Prostaglandin Endoperoxide H Synthases

Abstract

The cyclooxygenase (COX) activity of prostaglandin endoperoxide H synthases (PGHSs) converts arachidonic acid and O 2 to prostaglandin G 2 (PGG 2 ). PGHS peroxidase (POX) activity reduces PGG 2 to PGH 2 . 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 H 2 O 2 , 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 H 2 O 2 or 15-hydroperoxyeicosatetraenoic acid, a surrogate substrate for PGG 2 . 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 PGG 2 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 H 2 O 2 , 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.

Human Cyclooxygenase-2 Is a Sequence Homodimer That Functions as a Conformational Heterodimer

“…An examination of huPGHS-2 heterodimers having a defective POX active site indicated that the COX site of a monomer cannot function unless the POX site of that monomer is functional (25). This finding implies that the monomer of native huPGHS-2 to which heme is bound is the COX catalytic monomer.…”

“…Expression, Purification, and Assay of huPGHS-2-Procedures for the expression of huPGHS-2 in insect cells and purification of the enzyme were essentially the same as those reported previously (17,24,25). The purity of the recombinant huPGHS-2 was determined by SDS-PAGE and by Western blot analysis (24).…”

Leucine/Valine Residues Direct Oxygenation of Linoleic Acid by (10R)- and (8R)-Dioxygenases

“…Val-291 is one of these residues, which form a dome over the distal heme side of COX-1. The V291A mutant retained cyclooxygenase and peroxidase activities (27). 5,8-and 7,8-LDS also have valine residues in the homologous position, whereas 8,11-LDS and 10R-DOX have leucine residues (Fig.…”

“…PGG 2 and presumably 8R-HPODE bind to the distal side of the heme group, which can be delineated by hydrophobic amino acid residues (27). Val-291 is one of these residues, which form a dome over the distal heme side of COX-1.…”

“…12S ( Site-directed Mutagenesis of the Distal Heme Side of 10R-DOX-Leu-306 of 10R-DOX is homologous to Val-291 of PGHS, which is one of the hydrophobic residues forming the dome over the distal heme region of PGHS (27). Valine residues of 7,8-LDS and 5,8-LDS are found in the homologous position of Leu-306 (Fig.…”