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.
Highlights
EXPERIMENTAL PROCEDURESMaterials—Fatty acids were purchased from Cayman Chemical Co., Ann Arbor, MI. [1-14C]Arachidonic acid (40 – 60 mCi/mmol) was purchased from PerkinElmer Life Sciences
Prostaglandin endoperoxide H synthases (PGHSs) catalyze the committed step in the biosynthesis of prostaglandins and thromboxane, the conversion of arachidonic acid, two molecules of O2, and two electrons to prostaglandin endoperoxide H2 (PGH2)
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 PGG2 versus monohydroperoxy acid products (Val-349, Trp-387, and Leu534); and (e) all other active site residues, which individually make less but measurable contributions to optimal catalytic efficiency
Summary
Materials—Fatty acids were purchased from Cayman Chemical Co., Ann Arbor, MI. [1-14C]Arachidonic acid (40 – 60 mCi/mmol) was purchased from PerkinElmer Life Sciences. Inhibition of cyclooxygenase activity was measured by adding aliquots of microsomal suspensions to assay mixtures containing 100 M arachidonate and 200 M flurbiprofen. Inhibition of Cyclooxygenase Activity—To determine the rates of inactivation of native and mutant oPGHS-1 by aspirin, microsomal enzyme samples were incubated with or without 0.1 mM acetylsalicylate at 37 °C; aliquots were removed at 0, 10, 20, 30, 40, 60, and 90 min, and cyclooxygenase activity was measured as described above. For time-dependent inhibition studies, flurbiprofen at an appropriate concentration (e.g. 50 M) was preincubated with the enzyme (250 g of microsomal protein) at 37 °C for various times; cyclooxygenase measurements were performed upon adding enzymeinhibitor complex to the assay chamber. For RP-HPLC analyses of products, native or mutant oPGHS-1 (1 mg of microsomal protein) was reacted with 100 M arachidonic acid for 30 min at 37 °C, and products were collected as described previously [23]. Statistical significance of the kinetic data (Fig. 5) was determined using a two-sample t test assuming equal variances
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