skip to main content
DOE PAGES title logo U.S. Department of Energy
Office of Scientific and Technical Information

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. While 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 among AAmore » 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.« less

Authors:
 [1];  [1];  [1];  [1];  [1];  [1];  [1];  [1];  [1]
  1. Vanderbilt Univ., Nashville, TN (United States)
Publication Date:
Research Org.:
Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Oak Ridge Leadership Computing Facility (OLCF)
Sponsoring Org.:
USDOE Office of Science (SC); National Institutes of Health (NIH); National Science Foundation (NSF)
OSTI Identifier:
1565482
Resource Type:
Accepted Manuscript
Journal Name:
Proceedings of the National Academy of Sciences of the United States of America
Additional Journal Information:
Journal Volume: 112; Journal Issue: 40; Journal ID: ISSN 0027-8424
Publisher:
National Academy of Sciences
Country of Publication:
United States
Language:
English
Subject:
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; cyclooxygenase; endocannabinoids; allosteric regulation; chemical kinetics; Bayesian inference

Citation Formats

Mitchener, Michelle M., Hermanson, Daniel J., Shockley, Erin M., Brown, H. Alex, Lindsley, Craig W., Reese, Jeff, Rouzer, Carol A., Lopez, Carlos F., and Marnett, Lawrence J. Competition and allostery govern substrate selectivity of cyclooxygenase-2. United States: N. p., 2015. Web. doi:10.1073/pnas.1507307112.
Mitchener, Michelle M., Hermanson, Daniel J., Shockley, Erin M., Brown, H. Alex, Lindsley, Craig W., Reese, Jeff, Rouzer, Carol A., Lopez, Carlos F., & Marnett, Lawrence J. Competition and allostery govern substrate selectivity of cyclooxygenase-2. United States. doi:10.1073/pnas.1507307112.
Mitchener, Michelle M., Hermanson, Daniel J., Shockley, Erin M., Brown, H. Alex, Lindsley, Craig W., Reese, Jeff, Rouzer, Carol A., Lopez, Carlos F., and Marnett, Lawrence J. Mon . "Competition and allostery govern substrate selectivity of cyclooxygenase-2". United States. doi:10.1073/pnas.1507307112. https://www.osti.gov/servlets/purl/1565482.
@article{osti_1565482,
title = {Competition and allostery govern substrate selectivity of cyclooxygenase-2},
author = {Mitchener, Michelle M. and Hermanson, Daniel J. and Shockley, Erin M. and Brown, H. Alex and Lindsley, Craig W. and Reese, Jeff and Rouzer, Carol A. and Lopez, Carlos F. and Marnett, Lawrence J.},
abstractNote = {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. While 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 among 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.},
doi = {10.1073/pnas.1507307112},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
number = 40,
volume = 112,
place = {United States},
year = {2015},
month = {9}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record

Citation Metrics:
Cited by: 14 works
Citation information provided by
Web of Science

Save / Share:

Works referenced in this record:

Programming biological models in Python using PySB
journal, January 2013

  • Lopez, Carlos F.; Muhlich, Jeremy L.; Bachman, John A.
  • Molecular Systems Biology, Vol. 9, Issue 1
  • DOI: 10.1038/msb.2013.1

(R)-Profens are substrate-selective inhibitors of endocannabinoid oxygenation by COX-2
journal, September 2011

  • Duggan, Kelsey C.; Hermanson, Daniel J.; Musee, Joel
  • Nature Chemical Biology, Vol. 7, Issue 11
  • DOI: 10.1038/nchembio.663

13-Methylarachidonic Acid Is a Positive Allosteric Modulator of Endocannabinoid Oxygenation by Cyclooxygenase
journal, February 2015

  • Kudalkar, Shalley N.; Nikas, Spyros P.; Kingsley, Philip J.
  • Journal of Biological Chemistry, Vol. 290, Issue 12
  • DOI: 10.1074/jbc.M114.634014

Regulation of arachidonic acid release and cytosolic Phospholipase A 2 activation
journal, March 1999

  • Gijón, Miguel A.; Leslie, Christina C.
  • Journal of Leukocyte Biology, Vol. 65, Issue 3
  • DOI: 10.1002/jlb.65.3.330

The Structural Basis of Endocannabinoid Oxygenation by Cyclooxygenase-2
journal, April 2011

  • Vecchio, Alex J.; Malkowski, Michael G.
  • Journal of Biological Chemistry, Vol. 286, Issue 23
  • DOI: 10.1074/jbc.M111.230367

Global Kinetic Explorer: A new computer program for dynamic simulation and fitting of kinetic data
journal, April 2009

  • Johnson, Kenneth A.; Simpson, Zachary B.; Blom, Thomas
  • Analytical Biochemistry, Vol. 387, Issue 1
  • DOI: 10.1016/j.ab.2008.12.024

Lipid Profiling Reveals Arachidonate Deficiency in RAW264.7 Cells:  Structural and Functional Implications
journal, December 2006

  • Rouzer, Carol A.; Ivanova, Pavlina T.; Byrne, Mark O.
  • Biochemistry, Vol. 45, Issue 49
  • DOI: 10.1021/bi061723j

PyMC : Bayesian Stochastic Modelling in Python
journal, January 2010

  • Patil, Anand; Huard, David; Fonnesbeck, Christopher
  • Journal of Statistical Software, Vol. 35, Issue 4
  • DOI: 10.18637/jss.v035.i04

FitSpace Explorer: An algorithm to evaluate multidimensional parameter space in fitting kinetic data
journal, April 2009

  • Johnson, Kenneth A.; Simpson, Zachary B.; Blom, Thomas
  • Analytical Biochemistry, Vol. 387, Issue 1
  • DOI: 10.1016/j.ab.2008.12.025

Human Cyclooxygenase-2 Is a Sequence Homodimer That Functions as a Conformational Heterodimer
journal, April 2011

  • Dong, Liang; Vecchio, Alex J.; Sharma, Narayan P.
  • Journal of Biological Chemistry, Vol. 286, Issue 21
  • DOI: 10.1074/jbc.M111.231969

Localization and regulation of cytosolic phospholipase A2
journal, October 2000

  • Hirabayashi, Tetsuya; Shimizu, Takao
  • Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids, Vol. 1488, Issue 1-2
  • DOI: 10.1016/S1388-1981(00)00115-3

Prostaglandin H Synthase-2-catalyzed Oxygenation of 2-Arachidonoylglycerol Is More Sensitive to Peroxide Tone than Oxygenation of Arachidonic Acid
journal, September 2012

  • Musee, Joel; Marnett, Lawrence J.
  • Journal of Biological Chemistry, Vol. 287, Issue 44
  • DOI: 10.1074/jbc.M112.381202

Mechanism of Free Radical Oxygenation of Polyunsaturated Fatty Acids by Cyclooxygenases
journal, June 2003

  • Rouzer, Carol A.; Marnett, Lawrence J.
  • Chemical Reviews, Vol. 103, Issue 6
  • DOI: 10.1021/cr000068x

Structural Basis of Fatty Acid Substrate Binding to Cyclooxygenase-2
journal, May 2010

  • Vecchio, Alex J.; Simmons, Danielle M.; Malkowski, Michael G.
  • Journal of Biological Chemistry, Vol. 285, Issue 29
  • DOI: 10.1074/jbc.M110.119867

Differential Evolution Markov Chain with snooker updater and fewer chains
journal, October 2008


Prostaglandins and Leukotrienes: Advances in Eicosanoid Biology
journal, November 2001


Phospholipase A 2 inhibitors as potential therapeutic agents for the treatment of inflammatory diseases
journal, December 2009


Human cyclooxygenase-1 activity and its responses to COX inhibitors are allosterically regulated by nonsubstrate fatty acids
journal, April 2012

  • Zou, Hechang; Yuan, Chong; Dong, Liang
  • Journal of Lipid Research, Vol. 53, Issue 7
  • DOI: 10.1194/jlr.M026856

Properties of cell death models calibrated and compared using Bayesian approaches
journal, January 2013

  • Eydgahi, Hoda; Chen, William W.; Muhlich, Jeremy L.
  • Molecular Systems Biology, Vol. 9, Issue 1
  • DOI: 10.1038/msb.2012.69

Metabolism of the Endocannabinoids, 2-Arachidonylglycerol and Anandamide, into Prostaglandin, Thromboxane, and Prostacyclin Glycerol Esters and Ethanolamides
journal, September 2002

  • Kozak, Kevin R.; Crews, Brenda C.; Morrow, Jason D.
  • Journal of Biological Chemistry, Vol. 277, Issue 47
  • DOI: 10.1074/jbc.M206788200

Glycerylprostaglandin Synthesis by Resident Peritoneal Macrophages in Response to a Zymosan Stimulus
journal, May 2005

  • Rouzer, Carol A.; Marnett, Lawrence J.
  • Journal of Biological Chemistry, Vol. 280, Issue 29
  • DOI: 10.1074/jbc.M501021200

Oxygenation of the Endocannabinoid, 2-Arachidonylglycerol, to Glyceryl Prostaglandins by Cyclooxygenase-2
journal, August 2000

  • Kozak, Kevin R.; Rowlinson, Scott W.; Marnett, Lawrence J.
  • Journal of Biological Chemistry, Vol. 275, Issue 43
  • DOI: 10.1074/jbc.M007088200

Hydroperoxide Dependence and Cooperative Cyclooxygenase Kinetics in Prostaglandin H Synthase-1 and -2
journal, July 1999

  • Chen, Wei; Pawelek, Timothy R.; Kulmacz, Richard J.
  • Journal of Biological Chemistry, Vol. 274, Issue 29
  • DOI: 10.1074/jbc.274.29.20301

Analysis of endocannabinoids by Ag+ coordination tandem mass spectrometry
journal, March 2003


Simultaneous analysis of prostaglandin glyceryl esters and prostaglandins by electrospray tandem mass spectrometry
journal, August 2005

  • Kingsley, Philip J.; Rouzer, Carol A.; Saleh, Samir
  • Analytical Biochemistry, Vol. 343, Issue 2
  • DOI: 10.1016/j.ab.2005.05.005

Synthesis of Prostaglandin E 2 Ethanolamide from Anandamide by Cyclooxygenase-2
journal, August 1997

  • Yu, Ming; Ives, Danial; Ramesha, Chakkodabylu S.
  • Journal of Biological Chemistry, Vol. 272, Issue 34
  • DOI: 10.1074/jbc.272.34.21181

Zymosan-induced glycerylprostaglandin and prostaglandin synthesis in resident peritoneal macrophages: roles of cyclo-oxygenase-1 and -2
journal, September 2006

  • Rouzer, Carol A.; Tranguch, Susanne; Wang, Haibin
  • Biochemical Journal, Vol. 399, Issue 1
  • DOI: 10.1042/BJ20060615

Differential Allosteric Regulation of Prostaglandin H Synthase 1 and 2 by Arachidonic Acid
journal, May 1997

  • Swinney, David C.; Mak, Amy Y.; Barnett, Jim
  • Journal of Biological Chemistry, Vol. 272, Issue 19
  • DOI: 10.1074/jbc.272.19.12393

Differential Sensitivity and Mechanism of Inhibition of COX-2 Oxygenation of Arachidonic Acid and 2-Arachidonoylglycerol by Ibuprofen and Mefenamic Acid
journal, August 2009

  • Prusakiewicz, Jeffery J.; Duggan, Kelsey C.; Rouzer, Carol A.
  • Biochemistry, Vol. 48, Issue 31
  • DOI: 10.1021/bi900999z

Cytosolic phospholipase A2 is essential for both the immediate and the delayed phases of eicosanoid generation in mouse bone marrow-derived mast cells
journal, April 1999

  • Fujishima, H.; Sanchez Mejia, R. O.; Bingham, C. O.
  • Proceedings of the National Academy of Sciences, Vol. 96, Issue 9
  • DOI: 10.1073/pnas.96.9.4803