Information on EC 4.1.1.90 - peptidyl-glutamate 4-carboxylase

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The expected taxonomic range for this enzyme is: Coelomata

EC NUMBER
COMMENTARY hide
4.1.1.90
-
RECOMMENDED NAME
GeneOntology No.
peptidyl-glutamate 4-carboxylase
-
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
peptidyl-4-carboxyglutamate + 2,3-epoxyphylloquinone + H2O = peptidyl-glutamate + CO2 + O2 + phylloquinol
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
carboxylation
gamma-carboxylation
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Biosynthesis of secondary metabolites
-
-
Ubiquinone and other terpenoid-quinone biosynthesis
-
-
SYSTEMATIC NAME
IUBMB Comments
peptidyl-glutamate 4-carboxylase (2-methyl-3-phytyl-1,4-naphthoquinone-epoxidizing)
The enzyme can use various vitamin-K derivatives, including menaquinol, but does not contain iron. The mechanism appears to involve the generation of a strong base by oxygenation of vitamin K. It catalyses the post-translational carboxylation of glutamate residues of several proteins of the blood-clotting system. 9--12 glutamate residues are converted to 4-carboxyglutamate (Gla) in a specific domain of the target protein. The 4-pro-S hydrogen of the glutamate residue is removed [5] and there is an inversion of stereochemistry at this position [6].
CAS REGISTRY NUMBER
COMMENTARY hide
81181-72-8
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
SwissProt
Manually annotated by BRENDA team
-
UniProt
Manually annotated by BRENDA team
schematic of amino acid homology between human and the Drosophila gamma-glutamyl carboxylase shown
UniProt
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
Atlantic salmon
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
-
onset of mineralization in Abcc6-/-, Ggcx+/+ mice is delayed until between 3 and 4 months of age, suggesting that the genetic background plays a role in modifying the mineralization process. Mineralization in the Abcc6-/-, Ggcx+/- mice is accelerated in comparison with age-matched Abcc6-/-, Ggcx+/+ mice, with ca. 3fold difference at 3, 4, and 9 months of age
physiological function
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
4-benzoyl-L-phenylalanine + CO2 + O2 + vitamin K hydroquinone
? + vitamin K epoxide + H2O
show the reaction diagram
-
BPA
-
-
?
CDADWVEGYSMEYLSR + CO2 + O2 + vitamin K hydroquinone
? + vitamin K epoxide + H2O
show the reaction diagram
-
CDADWVEGYSMEYLSR
-
-
?
CGRPSLEQLAQEVTYA + CO2 + O2 + vitamin K hydroquinone
? + vitamin K epoxide + H2O
show the reaction diagram
-
CGRPSLEQLAQEVTYA
-
-
?
conantokin G + CO2 + O2 + vitamin K hydroquinone
? + vitamin K epoxide + H2O
show the reaction diagram
poorly carboxylated
-
-
?
conotoxin epsilon-TxIX + CO2 + O2 + vitamin KH2
?
show the reaction diagram
-
-
-
-
e-TxIX12 + CO2 + O2 + vitamin KH2
?
show the reaction diagram
residues 1-12 of e-TxIX
-
-
?
FLEEL + CO2 + O2 + ammonium sulfate
? + vitamin K epoxide + H2O
show the reaction diagram
-
carboxylase activity is measured by 14CO2 incorporation into the synthetic peptide substrate FLEEL
-
-
?
FLEEL + CO2 + O2 + phylloquinone
? + 2,3-epoxyphylloquinone + H2O
show the reaction diagram
-
-
-
-
?
FLEEL + CO2 + O2 + proFIX 19
? + vitamin K epoxide + H2O
show the reaction diagram
-
carboxylase activity is measured by 14CO2 incorporation into the synthetic peptide substrate FLEEL
-
-
?
FLEEL + CO2 + O2 + vitamin K hydroquinone
? + vitamin K epoxide + H2O
show the reaction diagram
FLEEL + CO2 + O2 + vitamin K1 hydroquinone
? + vitamin K1 epoxide + H2O
show the reaction diagram
-
a synthetic peptide substrate, assay for vitamin K–dependent carboxylase activity
-
-
?
FLEEL + CO2 + O2 + vitamin KH2
? + vitamin K epoxide + H2O
show the reaction diagram
FLEEV + CO2 + O2 + vitamin K hydroquinone
? + vitamin K epoxide + H2O
show the reaction diagram
-
-
-
-
?
gamma-carboxylated glutamyl containing vitamin K-dependent protein + vitamin K epoxide + H2O
?
show the reaction diagram
-
-
-
-
?
GKDRLTQMKRILKQRGNKARGEEELY + CO2 + O2 + vitamin K hydroquinone
? + vitamin K epoxide + H2O
show the reaction diagram
-
-
-
?
glutamyl containing vitamin K-dependent protein + CO2 + vitamin K hydroquinone + O2
?
show the reaction diagram
-
propeptide binding increases carboxylase affinity for the Glu substrate, and the coordinated binding of the vitamin K–dependent propeptide and Glu substrate increase carboxylase affinity for vitamin K and activity, possibly through a mechanism of substrate-assisted catalysis. The propeptide adjacent to the Gla domain is cleaved subsequently to carboxylation. The carboxylase uses the energy of vitamin K hydroquinone oxygenation to convert glutamyl residues to gamma-carboxylated glutamyl residues in vitamin K–dependent proteins. During carboxylation, the vitamin K hydroquinone cofactor is oxidized to a vitamin K epoxide product. The carboxylase itself is also a vitamin K–dependent protein and carboxylase carboxylation may be important in regulating the overall process of vitamin K–dependent protein carboxylation. All vitamin K–dependent proteins contain multiple glutamyl residues that undergo carboxylation, which is accomplished by a processive mechanism. A single binding event between carboxylase and vitamin K–dependent protein can give rise to all of the glutamyl to gamm-carboxylated glutamyl conversions in the vitamin K–dependent protein. Carboxylation is limited to the glutamyl residue residing within the Gla domain
-
-
?
L-glutamate + CO2 + O2 + vitamin K hydroquinone
gamma-carboxy L-glutamate + vitamin K epoxide + H2O
show the reaction diagram
L-glutamate + CO2 + O2 + vitamin K hydroquinone
gamma-carboxy-L-glutamate + vitamin K epoxide + H2O
show the reaction diagram
N-(bromoacetyl)-FLEELY + CO2 + O2 + vitamin KH2
? + vitamin K epoxide + H2O
show the reaction diagram
-
-
-
-
?
osteocalcin + reduced vitamin K + CO2 + O2
? + vitamin K epoxide + H2O
show the reaction diagram
-
-
-
?
peptidyl-4-carboxyglutamate + 2,3-epoxyphylloquinone + H2O
peptidyl-glutamate + CO2 + O2 + phylloquinone
show the reaction diagram
peptidyl-L-glutamate + CO2 + O2 + menaquinone
?
show the reaction diagram
-
-
-
-
?
peptidyl-L-glutamate + CO2 + O2 + phylloquinone
peptidyl-4-carboxy L-glutamate + 2,3-epoxyphylloquinone + H2O
show the reaction diagram
-
-
-
-
?
pro-e-TxIX/12 + CO2 + O2 + vitamin KH2
?
show the reaction diagram
residues -12 to -1 of e-TxIX precursor
-
-
?
pro-e-TxIX/24 + CO2 + O2 + vitamin KH2
?
show the reaction diagram
residues -12 to +12 of e-TxIX precursor
-
-
?
pro-e-TxIX/41 + CO2 + O2 + vitamin KH2
?
show the reaction diagram
residues -29 to +12 of e-TxIX precursor
-
-
?
pro-FIX19 + CO2 + O2 + vitamin K hydroquinone
? + vitamin K epoxide + H2O
show the reaction diagram
-
pro-FIX19: peptide comprising residues of human factor IX AVFLDHENANKILNRPKRY
-
-
?
pro-FIX19-16BPA + CO2 + O2 + vitamin K hydroquinone
? + vitamin K epoxide + H2O
show the reaction diagram
-
pro-FIX19-16BPA: peptide comprising residues TVBLDHENANKILNRPKRY
-
-
?
proFIX 19 + CO2 + O2 + vitamin K hydroquinone
? + vitamin K epoxide + H2O
show the reaction diagram
-
proFIX 19, peptide sequence: TVFLDHENANKILNRPKRY
-
-
-
ProFIX 19-6BPA + CO2 + O2 + FLEEL + vitamin KH2
? + vitamin K epoxide + H2O
show the reaction diagram
-
TVFLDHENANKIBNRPKR
-
-
?
ProFIX 19-7BPA + CO2 + O2 + FLEEL + vitamin KH2
? + vitamin K epoxide + H2O
show the reaction diagram
-
TVFLDHENfiNKBLNRPKR
-
-
?
proFIX/PT28 + CO2 + O2 + vitamin K hydroquinone
? + vitamin K epoxide + H2O
show the reaction diagram
-
proFIX/PT28, peptide sequence: TVFLDHENANKILNRPKRANTFLEEVRK
-
-
?
proFIX18 + CO2 + O2 + vitamin KH2
?
show the reaction diagram
residues -18 to -1 of proFactor IX
-
-
?
proFIX19 + CO2 + O2 + FLEEL + vitamin KH2
? + vitamin K epoxide + H2O
show the reaction diagram
-
TVFLDHENANKILNRPKRY
-
-
?
ProFIX19-13BPA + CO2 + O2 + FLEEL + vitamin KH2
? + vitamin K epoxide + H2O
show the reaction diagram
-
TVFLDBENWKILNRPKRY
-
-
?
ProFIX19-16BPA + CO2 + O2 + FLEEL + vitamin KH2
? + vitamin K epoxide + H2O
show the reaction diagram
-
TVBLDHENANKILNRPKRY
-
-
?
proFIX28 + CO2 + O2 + vitamin KH2
?
show the reaction diagram
residues -18 to +10 of proFactor IX
-
-
?
proFIXl9 + CO2 + O2 + vitamin KH2
? + vitamin K epoxide + H2O
show the reaction diagram
-
AVFLDHENANKILNRPKRY
-
-
?
proPT18 + CO2 + O2 + vitamin KH2
?
show the reaction diagram
residues -18 to -1 of proprothrombin. 28-residue peptides based on residues -18 to +10 of human proprothrombin and proFactor IX with Km values of 420 lM, 1.7 microM and 6 microM
-
-
?
proPT28 + CO2 + O2 + FLEEL + vitamin KH2
? + vitamin K epoxide + H2O
show the reaction diagram
-
HVFLAPQQARSLLQRVRRANTFLEEVRK
-
-
?
proPT28 + CO2 + O2 + vitamin K hydroquinone
? + vitamin K epoxide + H2O
show the reaction diagram
proPT28: synthetic peptide is designated by the following nomenclature: pro indicates the presence of the propeptide sequence, PT indicates prothrombin, the protein on which the peptide sequence is based, and 28 indicates the number of amino acid residues in the peptide
-
-
?
proPT28 + CO2 + O2 + vitamin KH2
?
show the reaction diagram
residues -18 to +10 of proprothrombin
-
-
?
proPTl8 + CO2 + O2 + FLEEL + vitamin KH2
? + vitamin K epoxide + H2O
show the reaction diagram
-
HVFLAPQQARSLLQRVRR
-
-
?
TVFLDHENANKILNRPKRANTBLEEVRK + CO2 + O2 + vitamin K hydroquinone
? + vitamin K epoxide + H2O
show the reaction diagram
-
carboxylase probe1, TVFLDHENANKILNRPKRANTBLEEVRK as a substrate
-
-
?
TVFLDHENANKILNRPKRYNTBLEEVRK + CO2 + O2 + vitamin K hydroquinone
? + vitamin K epoxide + H2O
show the reaction diagram
-
-
-
-
?
YVFLDHQDADANLILNRPKR + CO2 + O2 + vitamin KH2
? + vitamin K epoxide + H2O
show the reaction diagram
-
-
-
-
?
additional information
?
-
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
L-glutamate + CO2 + O2 + vitamin K hydroquinone
gamma-carboxy L-glutamate + vitamin K epoxide + H2O
show the reaction diagram
L-glutamate + CO2 + O2 + vitamin K hydroquinone
gamma-carboxy-L-glutamate + vitamin K epoxide + H2O
show the reaction diagram
-
-
-
-
?
peptidyl-4-carboxyglutamate + 2,3-epoxyphylloquinone + H2O
peptidyl-glutamate + CO2 + O2 + phylloquinone
show the reaction diagram
peptidyl-L-glutamate + CO2 + O2 + menaquinone
?
show the reaction diagram
-
-
-
-
?
peptidyl-L-glutamate + CO2 + O2 + phylloquinone
peptidyl-4-carboxy L-glutamate + 2,3-epoxyphylloquinone + H2O
show the reaction diagram
-
-
-
-
?
additional information
?
-
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
phylloquinone
-
-
vitamin K
vitamin K1
additional information
-
menadione does not work as a cofactor for gamma-glutamylcarboxylase
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2,3,5,6-Tetrachloro-4-pyridinol
-
TCP, anticoagulant action, effective in vitro inhibitor of the carboxylase
2-chloro-3-phytyl-1,4-naphthoquinone
5-mercapto-1-methylthiotetrazole
-
-
-
anti-carboxylase antiserum
-
effect of anti-carboxylase antiserum on carboxylase activity: under the conditions employed the carboxylation is inhibited by 80%, with parallel inhibition of CO2 incorporation into FLEEL and proPT28 (synthetic peptide)
-
Boc-(2S,4S)-4-methylglutamic acid-Glu-Val
-
competitive inhibitor, FLEEL as substrate
Boc-Ser(OPO4)-Ser(OPO4)-Leu-OMe
-
inhibits the enzyme apparently competitively with regard to other peptide substrate
bromoacetyl-FLEEL peptide
-
the His6-carboxylase is irreversibly inactivated. Up to 85% of the carboxylase activity is lost over a period of 120 min
CN-
-
enzyme is blocked by mM concentrations of CN-
Cu2+
-
free Cu2+ and Cu2+-complexes inhibit the reaction
ethanol
-
high concentrations
FFRCK
-
peptide, protease inhibitor
FLEEL
-
high FLEEL peptide substrate concentrations (from 1.2 up to 12 mM) inhibit GGCX activity
FPRCK
-
peptide, protease inhibitor
iodoacetic acid
poor inhibitor
methemoglobin
-
-
-
N-ethylmaleimide
p-chloromercuribenzoate
97% inhibition with 1.25 mM and at 5 mM the reaction is completely inhibited
p-hydroxymercuribenzoate
proFIX/PT28 (Bpa +4)
-
presence of proFIX/PT28 (Bpa +4) or its iodinated derivative 56% inactivation is observed
-
proFIX19-16BPA propeptide
-
-
-
protease inhibitor mixture
-
PIC, freshly prepared as a 10x PIC stock containing 20 mM dithiothreitol, 20 mM EDTA, FFRCK (1.25 microg/ml), FPRCK (1.25 microg/ml), leupeptin (5 microg/ml), pepstatin A (7 microg/ml), phenylmethylsulfonyl fluoride (340 microg/ml), aprotinin (20 microg/ml)
-
tetrachloropyridin
-
-
TVFLDHENANKILNRPKRANTBLEEVRK
-
the enzyme is photoirradiated on ice at 365 nm with TVFLDHENANKILNRPKRANTBLEEVRK, TVFLDHENANKILNRPKRYNTBLEEVRK and mono [127I]TVFLDHENANKILNRPKRYNTBLEEVRK for various times
TVFLDHENANKILNRPKRYNTBLEEVRK
-
the enzyme is photoirradiated on ice at 365 nm with TVFLDHENANKILNRPKRANTBLEEVRK, TVFLDHENANKILNRPKRYNTBLEEVRK and mono [127I]TVFLDHENANKILNRPKRYNTBLEEVRK for various times. Presence of TVFLDHENANKILNRPKRYNTBLEEVRK or its iodinated derivative 80% inactivation is observed
vitamin K
-
carboxylation of FLEEL by bovine liver carboxylase is inhibited by high concentrations of vitamin KH2. vitamin K (up to 400 mM) and vitamin K epoxide (up to 1 mM) are not inhibitory. R234A/H235A mutant, R406A/H408A mutant, and R513A/K515A mutant are more susceptible to inhibition by vitamin KH2 than wild type enzyme. R234A/H235A mutant and R406A/H408A mutant exhibit maximal activity at 111 mM vitamin KH2 and R513A/K515A mutant at 56 mM vitamin KH2
warfarin
additional information
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
ammonium sulfate
-
-
benzoylphenylalanine
-
Bpa, the four Bpa peptides enhance gamma-carboxylation by 1.5-2.3fold, and the rate enhancement profiles are very similar to that of proFIX19, showing that these propeptides are recognized by the carboxylase
-
decarboxylated plasma prothrombin
-
activates the enzyme
-
endogenous precursor
-
activates the enzyme
-
FLEEL
-
the enzyme activity increases with increasing FLEEL peptide concentration up to 1.2 mM
proFIX 19
-
-
-
proFIX19
-
enhances gamma-carboxylation of the synthetic FLEEL peptide by 2.2-2.3fold
-
proPT18
-
enhances gamma-carboxylation of the synthetic FLEEL peptide by 2.2-2.3fold
-
additional information
-
enzyme activity increases 2-3fold by a vitamin K deficiency or Warfarin treatment
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0017
28-residue peptides based on residues -18 to +10 of human proprothrombin
pH 7.0
-
0.3
CO2
-
pH 7.4
0.565
conotoxin epsilon-TxIX
pH 7.0
-
0.42 - 24.3
FLEEL
0.075
precursor analog containing 12 of the propeptide region
pH 7.0
-
0.074
precursor analog containing 29 amino acids of the propeptide region
pH 7.0
-
0.006
proFactor IX
pH 7.0
-
0.0068
TVFLDHENANKILNRPKRANTBLEEVRK
-
carboxylase probe1, TVFLDHENANKILNRPKRANTBLEEVRK as a substrate
0.052
vitamin K1
pH 7.0
0.0038 - 0.097
vitamin KH2
0.0023 - 2.06
YVFLDHQDADANLILNRPKR
additional information
additional information
-
Km-values measured by hyperbolic weighted least-squares analysis
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1
CO2
Bos taurus
-
pH 7.4
0.0019 - 1.7
FLEEL
0.018 - 0.66
vitamin KH2
0.002 - 0.645
YVFLDHQDADANLILNRPKR
additional information
additional information
Homo sapiens
-
kcat values relative to wild type are 51% (L394R), 1% (Y395A), and 2% (W399A), pH 7.4, 20°C
-
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.013 - 2.1
Boc-(2S,4S)-4-methylglutamic acid-Glu-Val
0.0174
proFIX19-16BPA propeptide
-
competitive inhibition experiments using prom28 substrate
-
additional information
additional information
-
Ki value for Boc-(2S,4S)-4-methylglutamic acid-Glu-Val is above 5 mM for W399A, pH 7.4, 20°C
-
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.0000026
-
H160A mutant
0.0000027
-
wild type
0.0000045
-
H381A mutant
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.2 - 7.4
-
assay at. The activity falls off sharply above pH 8 or below pH 7
8.5
-
assay at
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.5 - 8.5
-
-
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
-
coagulation assays
Manually annotated by BRENDA team
-
gamma-carboxylase gene targeting
Manually annotated by BRENDA team
-
vitamin K-dependent carboxylase mRNA expression in early rat embryonic development
Manually annotated by BRENDA team
-
-
Manually annotated by BRENDA team
-
expression of rat vitamin K-dependent carboxylase in adult and embryonic tissues
Manually annotated by BRENDA team
-
rat hepatoma cell line
Manually annotated by BRENDA team
-
expression of rat vitamin K-dependent carboxylase in adult and embryonic tissues
Manually annotated by BRENDA team
-
gamma-carboxylase gene targeting
Manually annotated by BRENDA team
-
expression of rat vitamin K-dependent carboxylase in adult and embryonic tissues
Manually annotated by BRENDA team
additional information
-
6-year-old Mexican American male
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
33000
cells transfected with the CAT-V5/His plasmid, Western Blot
60000
-
proteinase K digestion of the hGC-FLAG reveals a 60 kDa fragment, indicating the lumenal location of the FLAG tag and therefore the carboxyl-terminus of the carboxylase. In contrast, FLAG-hGC does not show a proteinase K–resistant fragment except for the residual undigested full-length carboxylase, which indicates the cytoplasmic location of the FLAG tag and therefore the amino-terminus of the carboxylase
77000
-
single major band on SDS gel electrophoresis. The eluted protein contains both stable vitamin K-dependent carboxylase and vitamin K epoxidase activity
85700
-
calculated from sequence
87540
-
calculated from sequence
95000
-
full-length carboxylase
99000
-
SDS-PAGE, Western Blot. CHO cells transfected with the cDNA for wild type FLAG-vitamin K-dependent gamma-glutamyl carboxylase
130000
SDS-PAGE, Sf21 cells transfected with the carboxylase cDNA-containing plasmid, Western Blot
additional information
-
determination of disulfide bond formation in purified two-chain carboxylase and P80L and P378L two-chain carboxylases by SDS-PAGE and Western Blot analyses
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
heterodimer
-
monomer
additional information
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
glycoprotein
-
-
additional information
-
enzyme catalyzes vitamin K-dependent posttranslational modification of glutamate to gamma-carboxyl glutamate
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
37
-
enzyme is not very stable at 37°C and lower temperatures are more desirable. At temperatures below 20°C, extended linear rates of incorporation of 14CO2 into exogenous peptide substrates can be observed
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
freeze-thawing, three times, stable
-
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-70°C, 6 months, stable
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
affinity chromatography
degree of purification is about 7000fold with reference to ammonium sulfate-fractionated microsomal protein from liver. Purification of carboxylase, solubilized microsomes: 281000 cpm/mg/h, flow-through of Affi-FIXQ/S: 200000 cpm/mg/h, bound to Affi-FIXQ/S: 140000000 cpm/mg/h, affinity-purified carboxylase: 1930000000 cpm/mg/h
-
difficult, different methods shown
-
inactivated His6-carboxylase-Ac-FLEEL purified under denaturing conditions by Ni-chelation chromatography followed by preparative polyacrylamide gel electrophoresis
-
partial purification of the enzyme by antibody affinity techniques. Purified 500fold by adsorption to an antiprothrombin column and elution with a dodeca peptide which competes with a prothrombin precursor enzyme recognition site. The purified enzyme is devoid of bound precursors, and has the same ratio of vitamin K epoxidase activity to carboxylase activity as the crude microsomal preparation
-
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in 293 cells
-
expressed in baculovirus-infected insect cell. Produced His6-tagged carboxylase as a recombinant protein using a baculovirus expression system
-
expressed in Chinese hamster ovary cells with an immunodetectable octapeptide inserted at the amino-terminal ends
-
expressed in Sf9 cells
-
expression in COS cells or expressed in Sf21 insect cells
expression of the cloned cDNA results in an increase in carboxylase activity in microsomes of transfected cells compared to mock-transfected cells
-
His6-tagged bovine liver carboxylase (His6-carboxylase) is produced in insect cells using a baculovirus expression system
-
mutational analysis is performed using an expression system lacking endogenous carboxylase. Construction of baculo viruses containing FLAG-tagged carboxylase mutants and expression in infected SF21 cells. Expresses the 758 amino acid human VKD carboxylase bearing a C-terminal extension of AAADYKDDDDK, where the last eight amino acids are the FLAG epitope
-
EXPRESSION
ORGANISM
UNIPROT
LITERATURE
GGCX mRNA in the urolithiasic group is lower than that in the normal control group, which is on average 7.86fold underexpressed in the urolithiasic group compared to the normal control group. Protein expression of GGCX in the urolithiasic group is weaker than that in the control groups
-
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
E567A/K569A
-
CBX567/568
E612A/D614A
-
CBX612/614
H177A/H178A
-
CBX177/178
H678A/E679A/R680A
-
CBX678/679/680
K217A/K218A
-
inactive, CBX217/218
K346A/R347A
-
CBX346/347
K438A/D439A/H440A
-
CBX438/439/440
K622A/E623A/K624A
-
CBX622/623/624
R189A/K190A/R191A
-
CBX189/190/191
R234A/H235A
-
vitamin K epoxidase activities are reduced in parallel with the carboxylase activities. Showed defects in the propeptide binding site. Slightly faster mobility than wild-type FLAG-CBX. CBX234/235
R359A/H360A/K361A
-
vitamin K epoxidase activities are reduced in parallel with the carboxylase activities. Showed defects in the propeptide binding site. CBX359/360/361
R406A/H408A
-
vitamin K epoxidase activities are reduced in parallel with the carboxylase activities. Showed defects in the propeptide binding site. CBX406/408
R416A/D417A
-
CBX416/417
R513A/K515A
-
vitamin K epoxidase activities are reduced in parallel with the carboxylase activities. The show normal affinity for the propeptide, FLEEL, proPT28, and vitamin K hydroquinone but exhibited a low catalytic rate for carboxylation. CBX513/515
R661A/R662A
-
CBX622/623/624
R671A/R672A/R673A
-
CBX671/672/673
R687A/K688A
-
CBX687/688
E373L/Q374L
-
transmembrane domain residues in the C-terminal peptide to test for polar/charge residues
G125L
-
two-chain carboxylase
G128L
-
two-chain carboxylase
G132L
-
two-chain carboxylase
G363L/T367L
-
transmembrane domain residues in the C-terminal peptide to test for polar/charge residues
H160A
-
His to Ala mutants all show full epoxidase activity
H287A
-
His to Ala mutants all show full epoxidase activity
H381A
-
His to Ala mutants all show full epoxidase activity
H404A
-
carboxylases W390A, S398A, and H404A have activities similar to that of wild type
K218A
-
K218A activity is not detectable. The addition of exogenous amines restores K218A activity while having little effect on wild type carboxylase
L128R
-
warfarin resistent mutant
L368/372P
-
mutation to disrupt the transmembrane helix
L394R
-
natural mutant, certain individuals with combined deficiencies of vitamin K-dependent proteins have a mutation, L394R, in their gamma-glutamyl carboxylase causing impaired glutamate binding
P378L
-
significantly decreases the disulfide formation in carboxylase
P80L
-
mutation of residue P80, which has activity similar to that of wild-type carboxylase, has a minor effect on disulfide formation
R325Q
-
the mutation is associated with the bone mineral density
R58G
-
warfarin resistent mutant
S398A
-
carboxylases W390A, S398A, and H404A have activities similar to that of wild type
V29L
-
warfarin resistent mutant
V45A
-
warfarin resistent mutant
W390A
-
carboxylases W390A, S398A, and H404A have activities similar to that of wild type
W399A
-
lower activity than wild type
Y395A
-
lower activity than wild type
additional information
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