title: Competition and allostery govern substrate selectivity of cyclooxygenase-2.
abstract: Cyclooxygenase-2 (COX-2) oxygenates arachidonic acid (AA) and its ester analog, 2-arachidonoylglycerol (2-AG), to prostaglandins (PGs) and prostaglandin glyceryl esters (PG-Gs), respectively. Although the efficiency of oxygenation of these substrates by COX-2 in vitro is similar, cellular biosynthesis of PGs far exceeds that of PG-Gs. Evidence that the COX enzymes are functional heterodimers suggests that competitive interaction of AA and 2-AG at the allosteric site of COX-2 might result in differential regulation of the oxygenation of the two substrates when both are present. Modulation of AA levels in RAW264.7 macrophages uncovered an inverse correlation between cellular AA levels and PG-G biosynthesis. In vitro kinetic analysis using purified protein demonstrated that the inhibition of 2-AG oxygenation by high concentrations of AA far exceeded the inhibition of AA oxygenation by high concentrations of 2-AG. An unbiased systems-based mechanistic model of the kinetic data revealed that binding of AA or 2-AG at the allosteric site of COX-2 results in a decreased catalytic efficiency of the enzyme toward 2-AG, whereas 2-AG binding at the allosteric site increases COX-2's efficiency toward AA. The results suggest that substrates interact with COX-2 via multiple potential complexes involving binding to both the catalytic and allosteric sites. Competition between AA and 2-AG for these sites, combined with differential allosteric modulation, gives rise to a complex interplay between the substrates, leading to preferential oxygenation of AA.
authors.affiliation: A. B. Hancock Memorial Laboratory for Cancer Research, Vanderbilt University School of Medicine, Nashville, TN 37232; Department of Chemistry, Vanderbilt University School of Medicine, Nashville, TN 37232; The Vanderbilt Institute of Chemical Biology, Vanderbilt University School of Medicine, Nashville, TN 37232; Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, TN 37232;
authors.forename: Michelle M
authors.initials: MM
authors.lastname: Mitchener
authors.affiliation: A. B. Hancock Memorial Laboratory for Cancer Research, Vanderbilt University School of Medicine, Nashville, TN 37232; Department of Chemistry, Vanderbilt University School of Medicine, Nashville, TN 37232; The Vanderbilt Institute of Chemical Biology, Vanderbilt University School of Medicine, Nashville, TN 37232; Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, TN 37232;
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Hermanson
authors.affiliation: The Vanderbilt Institute of Chemical Biology, Vanderbilt University School of Medicine, Nashville, TN 37232; Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, TN 37232; Department of Cancer Biology, Vanderbilt University School of Medicine, Nashville, TN 37232; Center for Quantitative Sciences, Vanderbilt University School of Medicine, Nashville, TN 37232;
authors.forename: Erin M
authors.initials: EM
authors.lastname: Shockley
authors.affiliation: The Vanderbilt Institute of Chemical Biology, Vanderbilt University School of Medicine, Nashville, TN 37232; Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, TN 37232; Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN 37232; Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, TN 37232;
authors.forename: H Alex
authors.initials: HA
authors.lastname: Brown
authors.affiliation: Department of Chemistry, Vanderbilt University School of Medicine, Nashville, TN 37232; The Vanderbilt Institute of Chemical Biology, Vanderbilt University School of Medicine, Nashville, TN 37232; Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, TN 37232; Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, TN 37232;
authors.forename: Craig W
authors.initials: CW
authors.lastname: Lindsley
authors.affiliation: Department of Pediatrics, Vanderbilt University School of Medicine, Nashville, TN 37232; Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN 37232;
authors.forename: Jeff
authors.initials: J
authors.lastname: Reese
authors.affiliation: A. B. Hancock Memorial Laboratory for Cancer Research, Vanderbilt University School of Medicine, Nashville, TN 37232; The Vanderbilt Institute of Chemical Biology, Vanderbilt University School of Medicine, Nashville, TN 37232; Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, TN 37232;
authors.forename: Carol A
authors.initials: CA
authors.lastname: Rouzer
authors.affiliation: The Vanderbilt Institute of Chemical Biology, Vanderbilt University School of Medicine, Nashville, TN 37232; Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, TN 37232; Department of Cancer Biology, Vanderbilt University School of Medicine, Nashville, TN 37232; Center for Quantitative Sciences, Vanderbilt University School of Medicine, Nashville, TN 37232; Department of Biomedical Informatics, Vanderbilt University School of Medicine, Nashville, TN 37232; Department of Biomedical Engineering, Vanderbilt University School of Medicine, Nashville, TN 37232; Vanderbilt University Center for Structural Biology, Vanderbilt University School of Medicine, Nashville, TN 37232.
authors.forename: Carlos F
authors.initials: CF
authors.lastname: Lopez
authors.affiliation: A. B. Hancock Memorial Laboratory for Cancer Research, Vanderbilt University School of Medicine, Nashville, TN 37232; Department of Chemistry, Vanderbilt University School of Medicine, Nashville, TN 37232; The Vanderbilt Institute of Chemical Biology, Vanderbilt University School of Medicine, Nashville, TN 37232; Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, TN 37232; Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN 37232; Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, TN 37232; larry.marnett@vanderbilt.edu.
authors.forename: Lawrence J
authors.initials: LJ
authors.lastname: Marnett
mesh_terms: D000465:Algorithms; D000494:Allosteric Regulation; D000495:Allosteric Site; D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism*; D001095:Arachidonic Acids / Q000378:metabolism*; D001667:Binding, Competitive; D020134:Catalytic Domain; D002460:Cell Line; D003198:Computer Simulation; D051546:Cyclooxygenase 2 / Q000737:chemistry / Q000378:metabolism*; D063388:Endocannabinoids / Q000378:metabolism*; D005989:Glycerides / Q000378:metabolism*; D007700:Kinetics; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D010084:Oxidation-Reduction; D011453:Prostaglandins / Q000378:metabolism*; D011485:Protein Binding; D055503:Protein Multimerization; D061987:Sf9 Cells; D018411:Spodoptera; D013379:Substrate Specificity; D015054:Zymosan / Q000494:pharmacology
chemical_list: D001095:Arachidonic Acids; D063388:Endocannabinoids; D005989:Glycerides; D011453:Prostaglandins; D016718:Arachidonic Acid; C094503:glyceryl 2-arachidonate; D015054:Zymosan; D051546:Cyclooxygenase 2
keywords: Bayesian inference; allosteric regulation; chemical kinetics; cyclooxygenase; endocannabinoids