The goal of this project is to develop a structural understanding of how prostaglandin H2synthase (PGHS) functions focusing on both the enzyme's recognition of inhibitors and fatty acid substrates and on the relationship between the enzyme's two distinct activities. PGHS catalyzes the conversion of arachidonic acid to prostaglandin H2 and this process is inhibited by nonsteroidal anti-inflammatory drugs (NSAIDs) such as aspirin, ibuprofen, naproxen, and indomethacin.
The specific aims of the project are: (1) exploration of active site plasticity: structures of PGHS complexed with conformationally constrained indomethacin analogs will be determined to investigate the ability of the active site to adopt alternate conformations in recognizing various NSAIDs, and to better understand the possible mechanism by which the cyclooxygenase and peroxidase active sites communicate with each other. (2) Analysis of fatty acid binding and cyclooxygenase catalysis. The nature of the Michaelis complex of arachidonic acid or related fatty acids bound to PGHS will be determined using crystals soaked in substrate solutions under anaerobic conditions. The structural data will be used to elucidate the spatial relationship of the substrate with the radical tryosine 385 in addition to any conformational constraints imposed by the enzyme on the substrate. The purpose of this specific aim is to examine the structures of fatty acids substrates and inhibitors bound to PGHS to test whether tyr-385 is situated appropriately to act as a hydrogen atom abstractor.
Specific Aim (3) Examination of the relationship between the peroxidase and cyclooxygenase catalytic activities: structures will be determined of peroxidase deficient forms of PGHS (using chemical modification techniques and/or reconstitution with non-iron metalloporphyrins. Comparison of these structures with that of the peroxidase competent form of the enzyme will contribute to the understanding of the peroxidase mechanism by revealing features of the active site necessary for substrate binding/catalysis. Finally, structures of different cyclooxygenase or peroxidase deficient forms of the enzyme will be determined to help clarify the relationship between the two activities.
| Gupta, Kushol; Kaub, Carl J; Carey, Kristen N et al. (2004) Manipulation of kinetic profiles in 2-aryl propionic acid cyclooxygenase inhibitors. Bioorg Med Chem Lett 14:667-71 |
| Gupta, Kushol; Selinsky, Barry S; Kaub, Carl J et al. (2004) The 2.0 A resolution crystal structure of prostaglandin H2 synthase-1: structural insights into an unusual peroxidase. J Mol Biol 335:503-18 |
| Selinsky, B S; Gupta, K; Sharkey, C T et al. (2001) Structural analysis of NSAID binding by prostaglandin H2 synthase: time-dependent and time-independent inhibitors elicit identical enzyme conformations. Biochemistry 40:5172-80 |
| Bevivino, A E; Loll, P J (2001) An expanded glutamine repeat destabilizes native ataxin-3 structure and mediates formation of parallel beta -fibrils. Proc Natl Acad Sci U S A 98:11955-60 |
