The goal of the work proposed in this application is to continue studies aimed defining the amino acid residues involved in the catalytic mechanism of prostaglandin endoperoxidase G/H synthase. PGG/H synthase catalyzes the first step in the conversion of arachidonic acid to prostacyclin and thromboxane A2, two agents which play important roles in hemostasis. PGG/H synthase is a hemoprotein which has two activities, including a cyclooxygenase and a hydroperoxidase activity. In work performed previously, we have deduced the amino acid sequences of the sheep and mouse PGG/H synthases and a portion of the human PGG/H synthase. Based on sequence comparisons among PGG/H synthases and other peroxidases, we have proposed that His226 and His3O9 are the distal and proximal heme ligands of PGG/H synthase. We have established that the """"""""active site"""""""" serine which is acetylated by aspirin is Ser530, and using site-directed mutagenesis, we have provided evidence that acetylation of Ser530 places a bulky group at the active site of the cyclooxygenase, thereby interfering with arachidonate binding. Using modification with tetranitromethane, we have also recently identified what is likely to be a tyrosine at the active site of the cyclooxygenase. We now propose: (a) to use site-directed mutagenesis to determine if His3O9 and His226 are specifically involved in heme binding to PGG/H synthase, and further, whether residues adjoining these histidines are important in heme binding. (b) to use site-directed mutagenesis to determine if the amino acids adjoining Ser530 play important roles in arachidonate binding. (c) to identify the tyrosine residue which is modified by tetranitromethane to cause inactivation of the cyclooxygenase.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
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Biochemistry Study Section (BIO)
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Michigan State University
Schools of Medicine
East Lansing
United States
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Smith, W L; Meade, E A; Dewitt, D L (1997) Interaction of PGH synthase isozymes-1 and -2 with nonsteroidal anti-inflammatory drugs. Adv Exp Med Biol 400A:189-96
Smith, W (1997) Molecular biology of prostanoid biosynthetic enzymes and receptors. Adv Exp Med Biol 400B:989-1011
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Otto, J C; Smith, W L (1996) Photolabeling of prostaglandin endoperoxide H synthase-1 with 3-trifluoro-3-(m-[125I]iodophenyl)diazirine as a probe of membrane association and the cyclooxygenase active site. J Biol Chem 271:9906-10
Smith, W L; Dewitt, D L (1996) Prostaglandin endoperoxide H synthases-1 and -2. Adv Immunol 62:167-215
Bhattacharyya, D K; Lecomte, M; Rieke, C J et al. (1996) Involvement of arginine 120, glutamate 524, and tyrosine 355 in the binding of arachidonate and 2-phenylpropionic acid inhibitors to the cyclooxygenase active site of ovine prostaglandin endoperoxide H synthase-1. J Biol Chem 271:2179-84
Kraemer, S A; Arthur, K A; Denison, M S et al. (1996) Regulation of prostaglandin endoperoxide H synthase-2 expression by 2,3,7,8,-tetrachlorodibenzo-p-dioxin. Arch Biochem Biophys 330:319-28
Goetzl, E J; An, S; Smith, W L (1995) Specificity of expression and effects of eicosanoid mediators in normal physiology and human diseases. FASEB J 9:1051-8
Otto, J C; Smith, W L (1995) Prostaglandin endoperoxide synthases-1 and -2. J Lipid Mediat Cell Signal 12:139-56
Laneuville, O; Breuer, D K; Xu, N et al. (1995) Fatty acid substrate specificities of human prostaglandin-endoperoxide H synthase-1 and -2. Formation of 12-hydroxy-(9Z, 13E/Z, 15Z)- octadecatrienoic acids from alpha-linolenic acid. J Biol Chem 270:19330-6

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