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Literature summary for 1.14.99.1 extracted from

  • Vecchio, A.J.; Simmons, D.M.; Malkowski, M.G.
    Structural basis of fatty acid substrate binding to cyclooxygenase-2 (2010), J. Biol. Chem., 285, 22152-22163.
    View publication on PubMedView publication on EuropePMC
Search for more literature for 1.14.99.1

Cloned(Commentary)

Cloned (Comment) Organism
expression of His-tagged wild-type and mutant COX-2 proteins Mus musculus

Crystallization (Commentary)

Crystallization (Comment) Organism
arachidonic acid, eicosapentaenoic acid, and docosahexaenoic acid bound to Co3+-protoporphyrin IX-reconstituted recombinant His-tagged isozyme COX-2 mutant N580A, 0.003 ml of 3 mg/ml protein in 25 mM Tris, pH 8.0, 150 mM NaCl, and 0.53% w/v beta-octylglucoside is mixed with 0.003 ml of reservoir solution containing 23-34% polyacrylic acid 5100, 100 mM HEPES, pH 7.5, 20 mM MgCl2, and 0.6% w/v beta-octylglucoside, equilibration over reservoir solution without beta-octylglucoside, 23°C, 3 days to 4 weeks, X-ray diffraction structure determination and analysis at 2.1 A, 2.4 A, and 2.65 A resolution, respectively Mus musculus

Protein Variants

Protein Variants Comment Organism
L531A site-directed mutagenesis, the mutant shows reduced Vmax and Km with arachidonate compared to the wild-type COX-2 Mus musculus
L531F site-directed mutagenesis, the mutant shows reduced Vmax and Km with arachidonate compared to the wild-type COX-2 Mus musculus
L531P site-directed mutagenesis, the mutant shows reduced Vmax and Km with arachidonate compared to the wild-type COX-2 Mus musculus
L531T site-directed mutagenesis, the mutant shows reduced Vmax and Km with arachidonate compared to the wild-type COX-2 Mus musculus
N580A site-directed mutagenesis, crystal structure determination with bound substrates, overview Mus musculus

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
 additional information Michaelis-Menten kinetics, overview Mus musculus
0.00295
-
 arachidonate pH 8.0, 25°C, COX-2 mutant L531P Mus musculus
0.00365
-
 arachidonate pH 8.0, 25°C, COX-2 mutant L531A Mus musculus
0.00514
-
 arachidonate pH 8.0, 25°C, COX-2 wild-type COX-2 and mutant N580A Mus musculus
0.00945
-
 (5Z,8Z,11Z,14Z,17Z)-eicosapentaenoic acid pH 8.0, 25°C, COX-2 mutant N580A Mus musculus
0.0368
-
 (4Z,7Z,10Z,13Z,16Z,19Z)-Docosahexaenoic acid pH 8.0, 25°C, COX-2 mutant N580A Mus musculus

Localization

Localization Comment Organism GeneOntology No. Textmining
membrane associated Mus musculus 16020
-

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
arachidonate + AH2 + O2 Mus musculus
-
 prostaglandin H2 + A + H2O
-
 ?

Organism

Organism UniProt Comment Textmining
Mus musculus Q05769
-
-

Posttranslational Modification

Posttranslational Modification Comment Organism
glycoprotein
-
 Mus musculus

Purification (Commentary)

Purification (Comment) Organism
recombinant His-tagged wild-type and mutant COX-2 proteins by nickel affinity chromatography and gel filtration Mus musculus

Renatured (Commentary)

Renatured (Comment) Organism
the purified recombinant apoenzyme is reconstituted with Co3+-protoporphyrin IX and fatty acid substrate to generate the appropriate enzyme-substrate complexes for X-ray crystallographic analyses Mus musculus

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
(4Z,7Z,10Z,13Z,16Z,19Z)-docosahexaenoic acid + AH2 + O2 the substrate is bound in the cyclooxygenase channel of COX-2, binding structure, overview. The carboxylate of docosahexaenoate interacts with Arg120 and Tyr355 at the base of the channel and the omega-end abuts the side chain of Ile377 near Gly533 in the hydrophobic groove above Ser530 Mus musculus ? + A + H2O
-
 ?
(5Z,8Z,11Z,14Z,17Z)-eicosapentaenoic acid + AH2 + O2 substrate binding structure and nonproductive conformation, overview Mus musculus ? + A + H2O
-
 ?
arachidonate + AH2 + O2
-
 Mus musculus prostaglandin H2 + A + H2O
-
 ?
arachidonate + AH2 + O2 substrate binding structure and nonproductive conformation, overview Mus musculus prostaglandin H2 + A + H2O
-
 ?
additional information although arachidonic acid is the preferred substrate, other fatty acids are oxygenated by the isozymes with varying efficiencies. The substrates bind in different conformations in each monomer constituting the homodimer in their respective structures such that one monomer exhibits nonproductive binding and the other productive binding of the substrate in the cyclooxygenase channel, Arg120 and Leu531 play a role, overview Mus musculus ?
-
 ?

Subunits

Subunits Comment Organism
homodimer
-
 Mus musculus

Synonyms

Synonyms Comment Organism
COX-1
-
 Mus musculus
COX-2
-
 Mus musculus
cyclooxygenase-2
-
 Mus musculus

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
25
-
 assay at Mus musculus

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
3.45
-
 (4Z,7Z,10Z,13Z,16Z,19Z)-Docosahexaenoic acid pH 8.0, 25°C, COX-2 mutant N580A Mus musculus
8.7
-
 (5Z,8Z,11Z,14Z,17Z)-eicosapentaenoic acid pH 8.0, 25°C, COX-2 mutant N580A Mus musculus
27
-
 arachidonate pH 8.0, 25°C, COX-2 mutant N580A Mus musculus

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
8
-
 assay at Mus musculus

Cofactor

Cofactor Comment Organism Structure
heme
-
 Mus musculus

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
9
-
 (4Z,7Z,10Z,13Z,16Z,19Z)-Docosahexaenoic acid pH 8.0, 25°C, COX-2 mutant N580A Mus musculus
920
-
 (5Z,8Z,11Z,14Z,17Z)-eicosapentaenoic acid pH 8.0, 25°C, COX-2 mutant N580A Mus musculus
5240
-
 arachidonate pH 8.0, 25°C, COX-2 mutant N580A Mus musculus