title: Fatty Acid Binding to the Allosteric Subunit of Cyclooxygenase-2 Relieves a Tonic Inhibition of the Catalytic Subunit.
abstract: Prostaglandin endoperoxide H synthase-2 (PGHS-2), also called cyclooxygenase-2 (COX-2), converts arachidonic acid to PGH2PGHS-2 is a conformational heterodimer composed of allosteric (Eallo) and catalytic (Ecat) subunits. Fatty acids (FAs) bind to Arg-120 of Ealloincreasing to different degrees, depending on the FA, the Vmaxof its Ecatpartner. We report here that movement of helical residues 120-122 and loop residues 123-129 of Eallounderlies the allosteric effects of FAs and allosteric COX-2 inhibitors, including naproxen and flurbiprofen. An S121P substitution in both PGHS-2 monomers yields a variant (S121P/S121P PGHS-2) that has 1.7-1.8 times the Vmaxof native PGHS-2 and is relatively insensitive to activation by FAs or inhibition by allosteric inhibitors. The S121P substitution in Eallois primarily responsible for these effects. In X-ray crystal structures, the Cα atoms of helical residues 119-122 of S121P/S121P PGHS-2 are displaced from their normal positions. Additionally, the S121P/S121P PGHS-2 variants in which Pro-127 and Ser-541 are replaced by cysteines spontaneously forms Cys-127 to Cys-541 cross-links between monomers. This is unlike the corresponding native PGHS-2 variant and suggests that S121P substitutions also unhinge the loop involving residues 123-129. We conclude the following: (a) the region involving residues 120-129 of unoccupied Eallotonically inhibits Ecat; (b) binding of an activating FA (e.g. arachidonic, palmitic, or oleic acid) to Ealloor an S121P substitution in Eallorepositions this region to increase Ecatactivity; and (c) allosteric COX inhibitors act by preventing FA binding to Ealloand additionally by relocating Ealloresidues to inhibit Ecat.
authors.affiliation: From the Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109 and.
authors.forename: Liang
authors.initials: L
authors.lastname: Dong
authors.affiliation: From the Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109 and.
authors.forename: Chong
authors.initials: C
authors.lastname: Yuan
authors.affiliation: the Department of Structural Biology, University at Buffalo, The State University of New York, and the Hauptman-Woodward Medical Research Institute, Buffalo, New York 14203.
authors.forename: Benjamin J
authors.initials: BJ
authors.lastname: Orlando
authors.affiliation: the Department of Structural Biology, University at Buffalo, The State University of New York, and the Hauptman-Woodward Medical Research Institute, Buffalo, New York 14203 malkowski@hwi.buffalo.edu.
authors.forename: Michael G
authors.initials: MG
authors.lastname: Malkowski
authors.affiliation: From the Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109 and smithww@umich.edu.
authors.forename: William L
authors.initials: WL
authors.lastname: Smith
mesh_terms: D000494:Allosteric Regulation; D019943:Amino Acid Substitution; D020134:Catalytic Domain; D051546:Cyclooxygenase 2 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D052246:Cyclooxygenase 2 Inhibitors / Q000737:chemistry*; D005227:Fatty Acids / Q000737:chemistry*; D005480:Flurbiprofen / Q000737:chemistry*; D006801:Humans; D020125:Mutation, Missense; D009288:Naproxen / Q000737:chemistry*; D017433:Protein Structure, Secondary
chemical_list: D052246:Cyclooxygenase 2 Inhibitors; D005227:Fatty Acids; D009288:Naproxen; D005480:Flurbiprofen; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human
keywords: arachidonic acid (AA) (ARA); cyclooxygenase (COX); eicosanoid; eicosapentaenoic acid; fatty acid; half-site; naproxen; oleic acid; palmitic acid; prostaglandin