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Information on EC 1.13.11.40 - arachidonate 8-lipoxygenase

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IUBMB Comments
From the coral Pseudoplexaura porosa.
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The enzyme appears in viruses and cellular organisms
EC NUMBER
COMMENTARY hide
1.13.11.40
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RECOMMENDED NAME
GeneOntology No.
arachidonate 8-lipoxygenase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
arachidonate + O2 = (5Z,9E,11Z,14Z)-(8R)-8-hydroperoxyicosa-5,9,11,14-tetraenoate
show the reaction diagram
from the coral Pseudoplexaura porosa
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
dioxygenation
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oxidation
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redox reaction
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reduction
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
arachidonic acid metabolism
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Arachidonic acid metabolism
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SYSTEMATIC NAME
IUBMB Comments
arachidonate:oxygen 8-oxidoreductase
From the coral Pseudoplexaura porosa.
CAS REGISTRY NUMBER
COMMENTARY hide
100900-72-9
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
marine mollusc
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Manually annotated by BRENDA team
starfish
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Manually annotated by BRENDA team
clam, synonym Concha fina
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Manually annotated by BRENDA team
gorgonian coral
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Manually annotated by BRENDA team
sea urchin
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Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
physiological function
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lipoxygenases (LOXs) are a family of enzymes that catalyze the highly specific hydroperoxidation of polyunsaturated fatty acids, such as arachidonic acid. Different stereo- or/and regioisomer hydroperoxidation products lead later to different metabolites that exert opposite physiological effects in the animal body and play a central role in inflammatory processes. The Gly-Ala switch of a single residue is crucial for the stereo- and regiocontrol in many lipoxygenases
additional information
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
(15S)-hydroperoxyeicosatetraenoic acid + O2
?
show the reaction diagram
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?
(15S)-hydroxyeicosatetraenoic acid + O2
?
show the reaction diagram
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?
(5Z,8Z,11Z,14Z)-nonadeca-5,8,11,14-tetraene-1,19-dioic acid + O2
?
show the reaction diagram
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8-LOX tolerates a carboxylic group in the substrate-binding pocket
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?
(8R)-8-hydroperoxy-5,9,11,14-eicosatetraenoic acid + O2
(8S,15S)-dihydroperoxy-5Z,9E,11Z,13E-eicosatetraenoic acid
show the reaction diagram
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?
(8S)-hydroxyeicosatetraenoic acid + O2
?
show the reaction diagram
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?
2 arachidonic acid + 2 O2
8-hydroxy,9-oxo-eicosa-5Z,11Z,14Z-trienoic acid + 9-oxo-[8,12-cis]-prosta-5Z,10,14Z-trienoic acid + H2O
show the reaction diagram
5-hydroperoxy fatty acid + O2
leukotriene A4
show the reaction diagram
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leukotriene A synthase activity, reaction rate is approximately 7% of arachidonate 8-lipoxygenation
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?
5-hydroperoxyeicosatetraenoic acid + ?
leukotriene A4
show the reaction diagram
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unstable product
?
alpha-linolenic acid + O2
?
show the reaction diagram
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?
arachidonate + O2
(5Z,9E,11Z,14Z)-(8R)-8-hydroperoxyicosa-5,9,11,14-tetraenoate
show the reaction diagram
arachidonate + O2
(5Z,9E,11Z,14Z)-(8S)-8-hydroperoxyeicosa-5,9,11,14-tetraenoate
show the reaction diagram
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?
arachidonate + O2
(5Z,9E,11Z,14Z)-(8S)-8-hydroperoxyicosa-5,9,11,14-tetraenoate
show the reaction diagram
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?
arachidonic acid + O2
(5Z,8E,11Z,14Z)-8-hydroperoxyicosa-5,8,11,14-tetraenoic acid + (5Z,8E,11Z,14Z)-8-hydroxyicosa-5,8,11,14-tetraenoic acid
show the reaction diagram
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arachidonic acid, enzyme activity only detectable after in vivo treatment with the phorbol ester tumor promoter TPA (12-O-tetradecanoylphorbol-13-acetate)
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?
arachidonic acid + O2
(8R)-8-hydroperoxy-5,9,11,14-eicosatetraenoic acid
show the reaction diagram
docosahexaenoic acid + O2
?
show the reaction diagram
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?
eicosapentaenoic acid + O2
?
show the reaction diagram
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?
gamma-linoleic acid + O2
?
show the reaction diagram
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?
linoleic acid + O2
?
show the reaction diagram
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poor substrate
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?
additional information
?
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NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
2 arachidonic acid + 2 O2
8-hydroxy,9-oxo-eicosa-5Z,11Z,14Z-trienoic acid + 9-oxo-[8,12-cis]-prosta-5Z,10,14Z-trienoic acid + H2O
show the reaction diagram
arachidonate + O2
(5Z,9E,11Z,14Z)-(8R)-8-hydroperoxyicosa-5,9,11,14-tetraenoate
show the reaction diagram
arachidonate + O2
(5Z,9E,11Z,14Z)-(8S)-8-hydroperoxyeicosa-5,9,11,14-tetraenoate
show the reaction diagram
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?
arachidonate + O2
(5Z,9E,11Z,14Z)-(8S)-8-hydroperoxyicosa-5,9,11,14-tetraenoate
show the reaction diagram
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?
additional information
?
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the inducible expression of 8-lipoxygenase inhibits cell growth, 15-LOX-2 and 8-LOX, although displaying different positional specificity, use common signaling pathways to induce growth inhibition in premalignant epithelial cells, overview
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Fe2+
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the catalytic iron in 8R-LOX is positioned by three invariant His384, His389, and His570 side chains and the terminal main chain. Fe2+ sits in the base of a large U-shaped cavity, positioned by invariant Leu385 on one side, and the iron and His384 and His389 on the other. Leu385 and the catalytic iron cradle the base of the U
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
AA861
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baicalein
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beta-mercaptoethanol
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Eicosa-5,8,11,14-tetraynoic acid
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glutathione
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50% inhibition
nordihydroguaiaretic acid
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additional information
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physico-chemical state of the substrate and the complex equilibrium between fatty acid monomers, acid soaps and micelles may impact the reaction specificity of LOX-isoforms
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
EDTA
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lecithin
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.039
(15S)-hydroperoxyeicosatetraenoic acid
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in 50 mM sodium phosphate buffer, pH 7.4, at 25°C
0.015
(15S)-hydroxyeicosatetraenoic acid
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in 50 mM sodium phosphate buffer, pH 7.4, at 25°C
0.0021
(8R)-8-hydroperoxy-5,9,11,14-eicosatetraenoic acid
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in 50 mM sodium phosphate buffer, pH 7.4, at 25°C
0.0057
(8S)-hydroxyeicosatetraenoic acid
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in 50 mM sodium phosphate buffer, pH 7.4, at 25°C
0.008 - 0.135
arachidonate
0.0012 - 0.008
arachidonic acid
additional information
additional information
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substrate inhibition steady state kinetics
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1.5
(15S)-hydroperoxyeicosatetraenoic acid
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in 50 mM sodium phosphate buffer, pH 7.4, at 25°C
1
(15S)-hydroxyeicosatetraenoic acid
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in 50 mM sodium phosphate buffer, pH 7.4, at 25°C
7
(8R)-8-hydroperoxy-5,9,11,14-eicosatetraenoic acid
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in 50 mM sodium phosphate buffer, pH 7.4, at 25°C
3.5
(8S)-hydroxyeicosatetraenoic acid
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in 50 mM sodium phosphate buffer, pH 7.4, at 25°C
0.3 - 210
arachidonate
3.2 - 66.7
arachidonic acid
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
55.2 - 7000
arachidonate
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
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specific activity of 8-LOX with (5Z,8Z,11Z,14Z)-nonadeca-5,8,11,14-tetraene-1,19-dioic acid as substrate strongly increases going from pH 6 to 7.2 but then drops down at more alkaline pH
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.4 - 8
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assay at
7.5 - 8
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7.5
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assay at
8
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8-LOX
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6 - 9.5
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TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
22
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assay at room temperature
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
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nervous tissue
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
PDB
SCOP
CATH
UNIPROT
ORGANISM
Plexaura homomalla;
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
76000
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mature protein predicted from the cDNA, difference from the size estimated by SDS-PAGE implies a post-translational modification of the enzyme
100000
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SDS-PAGE
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
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different values obtained from cDNA and SDS-PAGE imply a post-transitional modification
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
deletion mutant of 8R-LOX crystallized by sitting drop vapor diffusion, to 1.85 A resolution, belongs to space group P21 with four molecules in the asymmetric unit. U-shaped channel in 8R-LOX
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hanging drop vapour diffusion in 6.25% polyethylene glycol 8000, 100 mM imidazole acetate (pH 8.0), 100 mM CaCl2, and 5% sucrose at 22°C
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purified enzyme 8R-LOX containing the arachidonate substrate in subunit C
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purified enzyme in complex with arachidonic acid, anaerobic conditions, vapor diffusion with a well solution of 8% PEG-8000, 5% glycerol, 0.2 M CaCl2, 0.1 M imidazole acetate, pH 8.0, crystals are soaked for about 17 h in a solution consisting of 25% glycerol, 10% PEG-8000, 0.02 M CaCl2, 0.1 M imidazole acetate, pH 8.0, 1% dimethyl sulfoxide, and 1 mg/ml arachindonic acid, X-ray diffraction structure determination and analysis at 2.0 A resolution
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GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
leukotriene A synthase activity of the enzyme leveled off within 10 min, indicating suicide activation
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unstable in solution, activity is completely lost after standing on ice overnight
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-70°C, crude and purified enzyme preparations can be stored without appreciable loss of activity for at least 6 months
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-80°C, 10% glycerol
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
Ni-NTA-agarose column chromatography
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nickel-nitrilotriacetic acid-agarose chromatography, DE52 column chromatography, and Mono Q column chromatography
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on Ni column
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recombinant enzyme
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recombinant N-terminally His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3)
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli BL21(DE3) cells
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expressed in Escherichia coli strain BL21 (DE3)
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expression and establishment of a doxycycline-inducible Tet-On gene expression system in the premalignant mouse keratinocyte cell line 308
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expression in HeLa cells
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full-length cDNA cloned by PCR
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ligated into the pQE-9 plasmid and expressed as N-terminal His-tag fusion protein in Escherichia coli (XL-1 Blue)
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recombinant expression of N-terminally His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3)
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skin 8-lipoxygenase expressed in COS-7 cells by transient transfection of its cDNA, amplified by PCR, in pEF-BOS, also expressed in Escherichia coli system using pQE-32
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
H604F
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8-LOX mutant, induces strong pH-dependent alterations in the positional specificity but the pH-optimum remains the same. At acidic pH 8S-H(p)ETE is the exclusive arachidonic acid oxygenation product but with more alkaline pH increasing shares of 15S-H(p)ETE, above pH 9 15S-H(p)ETE is the major oxygenation product. Specific activity of 8-LOX mutant with (5Z,8Z,11Z,14Z)-nonadeca-5,8,11,14-tetraene-1,19-dioic acid as substrate continously declines when pH increases
Y603F
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8-LOX mutant, is relative insensitive towards pH alterations in the near physiological range (pH 6-8), at strong alkaline conditions (pH more than 9) significant shares of 15S-H(p)ETE are formed
Y603F/H604F
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8-LOX double mutant, induces strong pH-dependent alterations in the positional specificity but the pH-optimum remains the same. At acidic pH 8S-H(p)ETE is the exclusive arachidonic acid oxygenation product but with more alkaline pH increasing shares of 15S-H(p)ETE, above pH 9 15S-H(p)ETE is the major oxygenation product
A417G
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converts arachidonic acid mainly to 12-hydroxyeicosatetraenoic acid, mutant retains 38% of catalytic efficiency
A417S
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same oxygenase specificity and similar catalytic activity to wild-type 8S-LOX
A589M
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site-directed mutagenesis, the mutant shows reduced activity and altered protein fold compared to the wild-type enzyme
A620H
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site-directed mutagenesis, the mutant shows reduced activity and altered protein fold compared to the wild-type enzyme
D39A
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118% activity compared to the wild type enzyme, mutant with diminished fluorescence resonance energy transfer properties, consistent with a role for calcium in membrane binding
D39A/E47A
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106% activity compared to the wild type enzyme, a double mutant with calcium-binding residues from two of the three sites mutated exhibits no fluorescence resonance energy transfer signal
E47A
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65% of the activity of the wild type enzyme, mutant with diminished fluorescence resonance energy transfer properties, consistent with a role for calcium in membrane binding
G427A
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site-directed mutagenesis. In wild-type, molecular oxygen adds to C8 of arachidonic acid with an R stereochemistry. In the mutant, Ala427 pushes Leu385, blocks the region over C8, and opens an oxygen access channel now directed to C12, where molecular oxygen is added with an S stereochemistry. Thus, the specificity turns out to be dramatically inverted
I433A
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absence of the Ile side chain destabilizes the roof of the U-shaped channel, measurable activity only in the presence of CaCl2 and the detergent emolphogen
I433W
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has no measurable activity, presumably because the Trp side chain effectively blocks the arachidonic acid binding site
L432A
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less than 5% of the activity of the wild-type
L432F
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less than 5% of the activity of the wild-type
L432I
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less than 5% of the activity of the wild-type
L432V
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less than 20% of the activity of the wild-type
R182A
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site-directed mutagenesis, the mutant shows reduced activity and altered protein fold compared to the wild-type enzyme
W41A
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140% activity compared to the wild type enzyme, exhibits only less than 2% of the increase in fluorescence at 517 nm upon the addition of CaCl2 of the wild-type signal
W77A
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44% of the activity of the wild type enzyme, exhibits only 4% of the increase in fluorescence at 517 nm upon the addition of CaCl2 of the wild-type signal
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
medicine
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therapeutic effect of inhibitors of arachidonic acid metabolism on inflammatory skin diseases and epidermal tumors and on tumor development