Prostaglandin Endoperoxide H Synthase-1

Abstract

Prostaglandin endoperoxide H synthases (PGHSs) catalyze the committed step in the biosynthesis of prostaglandins and thromboxane, the conversion of arachidonic acid, two molecules of O(2), and two electrons to prostaglandin endoperoxide H(2) (PGH(2)). Formation of PGH(2) involves an initial oxygenation of arachidonate to yield PGG(2) catalyzed by the cyclooxygenase activity of the enzyme and then a reduction of the 15-hydroperoxyl group of PGG(2) to form PGH(2) catalyzed by the peroxidase activity. The cyclooxygenase active site is a hydrophobic channel that protrudes from the membrane binding domain into the core of the globular domain of PGHS. In the crystal structure of Co(3+)-heme ovine PGHS-1 complexed with arachidonic acid, 19 cyclooxygenase active site residues are predicted to make a total of 50 contacts with the substrate (Malkowski, M. G, Ginell, S., Smith, W. L., and Garavito, R. M. (2000) Science 289, 1933-1937); two of these are hydrophilic, and 48 involve hydrophobic interactions. We performed mutational analyses to determine the roles of 14 of these residues and 4 other closely neighboring residues in arachidonate binding and oxygenation. Mutants were analyzed for peroxidase and cyclooxygenase activity, and the products formed by various mutants were characterized. Overall, the results indicate that cyclooxygenase active site residues of PGHS-1 fall into five functional categories as follows: (a) residues directly involved in hydrogen abstraction from C-13 of arachidonate (Tyr-385); (b) residues essential for positioning C-13 of arachidonate for hydrogen abstraction (Gly-533 and Tyr-348); (c) residues critical for high affinity arachidonate binding (Arg-120); (d) residues critical for positioning arachidonate in a conformation so that when hydrogen abstraction does occur the molecule is optimally arranged to yield PGG(2) versus monohydroperoxy acid products (Val-349, Trp-387, and Leu-534); and (e) all other active site residues, which individually make less but measurable contributions to optimal catalytic efficiency.