The aspirin and heme-binding sites of ovine and murine prostaglandin endoperoxide synthases.

Abstract

Acetylation of Ser-530 of sheep prostaglandin endoperoxide (PGG/H) synthase by aspirin causes irreversible inactivation of the cyclooxygenase activity of the enzyme. To determine the catalytic function of the hydroxyl group of Ser-530, we used site-directed mutagenesis to replace Ser-530 with an alanine. Cos-1 cells transfected with expression vectors containing the native (Ser-530) or mutant (Ala-530) cDNAs for sheep PGG/H synthase expressed comparable cyclooxygenase and hydroperoxidase activities. Km values for arachidonate (8 microM) and ID50 values for reversible inhibition by the cyclooxygenase inhibitors, flurbiprofen (5 microM), flufenamate (20 microM), and aspirin (20 mM), were also the same for both native and mutant PGG/H synthases; however, only the native enzyme was irreversibly inactivated by aspirin. Thus, the "active site" Ser-530 of PGG/H synthase is not essential for catalysis or substrate binding. Apparently, acetylation of native PGG/H synthase by aspirin introduces a bulky sidechain at position 530 which interferes with arachidonate binding. In related studies, a cDNA for mouse PGG/H synthase was cloned and sequenced. A sequence of 35 residues with Ser-530 at the midpoint was identical in the two proteins. Thus, Ser-530 does lie in a highly conserved region, probably involved in cyclooxygenase catalysis. Sequence comparisons of mouse and sheep PGG/H synthase also provided information about the heme-binding site of the enzyme. The sheep HYPR sequence (residues 274-277), which had been proposed to form a portion of the distal heme-binding site, is not conserved in the mouse PGG/H synthase, suggesting that this region is not the distal heme-binding site. One sequence, TIWLREHNRV (residues 303-312 of the sheep enzyme), is very closely related to the sequence TLW(L)LREHNRL common to thyroid peroxidase and myeloperoxidase. The histidine in this latter sequence is the putative axial heme ligand of these peroxidases. We suggest that the histidine (His-309) of sheep PGG/H synthase sequence is the axial heme ligand of this enzyme.