Information on EC 2.7.7.15 - choline-phosphate cytidylyltransferase

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The expected taxonomic range for this enzyme is: Eukaryota, Bacteria

EC NUMBER
COMMENTARY hide
2.7.7.15
-
RECOMMENDED NAME
GeneOntology No.
choline-phosphate cytidylyltransferase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
CTP + phosphocholine = diphosphate + CDP-choline
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
nucleotidyl group transfer
-
-
-
-
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
choline biosynthesis III
-
-
phosphatidylcholine biosynthesis I
-
-
phosphatidylcholine biosynthesis II
-
-
plasmalogen biosynthesis
-
-
type IV lipoteichoic acid biosynthesis (S. pneumoniae)
-
-
phosphatidylethanolamine bioynthesis
-
-
Phosphonate and phosphinate metabolism
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Glycerophospholipid metabolism
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Metabolic pathways
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-
SYSTEMATIC NAME
IUBMB Comments
CTP:choline-phosphate cytidylyltransferase
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CAS REGISTRY NUMBER
COMMENTARY hide
9026-34-0
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
male Mongolian gerbil, induction of brain ischemia resulted in loss of enzyme activity in cytosol and membrane
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
two isozymes CTalpha and CTbeta2
-
-
Manually annotated by BRENDA team
castor bean, L- var. Hale
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-
Manually annotated by BRENDA team
strain R36A
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
metabolism
physiological function
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
CTP + choline phosphate
diphosphate + CDP-choline
show the reaction diagram
CTP + choline phosphate
diphosphate + CDPcholine
show the reaction diagram
CTP + ethanolamine phosphate
diphosphate + CDPethanolamine
show the reaction diagram
CTP + phosphocholine
diphosphate + CDP-choline
show the reaction diagram
CTP + phosphodimethylethanolamine
diphosphate + CDPdimethylethanolamine
show the reaction diagram
-
-
-
-
?
CTP + phosphomonomethylethanolamine
diphosphate + CDPmethylethanolamine
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
CTP + choline phosphate
diphosphate + CDP-choline
show the reaction diagram
CTP + choline phosphate
diphosphate + CDPcholine
show the reaction diagram
CTP + phosphocholine
diphosphate + CDP-choline
show the reaction diagram
additional information
?
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Co2+
Co2+ results in significant activity at a concentration of 25 mM, with 41% of the activity seen with 25 mM Mg2+, the cation preference of CCT1 is Mg2+ > Mn2+/Co2+ > Ca2+/Ni2+ > Zn2+
Ni2+
Ni2+ yields approximately 9% of the activity seen with Mg2+, the cation preference of CCT1 is Mg2+ > Mn2+/Co2+ > Ca2+/Ni2+ > Zn2+
Zn2+
Zn2+ results in catalytic activity of only 1% when compared to the activity in the presence of Mg2+, the cation preference of CCT1 is Mg2+ > Mn2+/Co2+ > Ca2+/Ni2+ > Zn2+
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphatidylcholine
4-chloromercuribenzoate
5,5'-dithiobis(2-nitrobenzoate)
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-
Blue H-B
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40% inhibition
Blue MX-R
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22% inhibition
C2 ceramide
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-
calpain
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calpain degrades CCTalpha, 0.6 pmol of calpain effectively cleaves 80% of purified CCTalpha (0.7 pmol) in the absence of calmodulin
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CDPcholine
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competitive to choline phosphate
ceramide
chlorpromazine
Dibucaine
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28% inhibition at 0.05 mM
Green H-4G
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85% inhibition
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Levafix E-5BNA
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40% inhibition
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lyso-phosphatidylcholine
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-
lysophosphatidylcholine
Lysosphingolipids
-
reversible
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N-ethylmaleimide
NaCl
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inhibitory above 200 mM, 39% inhibition at 300 mM, 67% inhibition at 500 mM
oxidized low density lipoprotein
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phosphate
phosphodimethylethanolamine
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competitive to phosphocholine
Phosphoethanolamine
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competitive to phosphocholine
phosphomonomethylethanolamine
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competitive to phosphocholine
propanolol
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28% inhibition at 0.05 mM
Sn-3-Lysophosphatidylcholine
-
-
-
Sphingolipids
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reversed by activating phospholipids
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sphingomyelin
-
causes inhibition when present in multilamellar vesicles
sphingosine
Tetracaine
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50% inhibition at 0.05 mM
Trifluoperazine
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5fold inhibition of microsomal enzyme at 0.025 mM, 50% inhibition at 0.75mM, addition of saturating amounts of rat liver phospholipid reverses inhibition, no inhibition of hepatocytes at 0.02 mM
Triton X-100
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Tumor necrosis factor alpha
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-
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Turquoise H-A
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40% inhibition
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Turquoise MX-G
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85% inhibition
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Urea
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50% inhibition of wild-type enzyme at 0.75 M, 50% inhibition of H92N mutant enzyme at 0.35 M, 50% inhibition of H92Q mutant enzyme at 0.2 M
additional information
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
activator protein-1
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potential regulator of CCTbeta2 expression in neuronal cells
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conduritol-B-epoxide
diphosphatidylglycerol
;
dithiothreitol
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20 mM
fatty acids
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activity slightly enhanced by addition of saturated fatty acids, markedly increased by addition of unsaturated fatty acids regardless of chain length and number of double bonds
glucosylceramide
Lipids
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phosphatidylcholine
6fold enhancement of activity at 0.025 mM; 6fold enhancement of activity at 0.025 mM
phosphatidylglycerol
phosphatidylinositol
;
retinoblastoma protein
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regulates CTalpha expression by different mechanisms depending on the phase of the cell cycle, the regulation of CTalpha expression requires both the Sp1 and the retinoblastoma control elements
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Sp1
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Sp1 stimulates CTalpha transcription, and Sp1-mediated transcription is stimulated by the retinoblastoma protein
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additional information
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.21 - 0.64
CDPcholine
0.167 - 3.2
choline phosphate
0.133 - 35.7
CTP
0.004 - 0.007
diphosphate
0.083 - 212
phosphocholine
4
phosphodimethylethanolamine
-
pH 7, 37C
1.43 - 69.4
Phosphoethanolamine
6.9
phosphomonomethylethanolamine
-
pH 7, 37C
0.14 - 2.1
phosphorylcholine
additional information
additional information
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
33.3
CTP
Rattus norvegicus
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with choline phosphate, 45000 MW protein
0.21 - 31.3
phosphocholine
additional information
additional information
Caenorhabditis elegans
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Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.09
CDPcholine
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pH 7.4, 37C
1
phosphodimethylethanolamine
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pH 7, 37C
16.7
Phosphoethanolamine
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pH 7, 37C
7.8
phosphomonomethylethanolamine
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pH 7, 37C
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.00296
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L-form
0.0048
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cell extract
0.021
H168A/Y173A mutant deletion construct 1-236, variable CTP concentration, 1.5 mM choline phosphate
0.025
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H-form
0.034
H168A mutant deletion construct 1-236, variable CTP concentration, 1.5 mM choline phosphate
0.055
H168A/Y173A mutant deletion construct 1-236, variable choline phosphate concentration, 8.8 mM CTP
0.069
H168A mutant deletion construct 1-236, variable choline phosphate concentration, 8.8 mM CTP
0.1096
-
-
0.116
Y173A mutant deletion construct 1-236, variable choline phosphate concentration, 8.8 mM CTP
0.167
Y173A mutant deletion construct 1-236, variable CTP concentration, 1.5 mM choline phosphate
0.35
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with phosphoethanolamine as substrate
1.724
wild-type deletion construct 1-236, variable choline phosphate concentration, 8.8 mM CTP
3.399
wild-type deletion construct 1-236, variable CTP concentration, 1.5 mM choline phosphate
3.45
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with phosphomonomethylethanolamine as substrate
10
wild-type protein in the presence of 0.01 mM phosphatidylcholine:oleate
10.7
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with phosphocholine as substrate
16.12
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with phosphodimethylethanolamine as substrate
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.3
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assay at
7 - 7.5
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7 - 7.5
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pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6 - 8
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pH 6: about 80% of activity maximum, pH 8: about 60% of activity maximum
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
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L-form and H-form
Manually annotated by BRENDA team
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contains only L-form
Manually annotated by BRENDA team
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adult human adenocarcinoma cell
Manually annotated by BRENDA team
isozyme CCTbeta2
Manually annotated by BRENDA team
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phaeochromocytoma cell
Manually annotated by BRENDA team
additional information
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
additional information
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30000
-
SDS-PAGE, gel filtration
41700
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SDS-PAGE
48000
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SDS-PAGE
49400
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calculated from amino acid sequence
50300
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gel filtration
60000
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SDS-PAGE
80000 - 110000
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diffuse band in SDS-PAGE using chemically cross-linked protein
97000
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gel filtration, glycerol density gradient centrifugation
155000
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gel filtration
200000
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L-form of enzyme, gel filtration, aggregates in the cytosol to form high molecular weight species (H-form) with a median value of 1200000
284000
-
Hep G2 cells, H-form of enzyme, glycerol density gradient centrifugation
840000
-
native PAGE
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
heterodimer
-
1 * 80000 + 1 * 30000
homodimer
monomer
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1 * 30000, SDS-PAGE
tetramer
additional information
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
lipoprotein
phosphoprotein
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
sitting drop vapor-diffusion method
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
calmodulin stabilizes CCTalpha from calpain proteolysis
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freezing and thawing once causes more than 90% loss of activity
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instability of the highly purified enzyme is partially overcome by addition of 1 mM DTT and 1% bovine serum albumin or 0.5 mM CTP and 2.5 mM magnesium acetate
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purified enzyme is unstable in presence of DTT. CTP-Mg2+ or bovine serum albumin do not significantly stabilize the enzyme
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removal of detergents leads to rapid loss of activity and self-aggregation, high tendency to bind to glass and plastic surfaces even in the presence of detergents
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Unstable at low protein concentration
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20C stable for several months in 50% glycerol/20 mM Tris pH 7.5
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-20C, enzyme concentrated to a small volume, 50% glycerol, 20% loss of activity after 2 weeks
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-20C, stable for at least 8 months in 70% ammonium sulfate
-20C, stable for more than 1 month in presence of 20% glycerol
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-70C, 50 mM Tris-HCl, 150 mM NaCl, 1.0 mM EDTA, 2.0 mM DTT, 0.025% NaN3, pH 7.4, 0.03% Triton X-100, 200 mM phosphate, stable for several months
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-70C, stable in presence of Triton X-100 and 0.2 M potassium phosphate
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0C, 80% loss of activity after 6 days
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4C, 20% loss of activity per day
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4C, 30% loss of activity after 1 week
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4C, increase of activity in fresh cytosol obtained by storage is due to an increase of lysophosphatidylethanolamine and a decrease in phosphatidylethanolamine
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
affinity chromatography, 2 forms: L-form and H-form, L-form: major species in fresh cytosol, H-form: consists of multiple copies of L-form
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CM- and Blue-Sepharose chromatography, gel filtartion
deletion mutant which is truncated after residue 236, called CCTalpha236
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dye-affinity chromatography with Green H-4G-Sepharose CL4B
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homogeneity
native and His-tag protein
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Ni-NTA column chromatography
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Ni-NTA column chromatography; Ni-NTA column chromatography
rapid purification method, 687fold purification
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recombinant enzymes using His-tag
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recombinant protein
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TALON Co2+ metal affinity chromatography
Talon metal affinity column chromatography; Talon metal affinity column chromatography
wild-type and truncated form
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wild-type and truncated forms
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wild-type enzyme and peptides after trypsin digestion
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
CCTbeta2 isoform
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deletion construct 1-236: expressed in T.ni cell using a baculovirus expression system, expressed in Escherichia coli BL21
deletion mutant which is truncated after residue 236, called CCTalpha236
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expressed as native and His-tag fusion protein in Sf9 insect cells, native protein expressed in Escherichia coli but expressed protein was found to be misfolded
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expressed in A-549 cell
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expressed in A-549 cells
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expressed in an adenoviral system
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expressed in CHO-58 cells
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expressed in Escherichia coli
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expressed in Escherichia coli Rosetta cells and expressed by baculovirus infection of Trichoplusia ni cells
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expressed in High 5 cells
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expressed in insect cells
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expressed in mice
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expressed in PC-12 cells
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expressed in Sf9 cells and Schneider 2 cells; expressed in Sf9 cells and Schneider 2 cells
full length enzyme and various truncated enzymes expressed in transgenic mice
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His-tagged version expressed in Sf9 cell using a baculovirus exression system
isozyme CCTalpha is expressed in baculovirus-infected cells
native and mutant enzyme expressed as His-tag fusion proteins in COS-1 cells
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wild-type and deletion mutants delta312-367, delta231-367 and delta257-367
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wild-type and truncated form
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wild-type and truncated forms
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EXPRESSION
ORGANISM
UNIPROT
LITERATURE
after stimulation by 12-O-tetradecanoylphorbol-13-acetate
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at low temperature
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in cultured primary neurons nerve growth factor increases the amount of isozyme CTbeta2, but not CTalpha, mRNA and protein
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
F260D
part of a putative amphipathic alpha helix
F260K
part of a putative amphipathic alpha helix
F260L
part of a putative amphipathic alpha helix
F260V
part of a putative amphipathic alpha helix
F269I
part of a putative amphipathic alpha helix
I256S
part of a putative amphipathic alpha helix
I257S
part of a putative amphipathic alpha helix
L246S
part of a putative amphipathic alpha helix
L246S/W249S/I256S/I257S
part of a putative amphipathic alpha helix
W249S
part of a putative amphipathic alpha helix
M27A
-
mutation in isoform CCTbeta1
K238R/K239R/Y240F
-
increased resistance to calpain cleavage, resistance to enzyme degradation by oxidized low density lipoprotein
Q243A
-
the mutation results in loss of calmodulin binding and leads to complete calpain resistance in vitro and in vivo
C113S
-
no significant effect of activity
C139S
-
no significant effect of activity
C354S
-
no significant effect of activity
C359S
-
no significant effect of activity
C37S
-
unable to form dimers
C68S
-
no significant effect of activity
C73S
-
no significant effect of activity
H168A
involved in catalysis
H168A/Y173A
involved in catalysis
I272R
-
part of the M-domain
I275R
-
part of the M-domain
K122R
-
decreased Vmax
L274R
-
part of the M-domain
R196K
-
decreased Vmax, 5fold increased Km for phosphorylcholine, 23fold increased Km for CTP
V267R
-
part of the M-domain
V267R/I272R
-
part of the M-domain
V267R/I272R/L274R
-
part of the M-domain
V267R/I272R/L274R/I275R
-
part of the M-domain
Y173A
involved in catalysis
Y192H
-
mutant can not be expressed in Escherichia coli
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
medicine
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