Abstract
The cyclooxygenase (COX) activity of prostaglandin endoperoxide H synthases (PGHSs) converts arachidonic acid and O2 to prostaglandin G2 (PGG2). PGHS peroxidase (POX) activity reduces PGG2 to PGH2. The first step in POX catalysis is formation of an oxyferryl heme radical cation (Compound I), which undergoes intramolecular electron transfer forming Intermediate II having an oxyferryl heme and a Tyr-385 radical required for COX catalysis. PGHS POX catalyzes heterolytic cleavage of primary and secondary hydroperoxides much more readily than H2O2, but the basis for this specificity has been unresolved. Several large amino acids form a hydrophobic "dome" over part of the heme, but when these residues were mutated to alanines there was little effect on Compound I formation from H2O2 or 15-hydroperoxyeicosatetraenoic acid, a surrogate substrate for PGG2. Ab initio calculations of heterolytic bond dissociation energies of the peroxyl groups of small peroxides indicated that they are almost the same. Molecular Dynamics simulations suggest that PGG2 binds the POX site through a peroxyl-iron bond, a hydrogen bond with His-207 and van der Waals interactions involving methylene groups adjoining the carbon bearing the peroxyl group and the protoporphyrin IX. We speculate that these latter interactions, which are not possible with H2O2, are major contributors to PGHS POX specificity. The distal Gln-203 four residues removed from His-207 have been thought to be essential for Compound I formation. However, Q203V PGHS-1 and PGHS-2 mutants catalyzed heterolytic cleavage of peroxides and exhibited native COX activity. PGHSs are homodimers with each monomer having a POX site and COX site. Cross-talk occurs between the COX sites of adjoining monomers. However, no cross-talk between the POX and COX sites of monomers was detected in a PGHS-2 heterodimer comprised of a Q203R monomer having an inactive POX site and a G533A monomer with an inactive COX site.
Highlights
The cyclooxygenase (COX) activity of prostaglandin endoperoxide H synthases (PGHSs) converts arachidonic acid and O2 to prostaglandin G2 (PGG2)
Molecular Dynamics (MD) Simulation of PGG2 Binding to the POX Site—We previously reported on the interaction between a PGG2 analog that does not include the endoperoxide group and ovPGHS-1 that was carried out by first docking pseudo
Peroxides function by oxidizing the heme group at the POX site of PGHSs, which in turn leads to oxidation of Tyr-385 in the COX site, and the Tyr-385 radical abstracts a hydrogen from arachidonic acid in the ratedetermining step in COX catalysis [1,2,3]
Summary
Materials—15-Hydroperoxyeicosatetraenoic acid (15-HPETE) was synthesized as described previously [33] or purchased from Cayman Chemical Company. Bound PGHS was eluted with three column volumes of 250 mM imidazole These latter eluates were pooled, concentrated, and desalted using a Sephadex G-25 (Sigma) spin column, which was pre-swelled in desalting buffer (20 mM Tris-HCl, pH 8.0, 5% glycerol, 150 NaCl and 0.02% C10E6). POX Activity Assays—POX reactions were conducted in 100 l of filtered and degassed buffer, 100 mM Tris-HCl, pH 8.0, and 100 mM NaCl. Heme-reconstituted PGHS (76 nM) containing 9.0 mM guaiacol was mixed with an equal volume of a peroxide substrate solution using a stopped-flow apparatus (SX-60 HiTech Instruments). In single wavelength experiments at least 10-fold higher substrate concentrations were used to ensure steady state kinetics Both the enzyme and substrate solutions were prepared in 20 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 0.02% C10E6. The MD was carried out with the SANDER module of AMBER7 [39] using explicit solvent (ϳ5000 waters) in a 80 ϫ 80 ϫ 80-Å3 box, with the ionization states of all residues set appropriate to pH 7
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