Biochemical and structural characterization of substrate binding to cyclooxygenase-2 insights into catalysis and inhibition
Vecchio, Alex J.
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This dissertation focuses on prostaglandin endoperoxide H synthases, also termed cyclooxygenase-1 and -2 (COX-1 and COX-2), which catalyze the committed-step in prostanoid biosynthesis. COX-1 and COX-2 are involved in an array of pathologies including thrombosis, vasoconstriction, inflammation, pain, fever, Alzheimer's disease, and cancer; and furthermore, are biomedically relevant because they are the targets of nonsteroidal anti-inflammatory drugs (NSAIDs) including aspirin, ibuprofen (Advil®, Motrin®), naproxen (Aleve®), celecoxib (Celebrex®), and rofecoxib (Vioxx®). Despite nearly three decades worth of research into cyclooxygenase biochemistry, unresolved mechanistic issues remain regarding cyclooxygenase function. Questions of why there are two cyclooxygenase isoforms, and what governs their distinctive tasks have plagued clinicians and biomedical researchers alike. Specifically, as COX- 1 and COX-2 functions diverge, their structures converge, seeming to challenge the biophysical dogma that "structure dictates function". The aim of this research was to understand cyclooxygenase function and the residues governing catalysis at the molecular level, by combining structural information with traditional biochemical techniques. To comprehend isoform differences, the approach was to specifically target COX-2, due to a lack of crystallographic knowledge for this isoform. We determined the X-ray crystal structures of the archetype substrate arachidonic acid as well as several selectively metabolized substrates in complex with recombinant murine COX-2 to, on average, 2.4 Å resolution. Results indicate that despite sequence conservation in the active sites of COX-1 and COX-2, structural differences arise during substrate binding and catalysis, explaining the functional disparities between cyclooxygenase isoforms.