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Information on EC 6.3.4.2 - CTP synthase (glutamine hydrolysing) and Organism(s) Saccharomyces cerevisiae and UniProt Accession P38627
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6.3.4.2 CTP synthase (glutamine hydrolysing)IUBMB Comments
The enzyme contains three functionally distinct sites: an allosteric GTP-binding site, a glutaminase site where glutamine hydrolysis occurs (cf. EC 3.5.1.2, glutaminase), and the active site where CTP synthesis takes place. The reaction proceeds via phosphorylation of UTP by ATP to give an activated intermediate 4-phosphoryl UTP and ADP [4,5]. Ammonia then reacts with this intermediate generating CTP and a phosphate. The enzyme can also use ammonia from the surrounding solution [3,6].
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The taxonomic range for the selected organisms is: Saccharomyces cerevisiae
The enzyme appears in selected viruses and cellular organisms
The enzyme appears in selected viruses and cellular organisms
Synonyms
ctps, ctp synthetase, ctp synthase, ctps1, ctpsyn, ctps2, cytidine triphosphate synthetase, ctp synthetase 1, cytidine 5'-triphosphate synthase, ecctps, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
CTP synthetase
CTPS
cytidine 5'-triphosphate synthetase
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-
-
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cytidine triphosphate synthetase
-
-
-
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synthetase, cytidine triphosphate
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-
-
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uridine triphosphate aminase
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-
-
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UTP-ammonia ligase
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-
-
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CTP synthetase
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-
-
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CTPS
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-
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-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
amination
-
-
-
-
SYSTEMATIC NAME
IUBMB Comments
UTP:ammonia ligase (ADP-forming)
The enzyme contains three functionally distinct sites: an allosteric GTP-binding site, a glutaminase site where glutamine hydrolysis occurs (cf. EC 3.5.1.2, glutaminase), and the active site where CTP synthesis takes place. The reaction proceeds via phosphorylation of UTP by ATP to give an activated intermediate 4-phosphoryl UTP and ADP [4,5]. Ammonia then reacts with this intermediate generating CTP and a phosphate. The enzyme can also use ammonia from the surrounding solution [3,6].
CAS REGISTRY NUMBER
COMMENTARY
9023-56-7
-
SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
ATP + UTP + glutamine
ADP + phosphate + CTP + L-glutamate
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CTP synthetase is a cytosolic-associated glutamine amidotransferase enzyme that catalyzes the ATP-dependent transfer of the amide nitrogen from glutamine to the C-4 position of UTP to form CTP. CTP is an essential precursor of all membrane phospholipids that are synthesized via the Kennedy, i.e. CDP-choline and CDP-ethanolamine branches, and CDP-diacylglycerol pathways. The URA7-encoded CTP synthetase is responsible for the majority of the CTP made in vivo. Regulation mechanisms, detailed overview
-
-
?
additional information
?
-
CTP synthetase plays a pivotal role in the synthesis of CTP and dCTP
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-
?
additional information
?
-
CTP synthetase plays a pivotal role in the synthesis of CTP and dCTP
-
-
?
additional information
?
-
CTP synthetase plays a pivotal role in the synthesis of CTP and dCTP
-
-
?
additional information
?
-
CTP synthetase plays a pivotal role in the synthesis of CTP and dCTP
-
-
?
additional information
?
-
-
regulation of CTP synthetase activity by CTP plays an important role in the regulation of phospholipid synthesis
-
-
?
additional information
?
-
-
the enzyme is allosterically regulated by CTP product inhibition and by reversible phosphorylation
-
-
?
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
ATP + UTP + glutamine
ADP + phosphate + CTP + L-glutamate
-
CTP synthetase is a cytosolic-associated glutamine amidotransferase enzyme that catalyzes the ATP-dependent transfer of the amide nitrogen from glutamine to the C-4 position of UTP to form CTP. CTP is an essential precursor of all membrane phospholipids that are synthesized via the Kennedy, i.e. CDP-choline and CDP-ethanolamine branches, and CDP-diacylglycerol pathways. The URA7-encoded CTP synthetase is responsible for the majority of the CTP made in vivo. Regulation mechanisms, detailed overview
-
-
?
additional information
?
-
CTP synthetase plays a pivotal role in the synthesis of CTP and dCTP
-
-
?
additional information
?
-
CTP synthetase plays a pivotal role in the synthesis of CTP and dCTP
-
-
?
additional information
?
-
CTP synthetase plays a pivotal role in the synthesis of CTP and dCTP
-
-
?
additional information
?
-
CTP synthetase plays a pivotal role in the synthesis of CTP and dCTP
-
-
?
additional information
?
-
-
regulation of CTP synthetase activity by CTP plays an important role in the regulation of phospholipid synthesis
-
-
?
additional information
?
-
-
the enzyme is allosterically regulated by CTP product inhibition and by reversible phosphorylation
-
-
?
COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Mg2+
Mg2+
-
dUTP-dependent activity is dependent on Mg2+, maximal activity at 4 mM
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
CTP
-
product inhibition of wild-type enzyme, mutant E161K is less sensitive to CTP product inhibition
CTP
-
IC50 for the native enzyme: 0.12 in presence of 0.5 mM ATP, 0.22 mM in presence of 1 mM ATP. IC50 for the phosphorylated enzyme: 0.21 mM in presence of 0.5 mM ATP, 0.31 mM in presence of 1 mM ATP
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
additional information
-
the URA7-encoded enzyme is phosphorylated by protein kinases A and C at Ser424, and these phosphorylations stimulate CTP synthetase activity and increase cellular CTP levels and the utilization of the Kennedy pathway
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.04
UTP
-
pH 8.0, 30°C, phosphorylated enzyme, in presence of 2 mM ATP
0.07
UTP
-
pH 8.0, mutant enzyme S354A, kinetic constant determined with 0.5 mM ATP
0.08
UTP
-
pH 8.0, 30°C, native enzyme or phosphorylated enzyme, in presence of 0.5 mM ATP
0.1
UTP
-
pH 8.0, mutant enzyme S330A, kinetic constant determined with 0.5 mM ATP
IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.21
CTP
Saccharomyces cerevisiae
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IC50 for the native enzyme: 0.12 in presence of 0.5 mM ATP, 0.22 mM in presence of 1 mM ATP. IC50 for the phosphorylated enzyme: 0.21 mM in presence of 0.5 mM ATP, 0.31 mM in presence of 1 mM ATP
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
pH RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
7.5 - 9
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pH 7.5: about 70% of maximal activity, pH 9.0: about 55% of maximal activity, dUTP-dependent activity
pI VALUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SOURCE
additional information
-
the URA7 mRNA is 2-fold more abundant than the URA8 transcript
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
physiological function
destabilization of the active tetrameric form of the enzyme increases filament formation. The sites responsible for feedback inhibition and allosteric activation control filament length, implying that multiple regions of the enzyme can influence filament structure. Blocking catalysis without disrupting the regulatory sites of the enzyme does not affect filament formation or length
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
tetramer
additional information
-
CTP synthetase oligomerizes to a tetramer in the presence of its substrates UTP and ATP
tetramer
-
UTP and ATP are responsible for the tetramerization and activation of the inactive dimeric form of the enzyme. UTP is absolutely required for the tatramerization of the enzyme when ATP is present at a saturating concentration
tetramer
-
the enzyme exists as an inactive dimer in the absence of ATP and UTP. In the presence of saturating concentrations of ATP and UTP, the CTP synthetase protein exists as an active tetramer. Increasing concentrations of ATP and UTP cause a dose-dependent conversion of the dimeric species to a tetramer. Tetramerization is dependent on UTP and Mg2+ ions. ATP facilitates the UTP-dependent teramerization of CTP synthetase by a mechanism that involves the ATP-dependent phosphorylation of UTP catalyzed by the enzyme
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
phosphoprotein
phosphoprotein
-
phosphorylation of CTP synthetase by protein kinase A results in the stimulation of CTP synthetase activity. The mechanism of stimulation involves an increase in Vmax of the reaction and an increase of the enzyme affinity for ATP
phosphoprotein
-
phosphorylation of the purified native CTP synthetase with protein kinase A and protein kinase C facilitates the nucleotide-dependent tetramerization. Dephosphorylation of native CTP-desynthetase with alkaline phosphatase prevents the nucleotide-dependent tetramerization of the enzyme
phosphoprotein
-
the enzyme is phosphorylated and stimulated by protein kinase C. Phosphorylation of CTP synthetase on Ser36, Ser330, Ser354, and Ser454 regulates the levels of CTP and phosphatidylcholine synthesis
phosphoprotein
-
the URA7-encoded enzyme is phosphorylated at Ser424 by protein kinases A and C, and these phosphorylations stimulate CTP synthetase activity and increase cellular CTP levels and the utilization of the Kennedy pathway
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
D70A
mutation in the ATP binding site, mutant exhibits approximately twofold increase in the number of cells containing filaments
E146A
mutation in the ATP binding site, mutant exhibits approximately twofold increase in the number of cells containing filaments
E161K
R381M
mutation in L11 lid, 3.1fold increase in the number of cells forming filaments
R381P
mutation in L11 lid, 3.1fold increase in the number of cells forming filaments
S330A
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CTP synthetase activity in cells bearing the mutant enzyme is elevated, mutation causes an elevation in the Vmax of the reaction. Mutation does not have a major effect on the oligomerization of CTP synthetase
S354A
-
CTP synthetase activity in extracts from cells bearing the mutant enzyme is reduced when compared with cells bearing the wild-type enzyme, decrease in Vmax of the reaction. The amount of inactive dimeric enzyme form is 98% greater compared to wild-type enzyme
S36A
S454A
-
CTP synthetase activity in extracts from cells bearing the mutant enzyme is reduced when compared with cells bearing the wild-type enzyme. Mutation does not have a major effect on the oligomerization of CTP synthetase
additional information
-
the ura7D/ura8D double mutant, that lacks CTP synthetase activity, shows a lethal phenotype, which can be rescued by functional expression of human CTPS1 and CTPS2 genes that encode CTP synthetase enzymes. In an ura8 mutant, CTP levels are 22% lower than in wild-type, whereas the CTP concentration in an ura7 mutant is 64% lower than in wild-type
E161K
-
specific activity of the mutant URA7-encoded and URA8-encoded enzymes are 2fold greater when compared with the wild-type enzymes. The mutant enzymes are less sensitive to CTP product inhibition with inhibitor constants for CTP of 8.4fold- and 5.5fold greater, respectively, than those of their wild-type counterparts. Cells expressing the E161K mutant enzymes on a multicopy plasmid exhibit an increase in resistance to the pyrimidine poison and cancer therapeutic drug cyclopentenylcytosine and accumulate elevated levels of CTP when compared with cells expressing the wild-type enzymes. Cells expressing the E161K mutation in the URA7-encoded CTP synthetase exhibit an 1.5fold increase in the utilization of the Kennedy pathway for phosphatidylcholine synthesis when compared with control cells. Cells bearing the mutation also exhibit an 1.5fold increase in the synthesis of phosphatidylcholine, 1.3fold for phosphatidylethanolamine and 2fold for phosphatidate and a 1.7fold decrease in synthesis of phosphatidylserine. Cells bearing the e161K mutation exhibit an 1.6fold increase in the ratio of total neutral lipids to phospholipids, an 1.4fold increase in triacylglycerol, a 1.7fold increase in free fatty acids, an1.8fold increase in ergosterol ester and a 1.3fold decrease in diacylgylcerol when compared with control cells
E161K
mutation in CTP binding site, filament formation is completely disrupted and the enzyme can only form foci. Mutation decreases the affinity of the enzyme for CTP
S36A
-
CTP synthetase activity in extracts from cells bearing the mutant enzyme is reduced when compared with cells bearing the wild-type enzyme, decrease in Vmax of the reaction. The amount of inactive dimeric enzyme form is 54% greater compared to wild-type enzyme
S36A
mutation in phosphorylation site, causes an approximately twofold decrease in the frequency of filament formation
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
expression in a ura7ura8 double mutant that lacks CTP synthetase activity
-
genes ura7D and ura8D, DNA and amino acid sequence determination and analysis, expression analysis
-
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
van Kuilenburg, A.B.; Meinsma, R.; Vreken, P.; Waterham, H.R.; van Gennip, A.H.
Identification of a cDNA encoding an isoform of human CTP synthetase
Biochim. Biophys. Acta
1492
548-552
2000
Escherichia coli (P0A7E5), Escherichia coli, Homo sapiens (P17812), Homo sapiens (Q9NRF8), Homo sapiens, Saccharomyces cerevisiae (P28274), Saccharomyces cerevisiae (P38627), Mus musculus (P70303), Mus musculus (P70698)
Park, T.S.; O'Brien, D.J.; Carman, G.M.
Phosphorylation of CTP synthetase on Ser36, Ser330, Ser354, and Ser454 regulates the levels of CTP and phosphatidylcholine synthesis in Saccharomyces cerevisiae
J. Biol. Chem.
278
20785-20794
2003
Saccharomyces cerevisiae
Pappas, A.; Yang, W.L.; Park, T.S.; Carman, G.M.
Nucleotide-dependent tetramerization of CTP synthetase from Saccharomyces cerevisiae
J. Biol. Chem.
273
15954-15960
1998
Saccharomyces cerevisiae
Ostrander, D.B.; O'Brien, D.J.; Gorman, J.A.; Carman, G.M.
Effect of CTP synthetase regulation by CTP on phospholipid synthesis in Saccharomyces cerevisiae
J. Biol. Chem.
273
18992-19001
1998
Saccharomyces cerevisiae
Pappas, A.; Park, T.S.; Carman, G.M.
Characterization of a novel dUTP-dependent activity of CTP synthetase from Saccharomyces cerevisiae
Biochemistry
38
16671-16677
1999
Saccharomyces cerevisiae
Yang, W.L.; Carman, G.M.
Phosphorylation and regulation of CTP synthetase from Saccharomyces cerevisiae by protein kinase A
J. Biol. Chem.
271
28777-28783
1996
Saccharomyces cerevisiae
Yang, W.L.; McDonough, V.M.; Ozier-Kalogeropoulos, O.; Adeline, M.T.; Flocco, M.T.; Carman, G.M.
Purification and characterization of CTP synthetase, the product of the URA7 gene in Saccharomyces cerevisiae
Biochemistry
33
10785-10793
1994
Saccharomyces cerevisiae
Chang, Y.F.; Carman, G.M.
CTP synthetase and its role in phospholipid synthesis in the yeast Saccharomyces cerevisiae
Prog. Lipid Res.
47
333-339
2008
Saccharomyces cerevisiae, Homo sapiens
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