Difference between revisions of "Transformation of AA to PGH2"

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[[Welcome to the In-Silico Model of Cutaneous Lipids Wiki | Return to overview]]
 
[[Welcome to the In-Silico Model of Cutaneous Lipids Wiki | Return to overview]]
 +
 +
Cyclooxygenase, COX, also known as prostaglandin endoperoxide H (PGH) synthase, has a broad substrate specificity and is reported to metabolise AA and other fatty acids into prostanoids such as PGs, TXs and prostacyclin <ref>Tsai, Al Wei, C. Baek, H. K. Kulmacz, R. J. Van Wart, H. E., ''Comparison of peroxidase reaction mechanisms of prostaglandin H synthase-1 containing heme and mangano protoporphyrin IX'', J Biol Chem (1997), 272, 8885-94.</ref><ref>Kulmacz, R. J. van der Donk, W. A. Tsai, A. L. , ''Comparison of the properties of prostaglandin H synthase-1 and -2'', Prog Lipid Res (2003), 42, 377-404</ref>. The preferred substrate is AA for both isoforms of COX, COX-1 and COX-2. The COX-1 isoform is constitutively expressed, whereas COX-2 expression is typically negligible in normal cells <ref>Gurram, B. Zhang, S. Li, M. Li, H. Xie, Y. Cui, H. Du, J. Fan, J. Wang, J. Peng, X. , ''Celecoxib conjugated fluorescent probe for identification and discrimination of cyclooxygenase-2 enzyme in cancer cells'', Anal Chem (2018), 90, 5187-5193.</ref> but can be induced in response to inflammatory stimuli, hormones, calcium and growth factors<ref>Fletcher, B. S. Kujubu, D. A. Perrin, D. M. Herschman, H. R. , ''Structure of the mitogen-inducible TIS10 gene and demonstration that the TIS10-encoded protein is a functional prostaglandin G/H synthase'', J Biol Chem (1992), 267, 4338-44.</ref><ref>Xie, W. L. Chipman, J. G. Robertson, D. L. Erikson, R. L. Simmons, D. L. , ''Expression of a mitogen-responsive gene encoding prostaglandin synthase is regulated by mRNA splicing'', Proc Natl Acad Sci U S A (1991), 88, 2692-6.</ref><ref>Ristimaki, A. Garfinkel, S. Wessendorf, J. Maciag, T. Hla, T. , ''Induction of cyclooxygenase-2 by interleukin-1 alpha. Evidence for post-transcriptional regulation'', J Biol Chem (1994), 269, 11769-75.</ref><ref>name, ''title'', journal (year)</ref><ref>Fu, J. Y. Masferrer, J. L. Seibert, K. Raz, A. Needleman, P., ''The induction and suppression of prostaglandin H2 synthase (cyclooxygenase) in human monocytes'', J Biol Chem (1990), 265, 16737-40.</ref><ref>Kujubu, D. A. Reddy, S. T. Fletcher, B. S. Herschman, H. R., ''Expression of the protein product of the prostaglandin synthase-2/TIS10 gene in mitogen-stimulated Swiss 3T3 cells'', J Biol Chem (1993), 268, 5425-30.</ref> <ref>Wang, D. An, S. J. Wang, W. H. McGiff, J. C. Ferreri, N. R., ''CaR-mediated COX-2 expression in primary cultured mTAL cells'', Am J Physiol Renal Physiol (2001), 281, F658-64.)</ref>. Interestingly, basal expression of COX-2 has been found to occur in the kidney, central nervous system, female reproductive organs and stomach <ref>Obermoser, V. Baecker, D. Schuster, C. Braun, V. Kircher, B. Gust, R. , ''Chlorinated cobalt alkyne complexes derived from acetylsalicylic acid as new specific antitumor agents'', Dalton Trans, 47, 4341-4351.</ref><ref>Kirkby, N. S. Chan, M. V. Zaiss, A. K. Garcia-Vaz, E. Jiao, J. Berglund, L. M. Verdu, E. F. Ahmetaj-Shala, B. Wallace, J. L. Herschman, H. R. Gomez, M. F. Mitchell, J. A., ''Systematic study of constitutive cyclooxygenase-2 expression: Role of NF-kappaB and NFAT transcriptional pathways'', Proc Natl Acad Sci U S A (2016), 113, 434-9.</ref>, and frequent expression of COX-2 can be found in the tumorigenic nests of most cancers <ref>Gurram, B. Zhang, S. Li, M. Li, H. Xie, Y. Cui, H. Du, J. Fan, J. Wang, J. Peng, X. , ''Celecoxib conjugated fluorescent probe for identification and discrimination of cyclooxygenase-2 enzyme in cancer cells'', Anal Chem (2018), 90, 5187-5193.</ref><ref>Raj, V. Bhadauria, A. S. Singh, A. K. Kumar, U. Rai, A. Keshari, A. K. Kumar, P. Kumar, D. Maity, B. Nath, S. Prakash, A. Ansari, K. M. Jat, J. L. Saha, S. , ''Novel 1,3,4-thiadiazoles inhibit colorectal cancer via blockade of IL-6/COX-2 mediated JAK2/STAT3 signals as evidenced through data-based mathematical modeling'', Cell, 135, 216-26.</ref>.
 +
 +
Both isoforms of COX catalyse a two-step reaction of cycloooxygenation and oxygenation, followed by a hydroperoxide reduction. The cyclooxygenase reaction occurs in the hydrophobic channel within the core of the protein and generates an unstable peroxide intermediate, PGG2. The subsequent peroxidase reaction produces PGH2 and occurs at the heme-containing active site near the protein surface. The two step reaction results in the insertion of molecular oxygen across the C-9 and C-11 double bonds. Both isoforms of COX are heme-containing glycoproteins, that are membrane-bound to the endoplasmic reticulum (ER) and function as homodimers <ref>Smith, W. L. DeWitt, D. L. Garavito, R. M., ''Cyclooxygenases: structural, cellular, and molecular biology'', Annu Rev Biochem (2000), 69, 145-82.</ref><ref>van der Donk, W. A. Tsai, A. L. Kulmacz, R. J., ''The cyclooxygenase reaction mechanism'', Biochemistry, 41, 15451-8.</ref><ref>Rouzer, C. A. Marnett, L. J., ''Mechanism of free radical oxygenation of polyunsaturated fatty acids by cyclooxygenases'', Chem Rev, 103, 2239-304.</ref><ref>Xiao, G. Chen, W. Kulmacz, R. J., ''Comparison of structural stabilities of prostaglandin H synthase-1 and -2'', J Biol Chem (1998), 273, 6801-11.</ref>. Interestingly, in certain tissues COX-1 has been found to colocalises preferentially, but not exclusively, with TXAS, PGFS and cPGES, whereas COX-2 has been found to colocalise with PGIS and mPGES in certain tissues <ref>Miyata, A. Yokoyama, C. Ihara, H. Bandoh, S. Takeda, O. Takahashi, E. Tanabe, T., ''Characterization of the human gene (TBXAS1) encoding thromboxane synthase'', Eur J Biochem (1994), 224, 273-9.</ref><ref>Ullrich, V. Zou, M. H. Bachschmid, M. , ''New physiological and pathophysiological aspects on the thromboxane A(2)-prostacyclin regulatory system'', Biochim Biophys Acta (2001), 1532, 1-14.</ref><ref>Smyth, E. M. Grosser, T. Wang, M. Yu, Y. FitzGerald, G. A., ''Prostanoids in health and disease'', J Lipid Res, 50 Suppl, S423-8.</ref><ref>Smith, W. L. DeWitt, D. L. Garavito, R. M., ''Cyclooxygenases: structural, cellular, and molecular biology'', Annu Rev Biochem (2000), 69, 145-82.</ref><ref>Ueno, N. Murakami, M. Tanioka, T. Fujimori, K. Tanabe, T.
 +
Urade, Y. Kudo, I. , ''Coupling between cyclooxygenase, terminal prostanoid synthase, and phospholipase A2'', J Biol Chem (2001), 276, 34918-27.</ref>.
 +
 +
 
== Reaction ==
 
== Reaction ==
  
Line 9: Line 16:
  
 
== Rate equation ==
 
== Rate equation ==
 +
[[File:R02.PNG|center|500px]]
 +
 +
== Enzyme Parameters ==
 +
=== K<sub>eq</sub> ===
 +
{|class="wikitable sortable"
 +
|+  style="text-align: left;" | Gibbs Free Energy Change
 +
|-
 +
! Value
 +
! Units
 +
! Species
 +
! Notes
 +
! Weight
 +
! Reference
 +
|-
 +
|(-30)
 +
|kcal/mol
 +
|Unspecified
 +
|Calculations with a Gaussian98 suite of programs
 +
Enzyme: COX (Unspecific)
 +
Substrate: Arachidonate
 +
Temperature: 298.15 K
 +
Pressure: 1 bar
 +
| 64
 +
|<ref name="Silva2003”>[http://link.springer.com/article/10.1007/s00214-003-0476-9 P. Silva, "A theoretical study of radical-only and combined radical/carbocationic mechanisms of arachidonic acid cyclooxygenation by prostaglandin H synthase" Theor Chem Acc (2003) 110: 345]</ref>
 +
|-
 +
 +
|}
 +
 +
{| class="wikitable"
 +
|+  style="text-align: left;" | Description of the COX-2 Keq distribution
 +
! Mode !! Confidence Interval  !! Location parameter (µ) !! Scale parameter (σ)
 +
|-
 +
| 4.18E+28 || 1.00E+01 || 6.67E+01 || 8.90E-01
 +
|-
 +
|}
  
 +
[[Image:Cox-2_keq.jpg|none|thumb|500px|The estimated probability distribution for COX-2 Keq. The value and weight of the literature values used to define the distribution are indicated by an orange dashed line. The x axis is plotted on a log-scale.  ]]
  
−
== COX-2 Enzyme Parameters ==
+
=== K<sub>ms</sub> ===
  
 
{|class="wikitable sortable"  
 
{|class="wikitable sortable"  
−
|+  style="text-align: left;" | Michaelis-Menten Constants (When AA is the substrate)
+
|+  style="text-align: left;" | Literature Information
 
|-
 
|-
 
! Value
 
! Value
Line 20: Line 63:
 
! Species
 
! Species
 
! Notes
 
! Notes
 +
! Weight
 
! Reference
 
! Reference
 
|-
 
|-
−
|1.62E-02 ± 0.22
+
|1.62E-02 ± 0.22E-02
 
|<math> mM </math>
 
|<math> mM </math>
 
|Human  
 
|Human  
−
|Embryonic kidney cells
+
|Expression Vector: Embryonic kidney cells
 +
Enzyme: Cyclooxygenase-2
 +
pH: Not specified
 +
Temperature: Not specified
 +
|128
 
|<ref name="Kim2005"> [http://www.ncbi.nlm.nih.gov/pubmed/16373578 S.F. Kim Inducible nitric oxide synthase binds, S-nitrosylates, and activates cyclooxygenase-2.'' Science 2005,310(5756):1966-70)]</ref>
 
|<ref name="Kim2005"> [http://www.ncbi.nlm.nih.gov/pubmed/16373578 S.F. Kim Inducible nitric oxide synthase binds, S-nitrosylates, and activates cyclooxygenase-2.'' Science 2005,310(5756):1966-70)]</ref>
 
|-
 
|-
Line 31: Line 79:
 
|mM
 
|mM
 
|Mouse
 
|Mouse
−
|In vitro, 37 °C, pH 8.0, 1–200 µM of substrate.
+
|Expression Vector: Baculovirus (Insect Cell)
 +
Enzyme: COX-2
 +
pH: 8.0
 +
Temperature: 37 °C
 +
1–200 µM of substrate.
 +
|128
 
|<ref name="Vecchoi2010"> [www.ncbi.nlm.nih.gov/pubmed/20463020 Vecchio A. J. "Structural basis of fatty acid substrate binding to cyclooxygenase-2." J. Biol. Chem. 285 22152-63 (2010)]</ref>
 
|<ref name="Vecchoi2010"> [www.ncbi.nlm.nih.gov/pubmed/20463020 Vecchio A. J. "Structural basis of fatty acid substrate binding to cyclooxygenase-2." J. Biol. Chem. 285 22152-63 (2010)]</ref>
 
|-
 
|-
Line 37: Line 90:
 
|mM
 
|mM
 
|Human
 
|Human
−
|Wild Type Enzyme Assay, 30 °C
+
|Expression Vector: E. Coli
 +
Enzyme: Wild Type Cyclooxygenase-2 Enzyme  
 +
pH: 8.5
 +
Temperature:30 °C
 +
|64
 
|<ref name="Rogge2003"> [http://pubs.acs.org/doi/pdf/10.1021/bi035717o Rogge C. "Identification of Tyr504 as an Alternative Tyrosyl Radical Site in Human
 
|<ref name="Rogge2003"> [http://pubs.acs.org/doi/pdf/10.1021/bi035717o Rogge C. "Identification of Tyr504 as an Alternative Tyrosyl Radical Site in Human
 
Prostaglandin H Synthase-2" Biochemistry 2004, 43, 1560-1568]</ref>
 
Prostaglandin H Synthase-2" Biochemistry 2004, 43, 1560-1568]</ref>
Line 44: Line 101:
 
|mM
 
|mM
 
|Human
 
|Human
−
|Wild Type cycloxygenase activty, 100 �M arachidonate substrate, pH 7.2.
+
|Expression Vector: Baculuvirus
 +
Enzyme: Wild Type Cycloxygenase-2
 +
pH: 7.2
 +
Temperature: 30 °C
 +
100 uM arachidonate substrate,
 +
|128
 
|<ref name="Bambai2004"> [http://www.jbc.org/content/279/6/4084.full.pdf Bambai B. "Role of Asn-382 and Thr-383 in Activation and Inactivation of
 
|<ref name="Bambai2004"> [http://www.jbc.org/content/279/6/4084.full.pdf Bambai B. "Role of Asn-382 and Thr-383 in Activation and Inactivation of
 
Human Prostaglandin H Synthase Cyclooxygenase Catalysis" February 6, 2004 The Journal of Biological Chemistry, 279, 4084-4092.]</ref>
 
Human Prostaglandin H Synthase Cyclooxygenase Catalysis" February 6, 2004 The Journal of Biological Chemistry, 279, 4084-4092.]</ref>
 
|-
 
|-
 
|}
 
|}
 +
 +
 +
{| class="wikitable"
 +
|+  style="text-align: left;" | Description of the COX-2 Kms distribution
 +
|-
 +
! Mode (mM) !! Confidence Interval  !! Location parameter (μ) !! Scale parameter (σ)
 +
|-
 +
| 4.90E-03 || 5.70E+00 || -4.72E+00 || 7.77E-01
 +
|}
 +
 +
 +
[[Image:5.jpg|none|thumb|500px|The estimated probability distribution for COX-2 Kms. The value and weight of the literature values used to define the distribution are indicated by an orange dashed line. The x axis is plotted on a log-scale.  ]]
 +
 +
=== K<sub>mp</sub> (Dependent parameter) ===
 +
 +
This is a “Dependent parameter”, meaning that the log-normal distribution for this parameter was calculated using multivariate distributions  (discussed in [[Quantification of parameter uncertainty | Protocol for defining informative priors for ensemble modelling in systems biology]]). As a result, no confidence interval factor or literature values were cited for this parameter. 
 +
 +
{| class="wikitable"
 +
|+  style="text-align: left;" | Description of the COX-2 Kmp distribution
 +
! Mode !! Location parameter (µ) !! Scale parameter (σ)
 +
|-
 +
| 4.70E-03 || -4.75E+00 || 7.87E-01
 +
|}
 +
 +
[[Image:6.jpg|none|thumb|500px|The estimated probability distribution for COX-2 Kmp. The value and weight of the literature values used to define the distribution are indicated by an orange dashed line. The x axis is plotted on a log-scale.  ]]
 +
 +
=== k<sub>cat</sub> ===
  
 
{|class="wikitable sortable"  
 
{|class="wikitable sortable"  
−
|+  style="text-align: left;" | Enzyme Turnover Numbers (When AA is the substrate)
+
|+  style="text-align: left;" | Literature Information
 
|-
 
|-
 
! Value
 
! Value
Line 57: Line 146:
 
! Species
 
! Species
 
! Notes
 
! Notes
 +
! Weight
 
! Reference
 
! Reference
 
|-
 
|-
Line 62: Line 152:
 
|per minute
 
|per minute
 
|Mouse
 
|Mouse
−
|In vitro, 37 °C, pH 8.0, 1–200 µM of substrate.
+
|Expression Vector: Baculovirus (Insect Cell)
 +
Enzyme: COX-2
 +
pH: 8.0
 +
Temperature: 37 °C
 +
1–200 µM of substrate.
 +
|128
 
|<ref name="Vecchoi2010"> [www.ncbi.nlm.nih.gov/pubmed/20463020 Vecchio A. J. "Structural basis of fatty acid substrate binding to cyclooxygenase-2." J. Biol. Chem. 285 22152-63 (2010)]</ref>
 
|<ref name="Vecchoi2010"> [www.ncbi.nlm.nih.gov/pubmed/20463020 Vecchio A. J. "Structural basis of fatty acid substrate binding to cyclooxygenase-2." J. Biol. Chem. 285 22152-63 (2010)]</ref>
 
|-
 
|-
 +
|}
  
 +
{| class="wikitable"
 +
|+  style="text-align: left;" | Description of the COX-2 k<sub>cat</sub> distribution
 +
! Mode (min-1) !! Confidence Interval  !! Location parameter (µ) !! Scale parameter (σ)
 +
|-
 +
| 1.62E+03 || 1.01E+00 || 7.39E+00 || 1.48E-02
 
|}
 
|}
 +
 +
[[Image:Cox2_kcat.jpg|none|thumb|500px|The estimated probability distribution for COX-2 kcat. The value and weight of the literature values used to define the distribution are indicated by an orange dashed line. The x axis is plotted on a log-scale.  ]]
 +
 +
=== Enzyme concentration ===
 +
 +
{|class="wikitable sortable"
 +
|+  style="text-align: left;" | Literature Information
 +
|-
 +
! Value
 +
! Units
 +
! Species
 +
! Notes
 +
! Weight
 +
! Reference
 +
|-
 +
|13.7
 +
|<math> ppm </math>
 +
|Human
 +
|Expression Vector: Platlet
 +
Enzyme: Cyclooxygenase-2 (PGTS2)
 +
pH: 7.5
 +
Temperature: 37 °C
 +
|1024
 +
|<ref name="Kim2014"> [http://www.nature.com/nature/journal/v509/n7502/pdf/nature13302.pdf M. Kim ''A draft map of the human proteome'' Nature, 2014 509, 575–581]</ref>
 +
|-
 +
|4.11
 +
|<math> ppm </math>
 +
|Human
 +
|Expression Vector: Stomach
 +
Enzyme: Cyclooxygenase-2 (PGTS2)
 +
pH: 7.5
 +
Temperature: 37 °C
 +
|1024
 +
|<ref name="Wilhelm2014"> [http://www.nature.com/nature/journal/v509/n7502/pdf/nature13319.pdf M. Wilhelm ''Mass-spectrometry-based draft of the
 +
human proteome'' Nature, 2014 509, 582–587]</ref>
 +
|-
 +
|1.49
 +
|<math> ppm </math>
 +
|Human
 +
|Expression Vector: Oral Cavity
 +
Enzyme: Cyclooxygenase-2 (PGTS2)
 +
pH: 7.5
 +
Temperature: 37 °C
 +
|1024
 +
|<ref name="Wilhelm2014"> [http://www.nature.com/nature/journal/v509/n7502/pdf/nature13319.pdf M. Wilhelm ''Mass-spectrometry-based draft of the
 +
human proteome'' Nature, 2014 509, 582–587]</ref>
 +
|-
 +
|}
 +
 +
The abundance of COX-2 (ppm) was converted to COX-2 (mM). As a result, the concentration of COX-2 in unstimulated tissue was estimated as 2.27 x10-5 mM. The upregulation of COX-2 in HaCaT keratinocytes was estimated using western blotting in (Kiezel-Tsugunova, 2017), figure * shows an example. Using this information, in silico experiments which included an upregulation of COX-2, included the concentration of COX-2 reaching 100 times higher concentration than the estimated unstimulated concentration. Therefore, the COX-2 induction event includes the concentration of COX-2 eventually reaching a concentration of 2.27 x10-3 mM after 6h post irradiation.
 +
 +
[[Image:MKT_COX-2.png|none|thumb|500px| The relative expression of COX-2 protein in HaCaT keratinocytes (Kiezel-Tsugunova, 2017).]]
 +
 +
== COX Enzyme Parameters with other substrates ==
  
 
{|class="wikitable sortable"  
 
{|class="wikitable sortable"  
Line 80: Line 235:
 
|mM
 
|mM
 
|Mouse
 
|Mouse
−
|In vitro, 37 °C, pH 8.0, 1–200 µM of substrate.
+
|Expression Vector: Baculovirus (Insect Cell)
 +
Enzyme: COX-2
 +
pH: 8.0
 +
Temperature: 37 °C
 +
1–200 µM of substrate.
 
|<ref name="Vecchoi2010"> [www.ncbi.nlm.nih.gov/pubmed/20463020 Vecchio A. J. "Structural basis of fatty acid substrate binding to cyclooxygenase-2." J. Biol. Chem. 285 22152-63 (2010)]</ref>
 
|<ref name="Vecchoi2010"> [www.ncbi.nlm.nih.gov/pubmed/20463020 Vecchio A. J. "Structural basis of fatty acid substrate binding to cyclooxygenase-2." J. Biol. Chem. 285 22152-63 (2010)]</ref>
 
|-
 
|-
Line 97: Line 256:
 
|per minute
 
|per minute
 
|Mouse
 
|Mouse
−
|In vitro, 37 °C, pH 8.0, 1–200 µM of substrate.
+
|Expression Vector: Baculovirus (Insect Cell)
 +
Enzyme: COX-2
 +
pH: 8.0
 +
Temperature: 37 °C
 +
1–200 µM of substrate.
 
|<ref name="Vecchoi2010"> [www.ncbi.nlm.nih.gov/pubmed/20463020 Vecchio A. J. "Structural basis of fatty acid substrate binding to cyclooxygenase-2." J. Biol. Chem. 285 22152-63 (2010)]</ref>
 
|<ref name="Vecchoi2010"> [www.ncbi.nlm.nih.gov/pubmed/20463020 Vecchio A. J. "Structural basis of fatty acid substrate binding to cyclooxygenase-2." J. Biol. Chem. 285 22152-63 (2010)]</ref>
 
|-
 
|-
Line 114: Line 277:
 
|mM
 
|mM
 
|Mouse
 
|Mouse
−
|In vitro, 37 °C, pH 8.0, 1–200 µM of substrate.
+
|Expression Vector: Baculovirus (Insect Cell)
 +
Enzyme: COX-2
 +
pH: 8.0
 +
Temperature: 37 °C
 +
1–200 µM of substrate.
 
|<ref name="Vecchoi2010"> [www.ncbi.nlm.nih.gov/pubmed/20463020 Vecchio A. J. "Structural basis of fatty acid substrate binding to cyclooxygenase-2." J. Biol. Chem. 285 22152-63 (2010)]</ref>
 
|<ref name="Vecchoi2010"> [www.ncbi.nlm.nih.gov/pubmed/20463020 Vecchio A. J. "Structural basis of fatty acid substrate binding to cyclooxygenase-2." J. Biol. Chem. 285 22152-63 (2010)]</ref>
 
|-
 
|-
Line 131: Line 298:
 
|per minute
 
|per minute
 
|Mouse
 
|Mouse
−
|In vitro, 37 °C, pH 8.0, 1–200 µM of substrate.
+
|Expression Vector: Baculovirus (Insect Cell)
−
|<ref name="Vecchoi2010"> [www.ncbi.nlm.nih.gov/pubmed/20463020 Vecchio A. J. "Structural basis of fatty acid substrate binding to cyclooxygenase-2." J. Biol. Chem. 285 22152-63 (2010)]</ref>
+
Enzyme: COX-2
−
|-
+
pH: 8.0
−
|}
+
Temperature: 37 °C
−
 
+
1–200 µM of substrate.
−
{|class="wikitable sortable"
 
−
|+  style="text-align: left;" | Enzyme Concentration
 
−
|-
 
−
! Value
 
−
! Units
 
−
! Species
 
−
! Notes
 
−
! Reference
 
−
|-
 
−
|1.45e-02
 
−
|mM
 
−
|Mouse
 
−
|In vitro, 37 °C, pH 8.0, 1–200 µM of substrate.
 
 
|<ref name="Vecchoi2010"> [www.ncbi.nlm.nih.gov/pubmed/20463020 Vecchio A. J. "Structural basis of fatty acid substrate binding to cyclooxygenase-2." J. Biol. Chem. 285 22152-63 (2010)]</ref>
 
|<ref name="Vecchoi2010"> [www.ncbi.nlm.nih.gov/pubmed/20463020 Vecchio A. J. "Structural basis of fatty acid substrate binding to cyclooxygenase-2." J. Biol. Chem. 285 22152-63 (2010)]</ref>
 
|-
 
|-

Latest revision as of 15:41, 20 August 2019

Return to overview

Cyclooxygenase, COX, also known as prostaglandin endoperoxide H (PGH) synthase, has a broad substrate specificity and is reported to metabolise AA and other fatty acids into prostanoids such as PGs, TXs and prostacyclin [1][2]. The preferred substrate is AA for both isoforms of COX, COX-1 and COX-2. The COX-1 isoform is constitutively expressed, whereas COX-2 expression is typically negligible in normal cells [3] but can be induced in response to inflammatory stimuli, hormones, calcium and growth factors[4][5][6][7][8][9] [10]. Interestingly, basal expression of COX-2 has been found to occur in the kidney, central nervous system, female reproductive organs and stomach [11][12], and frequent expression of COX-2 can be found in the tumorigenic nests of most cancers [13][14].

Both isoforms of COX catalyse a two-step reaction of cycloooxygenation and oxygenation, followed by a hydroperoxide reduction. The cyclooxygenase reaction occurs in the hydrophobic channel within the core of the protein and generates an unstable peroxide intermediate, PGG2. The subsequent peroxidase reaction produces PGH2 and occurs at the heme-containing active site near the protein surface. The two step reaction results in the insertion of molecular oxygen across the C-9 and C-11 double bonds. Both isoforms of COX are heme-containing glycoproteins, that are membrane-bound to the endoplasmic reticulum (ER) and function as homodimers [15][16][17][18]. Interestingly, in certain tissues COX-1 has been found to colocalises preferentially, but not exclusively, with TXAS, PGFS and cPGES, whereas COX-2 has been found to colocalise with PGIS and mPGES in certain tissues [19][20][21][22][23].


Reaction

R2 AA-PGH2.jpg

Chemical equation

AA \rightleftharpoons PGH2

Rate equation

R02.PNG

Enzyme Parameters

Keq

Gibbs Free Energy Change
Value Units Species Notes Weight Reference
(-30) kcal/mol Unspecified Calculations with a Gaussian98 suite of programs

Enzyme: COX (Unspecific) Substrate: Arachidonate Temperature: 298.15 K Pressure: 1 bar

64 [24]
Description of the COX-2 Keq distribution
Mode Confidence Interval Location parameter (µ) Scale parameter (σ)
4.18E+28 1.00E+01 6.67E+01 8.90E-01
The estimated probability distribution for COX-2 Keq. The value and weight of the literature values used to define the distribution are indicated by an orange dashed line. The x axis is plotted on a log-scale.

Kms

Literature Information
Value Units Species Notes Weight Reference
1.62E-02 ± 0.22E-02 mM Human Expression Vector: Embryonic kidney cells

Enzyme: Cyclooxygenase-2 pH: Not specified Temperature: Not specified

128 [25]
5.14E-03 ± 2.90E-04 mM Mouse Expression Vector: Baculovirus (Insect Cell)

Enzyme: COX-2 pH: 8.0 Temperature: 37 °C 1–200 µM of substrate.

128 [26]
2.1E-03 ± 4.00E-04 mM Human Expression Vector: E. Coli

Enzyme: Wild Type Cyclooxygenase-2 Enzyme pH: 8.5 Temperature:30 °C

64 [27]
2.1E-03 ± 4.00E-04 mM Human Expression Vector: Baculuvirus

Enzyme: Wild Type Cycloxygenase-2 pH: 7.2 Temperature: 30 °C 100 uM arachidonate substrate,

128 [28]


Description of the COX-2 Kms distribution
Mode (mM) Confidence Interval Location parameter (μ) Scale parameter (σ)
4.90E-03 5.70E+00 -4.72E+00 7.77E-01


The estimated probability distribution for COX-2 Kms. The value and weight of the literature values used to define the distribution are indicated by an orange dashed line. The x axis is plotted on a log-scale.

Kmp (Dependent parameter)

This is a “Dependent parameter”, meaning that the log-normal distribution for this parameter was calculated using multivariate distributions (discussed in Protocol for defining informative priors for ensemble modelling in systems biology). As a result, no confidence interval factor or literature values were cited for this parameter.

Description of the COX-2 Kmp distribution
Mode Location parameter (µ) Scale parameter (σ)
4.70E-03 -4.75E+00 7.87E-01
The estimated probability distribution for COX-2 Kmp. The value and weight of the literature values used to define the distribution are indicated by an orange dashed line. The x axis is plotted on a log-scale.

kcat

Literature Information
Value Units Species Notes Weight Reference
1620 ± 24 per minute Mouse Expression Vector: Baculovirus (Insect Cell)

Enzyme: COX-2 pH: 8.0 Temperature: 37 °C 1–200 µM of substrate.

128 [26]
Description of the COX-2 kcat distribution
Mode (min-1) Confidence Interval Location parameter (µ) Scale parameter (σ)
1.62E+03 1.01E+00 7.39E+00 1.48E-02
The estimated probability distribution for COX-2 kcat. The value and weight of the literature values used to define the distribution are indicated by an orange dashed line. The x axis is plotted on a log-scale.

Enzyme concentration

Literature Information
Value Units Species Notes Weight Reference
13.7 ppm Human Expression Vector: Platlet

Enzyme: Cyclooxygenase-2 (PGTS2) pH: 7.5 Temperature: 37 °C

1024 [29]
4.11 ppm Human Expression Vector: Stomach

Enzyme: Cyclooxygenase-2 (PGTS2) pH: 7.5 Temperature: 37 °C

1024 [30]
1.49 ppm Human Expression Vector: Oral Cavity

Enzyme: Cyclooxygenase-2 (PGTS2) pH: 7.5 Temperature: 37 °C

1024 [30]

The abundance of COX-2 (ppm) was converted to COX-2 (mM). As a result, the concentration of COX-2 in unstimulated tissue was estimated as 2.27 x10-5 mM. The upregulation of COX-2 in HaCaT keratinocytes was estimated using western blotting in (Kiezel-Tsugunova, 2017), figure * shows an example. Using this information, in silico experiments which included an upregulation of COX-2, included the concentration of COX-2 reaching 100 times higher concentration than the estimated unstimulated concentration. Therefore, the COX-2 induction event includes the concentration of COX-2 eventually reaching a concentration of 2.27 x10-3 mM after 6h post irradiation.

The relative expression of COX-2 protein in HaCaT keratinocytes (Kiezel-Tsugunova, 2017).

COX Enzyme Parameters with other substrates

Michaelis-Menten Constants (When EPA is the substrate)
Value Units Species Notes Reference
9.45E-03 ± 7.30E-04 mM Mouse Expression Vector: Baculovirus (Insect Cell)

Enzyme: COX-2 pH: 8.0 Temperature: 37 °C 1–200 µM of substrate.

[26]
Enzyme Turnover Numbers (When EPA is the substrate)
Value Units Species Notes Reference
522 ± 12.6 per minute Mouse Expression Vector: Baculovirus (Insect Cell)

Enzyme: COX-2 pH: 8.0 Temperature: 37 °C 1–200 µM of substrate.

[26]
Michaelis-Menten Constants (When DHA is the substrate)
Value Units Species Notes Reference
3.68E-02 ± 4.25E-03 mM Mouse Expression Vector: Baculovirus (Insect Cell)

Enzyme: COX-2 pH: 8.0 Temperature: 37 °C 1–200 µM of substrate.

[26]
Enzyme Turnover Numbers (When DHA is the substrate)
Value Units Species Notes Reference
207 ± 9 per minute Mouse Expression Vector: Baculovirus (Insect Cell)

Enzyme: COX-2 pH: 8.0 Temperature: 37 °C 1–200 µM of substrate.

[26]

References

  1. ↑ Tsai, Al Wei, C. Baek, H. K. Kulmacz, R. J. Van Wart, H. E., Comparison of peroxidase reaction mechanisms of prostaglandin H synthase-1 containing heme and mangano protoporphyrin IX, J Biol Chem (1997), 272, 8885-94.
  2. ↑ Kulmacz, R. J. van der Donk, W. A. Tsai, A. L. , Comparison of the properties of prostaglandin H synthase-1 and -2, Prog Lipid Res (2003), 42, 377-404
  3. ↑ Gurram, B. Zhang, S. Li, M. Li, H. Xie, Y. Cui, H. Du, J. Fan, J. Wang, J. Peng, X. , Celecoxib conjugated fluorescent probe for identification and discrimination of cyclooxygenase-2 enzyme in cancer cells, Anal Chem (2018), 90, 5187-5193.
  4. ↑ Fletcher, B. S. Kujubu, D. A. Perrin, D. M. Herschman, H. R. , Structure of the mitogen-inducible TIS10 gene and demonstration that the TIS10-encoded protein is a functional prostaglandin G/H synthase, J Biol Chem (1992), 267, 4338-44.
  5. ↑ Xie, W. L. Chipman, J. G. Robertson, D. L. Erikson, R. L. Simmons, D. L. , Expression of a mitogen-responsive gene encoding prostaglandin synthase is regulated by mRNA splicing, Proc Natl Acad Sci U S A (1991), 88, 2692-6.
  6. ↑ Ristimaki, A. Garfinkel, S. Wessendorf, J. Maciag, T. Hla, T. , Induction of cyclooxygenase-2 by interleukin-1 alpha. Evidence for post-transcriptional regulation, J Biol Chem (1994), 269, 11769-75.
  7. ↑ name, title, journal (year)
  8. ↑ Fu, J. Y. Masferrer, J. L. Seibert, K. Raz, A. Needleman, P., The induction and suppression of prostaglandin H2 synthase (cyclooxygenase) in human monocytes, J Biol Chem (1990), 265, 16737-40.
  9. ↑ Kujubu, D. A. Reddy, S. T. Fletcher, B. S. Herschman, H. R., Expression of the protein product of the prostaglandin synthase-2/TIS10 gene in mitogen-stimulated Swiss 3T3 cells, J Biol Chem (1993), 268, 5425-30.
  10. ↑ Wang, D. An, S. J. Wang, W. H. McGiff, J. C. Ferreri, N. R., CaR-mediated COX-2 expression in primary cultured mTAL cells, Am J Physiol Renal Physiol (2001), 281, F658-64.)
  11. ↑ Obermoser, V. Baecker, D. Schuster, C. Braun, V. Kircher, B. Gust, R. , Chlorinated cobalt alkyne complexes derived from acetylsalicylic acid as new specific antitumor agents, Dalton Trans, 47, 4341-4351.
  12. ↑ Kirkby, N. S. Chan, M. V. Zaiss, A. K. Garcia-Vaz, E. Jiao, J. Berglund, L. M. Verdu, E. F. Ahmetaj-Shala, B. Wallace, J. L. Herschman, H. R. Gomez, M. F. Mitchell, J. A., Systematic study of constitutive cyclooxygenase-2 expression: Role of NF-kappaB and NFAT transcriptional pathways, Proc Natl Acad Sci U S A (2016), 113, 434-9.
  13. ↑ Gurram, B. Zhang, S. Li, M. Li, H. Xie, Y. Cui, H. Du, J. Fan, J. Wang, J. Peng, X. , Celecoxib conjugated fluorescent probe for identification and discrimination of cyclooxygenase-2 enzyme in cancer cells, Anal Chem (2018), 90, 5187-5193.
  14. ↑ Raj, V. Bhadauria, A. S. Singh, A. K. Kumar, U. Rai, A. Keshari, A. K. Kumar, P. Kumar, D. Maity, B. Nath, S. Prakash, A. Ansari, K. M. Jat, J. L. Saha, S. , Novel 1,3,4-thiadiazoles inhibit colorectal cancer via blockade of IL-6/COX-2 mediated JAK2/STAT3 signals as evidenced through data-based mathematical modeling, Cell, 135, 216-26.
  15. ↑ Smith, W. L. DeWitt, D. L. Garavito, R. M., Cyclooxygenases: structural, cellular, and molecular biology, Annu Rev Biochem (2000), 69, 145-82.
  16. ↑ van der Donk, W. A. Tsai, A. L. Kulmacz, R. J., The cyclooxygenase reaction mechanism, Biochemistry, 41, 15451-8.
  17. ↑ Rouzer, C. A. Marnett, L. J., Mechanism of free radical oxygenation of polyunsaturated fatty acids by cyclooxygenases, Chem Rev, 103, 2239-304.
  18. ↑ Xiao, G. Chen, W. Kulmacz, R. J., Comparison of structural stabilities of prostaglandin H synthase-1 and -2, J Biol Chem (1998), 273, 6801-11.
  19. ↑ Miyata, A. Yokoyama, C. Ihara, H. Bandoh, S. Takeda, O. Takahashi, E. Tanabe, T., Characterization of the human gene (TBXAS1) encoding thromboxane synthase, Eur J Biochem (1994), 224, 273-9.
  20. ↑ Ullrich, V. Zou, M. H. Bachschmid, M. , New physiological and pathophysiological aspects on the thromboxane A(2)-prostacyclin regulatory system, Biochim Biophys Acta (2001), 1532, 1-14.
  21. ↑ Smyth, E. M. Grosser, T. Wang, M. Yu, Y. FitzGerald, G. A., Prostanoids in health and disease, J Lipid Res, 50 Suppl, S423-8.
  22. ↑ Smith, W. L. DeWitt, D. L. Garavito, R. M., Cyclooxygenases: structural, cellular, and molecular biology, Annu Rev Biochem (2000), 69, 145-82.
  23. ↑ Ueno, N. Murakami, M. Tanioka, T. Fujimori, K. Tanabe, T. Urade, Y. Kudo, I. , Coupling between cyclooxygenase, terminal prostanoid synthase, and phospholipase A2, J Biol Chem (2001), 276, 34918-27.
  24. ↑ P. Silva, "A theoretical study of radical-only and combined radical/carbocationic mechanisms of arachidonic acid cyclooxygenation by prostaglandin H synthase" Theor Chem Acc (2003) 110: 345
  25. ↑ S.F. Kim Inducible nitric oxide synthase binds, S-nitrosylates, and activates cyclooxygenase-2. Science 2005,310(5756):1966-70)
  26. ↑ 26.0 26.1 26.2 26.3 26.4 26.5 [www.ncbi.nlm.nih.gov/pubmed/20463020 Vecchio A. J. "Structural basis of fatty acid substrate binding to cyclooxygenase-2." J. Biol. Chem. 285 22152-63 (2010)]
  27. ↑ [http://pubs.acs.org/doi/pdf/10.1021/bi035717o Rogge C. "Identification of Tyr504 as an Alternative Tyrosyl Radical Site in Human Prostaglandin H Synthase-2" Biochemistry 2004, 43, 1560-1568]
  28. ↑ [http://www.jbc.org/content/279/6/4084.full.pdf Bambai B. "Role of Asn-382 and Thr-383 in Activation and Inactivation of Human Prostaglandin H Synthase Cyclooxygenase Catalysis" February 6, 2004 The Journal of Biological Chemistry, 279, 4084-4092.]
  29. ↑ M. Kim A draft map of the human proteome Nature, 2014 509, 575–581
  30. ↑ 30.0 30.1 [http://www.nature.com/nature/journal/v509/n7502/pdf/nature13319.pdf M. Wilhelm Mass-spectrometry-based draft of the human proteome Nature, 2014 509, 582–587]

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