Prostaglandin endoperoxide synthase (PGHS) catalyzes the committed step in the conversion of arachidonic acid to prostaglandins and thromboxane. Prostanoids are """"""""local"""""""" hormones which have physiologically important roles in virtually all mammalian tissues. PGHS exhibits both a cyclooxygenase activity involved in the synthesis of PGG2 from arachidonate and a peroxidase activity involved in the reduction of PGG2 to PGH2. The cyclooxygenase activity of PGHS is a target of nonsteroidal anti-inflammatory drugs (NSAIDs). Thus, information about how the enzyme functions and how it interacts with NSAIDs has potential therapeutic importance. The long-range goal of our research is to determine the mechanisms of cyclooxygenase and peroxidase catalysis. Current work is concentrated on identifying amino acids essential for catalysis by, PGHS. During the past budget period, histidine and tyrosine residues essential for cyclooxygenase and peroxidase activities were identified. In addition, the mechanism by which aspirin inhibits cyclooxygenase activity was determined. Finally, the sites of N-glycosylation of PGHS-1 were identified, and the importance of N-glycosylation in the expression of PGHS activity was established. We now propose: (a) to determine if the tyrosyl radical observed during catalysis initiates suicide inactivation of cyclooxygenase activity and to identify the tyrosine residue which is the source of the radical; these studies will involve a combination of site-directed mutagenesis and EPR spectroscopy. (b) to characterize more completely the structure of the cyclooxygenase active site; photoaffinity probes will be employed to identify active site residues; in addition, chimeras of murine PGHS-1 and PGHS-2 isozymes will be used to determine the importance of the C-terminal domain, encoded by the last exon and containing the aspirin acetylation site, in governing isozyme specificities toward NSAIDs; in addition, in vitro expression systems will be utilized to determine the specificities of the human PGHS-1 and -2 isozymes toward NSAIDs. (c) to determine the inside/outside orientation of various domains of PGH synthase within the endoplasmic reticulum; these studies Will employ a combination of several anti-peptide antibodies and a photoactive probe reactive with amino acids located in transmembrane regions.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
2R01DK042509-04
Application #
3243619
Study Section
Medical Biochemistry Study Section (MEDB)
Project Start
1990-06-01
Project End
1998-05-31
Budget Start
1993-06-01
Budget End
1994-05-31
Support Year
4
Fiscal Year
1993
Total Cost
Indirect Cost
Name
Michigan State University
Department
Type
Schools of Medicine
DUNS #
193247145
City
East Lansing
State
MI
Country
United States
Zip Code
48824
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
Smith, W L; Garavito, R M; DeWitt, D L (1996) Prostaglandin endoperoxide H synthases (cyclooxygenases)-1 and -2. J Biol Chem 271:33157-60
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
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
Regier, M K; Otto, J C; DeWitt, D L et al. (1995) Localization of prostaglandin endoperoxide synthase-1 to the endoplasmic reticulum and nuclear envelope is independent of its C-terminal tetrapeptide-PTEL. Arch Biochem Biophys 317:457-63

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