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Competition and allostery govern substrate selectivity of cyclooxygenase-2.
Proc Natl Acad Sci U S A. 2015 Oct 06; 112(40):12366-71.PN

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+Show Affiliations

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;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;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;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;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;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;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;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.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.

Pub Type(s)

Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.

Language

eng

PubMed ID

26392530

Citation

Mitchener, Michelle M., et al. "Competition and Allostery Govern Substrate Selectivity of Cyclooxygenase-2." Proceedings of the National Academy of Sciences of the United States of America, vol. 112, no. 40, 2015, pp. 12366-71.
Mitchener MM, Hermanson DJ, Shockley EM, et al. Competition and allostery govern substrate selectivity of cyclooxygenase-2. Proc Natl Acad Sci U S A. 2015;112(40):12366-71.
Mitchener, M. M., Hermanson, D. J., Shockley, E. M., Brown, H. A., Lindsley, C. W., Reese, J., Rouzer, C. A., Lopez, C. F., & Marnett, L. J. (2015). Competition and allostery govern substrate selectivity of cyclooxygenase-2. Proceedings of the National Academy of Sciences of the United States of America, 112(40), 12366-71. https://doi.org/10.1073/pnas.1507307112
Mitchener MM, et al. Competition and Allostery Govern Substrate Selectivity of Cyclooxygenase-2. Proc Natl Acad Sci U S A. 2015 Oct 6;112(40):12366-71. PubMed PMID: 26392530.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Competition and allostery govern substrate selectivity of cyclooxygenase-2. AU - Mitchener,Michelle M, AU - Hermanson,Daniel J, AU - Shockley,Erin M, AU - Brown,H Alex, AU - Lindsley,Craig W, AU - Reese,Jeff, AU - Rouzer,Carol A, AU - Lopez,Carlos F, AU - Marnett,Lawrence J, Y1 - 2015/09/21/ PY - 2015/9/23/entrez PY - 2015/9/24/pubmed PY - 2016/3/16/medline KW - Bayesian inference KW - allosteric regulation KW - chemical kinetics KW - cyclooxygenase KW - endocannabinoids SP - 12366 EP - 71 JF - Proceedings of the National Academy of Sciences of the United States of America JO - Proc Natl Acad Sci U S A VL - 112 IS - 40 N2 - 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. SN - 1091-6490 UR - https://www.unboundmedicine.com/medline/citation/26392530/Competition_and_allostery_govern_substrate_selectivity_of_cyclooxygenase_2_ L2 - http://www.pnas.org/cgi/pmidlookup?view=long&pmid=26392530 DB - PRIME DP - Unbound Medicine ER -