC and quantified by on-line liquid scintillation. The amounts of homolytic reaction (15-KETE + 13-OH-14,15-EET; filled circles) and heterolytic reaction (15-HETE; open circles) were fit to the Michaelis-Menten equation by linear regression.

Fig.4

Pre-steady-state kinetics of PGIS reaction with 15-HPETE. (A) rapid scan absorbance data of the first 16.4 s for the reaction of PGIS (3.75 μM) with 15-HPETE (56 μM). Spectra were recorded at the indicated time. Arrows show the direction of spectral changes with increasing time. Inset: Resolved spectral intermediates by global analysis using a sequential model (A→ B → C). The fitted rate constants were: k₁= 0.53 s⁻¹, and k₂= 0.07 s⁻¹. The spectrum for A is shown with a solid line, B with a dotted line, and C with a dashed line. (B) Left panel, time course of absorbance at 418 nm during the reaction of PGIS (3 μM) with 15-HPETE (32 to 414 μM). Right panel, plots of k_obs for the first phase (filled circles) and the second phase (open circles) of A₄₁₈ changes versus 15-HPETE concentration.

Fig.5

Pre-steady-state kinetics of 15-HPETE catalyzed by TXAS. (A) rapid scan absorbance data at 0.08, 4.18, 8.27, 12.4, 16.5, 20.6, 24.7 and 28.8 s during the reaction of TXAS (4 μM) with 15-HPETE (53 μM). Arrows show the direction of spectral changes with increasing time. Inset: Resolved spectral intermediates from singular value decomposition and global analyses by an irreversible two-step sequential mechanism (A→ B → C) with k₁= 0.2 s⁻¹ and k₂= 0.05 s⁻¹. Species A, solid line; species B, dotted line; species C, dashed line. (B) Left panel, time course of absorbance changes at 418 nm during the reaction of TXAS (3.0 μM) with 15-HPETE (27 to 360 μM). Right panel, plots of k_obs for the first phase (filled circles) and the second phase (open circles) versus 15-HPETE concentration.

Fig.6

EPR spectra of PGIS and TXAS (55 μM) before and after reacting with a 20-fold excess of 15-HPETE for 3-5 s at 23°C. EPR conditions were: microwave power, 1 mW; microwave frequency, 9.6 GHz; modulation amplitude, 10.87 G, and temperature, 10 K.

Scheme I

Proposed metabolism of 15-HPETE, catalyzed by PGIS or TXAS.

Scheme II