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Information on EC 6.3.4.2 - CTP synthase (glutamine hydrolysing) and Organism(s) Homo sapiens and UniProt Accession Q9NRF8

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EC Tree
     6 Ligases
         6.3 Forming carbon-nitrogen bonds
             6.3.4 Other carbon-nitrogen ligases
                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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Homo sapiens
UNIPROT: Q9NRF8
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Word Map
The taxonomic range for the selected organisms is: Homo sapiens
The enzyme appears in selected viruses and cellular organisms
Reaction Schemes
hide(Overall reactions are displayed. Show all >>)
Synonyms
ctps, ctp synthetase, ctp synthase, ctps1, ctpsyn, ctps2, cytidine triphosphate synthetase, ctp synthetase 1, cytidine 5'-triphosphate synthase, ecctps, more
SYNONYM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
cytidine triphosphate synthetase
-
CTP synthase
-
-
CTP synthetase
CTP synthetase 1
-
-
CTPS2
-
-
cytidine 5'-triphosphate synthetase
-
-
-
-
cytidine triphosphate synthetase
cytidine triphosphate synthetase 1
-
-
synthetase, cytidine triphosphate
-
-
-
-
uridine triphosphate aminase
-
-
-
-
UTP-ammonia ligase
-
-
-
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
amination
-
-
-
-
PATHWAY SOURCE
PATHWAYS
-
-, -, -
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 hide
9023-56-7
-
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
ATP + UTP + Gln + H2O
ADP + CTP + Glu + phosphate
show the reaction diagram
-
-
-
?
ATP + UTP + L-Gln
ADP + phosphate + CTP + L-Glu
show the reaction diagram
-
-
-
?
ATP + UTP + NH4+
ADP + phosphate + CTP
show the reaction diagram
-
-
?
ATP + UTP + Gln + H2O
ADP + CTP + Glu + phosphate
show the reaction diagram
-
-
-
-
?
ATP + UTP + glutamine
ADP + phosphate + CTP + L-glutamate
show the reaction diagram
ATP + UTP + L-Gln
ADP + phosphate + CTP + L-Glu
show the reaction diagram
-
-
-
?
ATP + UTP + NH3
ADP + phosphate + CTP
show the reaction diagram
ATP + UTP + NH4+
ADP + phosphate + CTP
show the reaction diagram
additional information
?
-
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
ATP + UTP + glutamine
ADP + phosphate + CTP + L-glutamate
show the reaction diagram
-
the CTPS1-encoded enzyme is regulated by reversible phosphorylation at Thr455, regulation mechanisms, overview
-
-
?
ATP + UTP + NH3
ADP + phosphate + CTP
show the reaction diagram
-
CTPS1 is involved in microtubule network formation and/or stabilzation, overview
-
-
?
additional information
?
-
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
cyclopentenyl cytosine
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.072 - 0.1
L-Gln
0.19 - 0.8
UTP
0.002
ATP
-
-
0.027
L-Gln
wild-type, pH 8.1, 37°C
0.59 - 1.9
UTP
additional information
additional information
-
IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.04 - 0.072
CTP
0.042 - 0.057
CTP
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.0025
-
specific activity of Escherichia coli expressed enzyme, after a 20 min incubation with protein kinase C, the activity of the CTP synthetase was stimulated 95fold
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
8.1
assay at
8
-
assay at
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
37
assay at
37
-
assay at
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
-
significantly higher activity is detected in leukaemic cells of children suffering from acute lymphocytic leukemia than in lymphocytes of healthy controls
Manually annotated by BRENDA team
-
highest activity is found in thombocytes, followed by monocytes, lymphocytes, granulocytes and erythrocytes
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
physiological function
-
CTP synthase protein molecules form filamentous structures termed cytoophidia or CTP synthase filaments in the cytoplasm and nucleus
UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
SEQUENCE
LOCALIZATION PREDICTION?
PYRG2_HUMAN
586
0
65678
Swiss-Prot
other Location (Reliability: 3)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
65678
x * 65678, calculation from nucleotide sequence
70000
-
x * 70000, SDS-PAGE
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
x * 65678, calculation from nucleotide sequence
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
phosphoprotein
phosphoprotein
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
crystal structure analysis discloses a homotetrameric structure and each active site is formed by three different subunits. Sulfate ions bound to the active sites indicate the positions of phosphate-binding sites for the substrates ATP and UTP and the feedback inhibitor CTP. Together with earlier structures of bacterial CTPS, the human CTPS structure provides an extended understanding of the structure–function relationship of CTPS-family members
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the protein is crystallized by the hanging-drop method, equilibrating 1 ml protein solution and 1 ml of a solution containing 0.1 M Tris pH 8.8, 1.2 M (NH4)2SO4 and 50 mM malonic acid against 0.5 ml of a well solution containing 0.1 M Tris pH 8.8 and 1.2 M (NH4)2SO4. Crystals with a maximum dimension of 0.03 nm formed in 3 d
-
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
E579A
HEK 293 cells: no effect on phosphorylation of CTPS1 by glycogen synthase kinase 3
S568A
2fold increase in Km value for UTP. Mutation of S568A significantly increases CTPS2 activity. The S568A mutation has a greater effect on the glutamine than ammonia-dependent activity
S571A
4fold increase in Km value for UTP
S571I
Ser-571 is the major site phosphorylated by glycogen synthase kinase 3 in intact human embryonic kidney 293 cells
S571I/S574A
phosphorylation by glycogen synthase kinase 3 does not show altered incorporation of phosphate compared with S571I alone
S571I/S574A/S575A
phosphorylation by glycogen synthase kinase 3 shows a slight decrease in the amount of phosphate incorporated into CTPS1 compared with S571I/S575A, suggesting that Ser-574 may serve as a minor secondary site for glycogen synthase kinase 3 phosphorylation
S571I/S575A
phosphorylation by glycogen synthase kinase 3 shows slightly elevated amounts of phosphate incorporated into CTPS1 compared with S571I, suggesting that without the ability to phosphorylate Ser-571 or Ser-575 in vitro, glycogen synthase kinase 3 may phosphorylate an alternative site, albeit to a much lesser extent
S574A
greatly reduces phosphorylation by glycogen synthase kinase 3
S574A/S575A
greatly reduces phosphorylation by glycogen synthase kinase 3
S575A
greatly reduces phosphorylation by glycogen synthase kinase 3. Mutation of Ser-575 prevents the phosphorylation of Ser-571, suggesting that phosphorylation of Ser-575 is necessary for priming the glycogen synthase kinase 3 phosphorylation of Ser-571
S462A/T455A
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S462A and T455A mutations result in a decreased CTP synthetase 1 phosphorylation which appear to be much less than of the individual mutant enzymes S462A or T455A
S575A
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site-directed mutagenesis the mutant does not interact with peptidyl prolyl isomerase Pin1
T455A
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T455A mutation causes a 78% decrease in protein kinase A phosphorylation
additional information
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
His6-tagged human CTP synthetase 1 is purified by loading onto a Ni2+-NTA column
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His6-tagged wild type and mutant human CTP synthetase 1 enzymes are expressed and purified from yeast cell extracts with Ni2+-NTA resin
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His6-tagged wild type human CTP synthetase 1 is expressed and purified from Escherichia coli by Ni2+-NTA chromatography and with PorosHQ-ion-exchange chromatography
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Ni2+-IDA resin column chromatography
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protein is purified by using HisTrap HP and Superdex 75 columns
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CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
a carboxyl-terminal FLAG sequence is introduced after insertion of human CTPS1 into pCDNA 4 myc/his, expression by transient transfection of human embryonic kidney (HEK) 293 cells
cDNA encoding the synthetase domain of human CTPS (isoform 1) is subcloned into the pNIC-Bsa4 vector, the resulting construct codes for a protein with an N-terminal hexahistidine tag with an integrated TEV protease-cleavage site, the entire plasmid is transformed in Escherichia coli BL-21
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expressed in Escherichia coli BL21(DE3) cells
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expression of the human type II CTP synthetase gene in the CTP synthetase-deficient mutant Escherichia coli JF618 completely abolishes the need for exogenous cytidine for their survival
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expression of wild-type and mutant enzymes in HEK-293 cells, co-expression with GST-tagged Xenopus laevis peptidyl prolyl isomerase Pin1
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functional expression of human CTPS1 and CTPS2 genes that encode CTP synthetase enzymes in enzyme-deficient Saccharomyces cerevisiae ura7D/ura8D double mutant, complementation
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human CTP synthetase 1 is expressed and purified from a Saccharomyces cerevisiae ura7delta ura8delta double mutant that lacks CTP synthetase activity
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protein is expressed as a His6-tagged wild type human CTP synthetase 1 fusion protein in Escherichia coli
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protein is expressed as a His6-tagged wild type human CTP synthetase 1 fusion protein in Saccharomyces cerevisiae
-
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
drug development
-
structure serves as a basis for structure-based design of anti-proliferative inhibitors
medicine
-
glutamine deficiency induces CTPS cytoophidia in human Huh-7 cells. CTPS cytoophidia are found in various human cancers and some noncancerous tissues. Among 203 tissue samples of hepatocellular carcinoma, 28% exhibited many cytoophidia, whereas no cytoophidia are detected in adjacent noncancerous hepatocytes for all samples
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Verschuur, A.C.; van Gennip, A.H.; Muller, E.J.; Voute, P.A.; Vreken, P.; Van Kuilenburg, A.B.
Cytidine triphosphate synthase activity and mRNA expression in normal human blood cells
Biol. Chem.
380
41-46
1999
Homo sapiens
Manually annotated by BRENDA team
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)
Manually annotated by BRENDA team
Verschuur, A.C.; Van Gennip, A.H.; Leen, R.; Meinsma, R.; Voute, P.A.; van Kuilenburg, A.B.
In vitro inhibition of cytidine triphosphate synthetase activity by cyclopentenyl cytosine in paediatric acute lymphocytic leukaemia
Br. J. Haematol.
110
161-169
2000
Homo sapiens
Manually annotated by BRENDA team
van Kuilenburg, A.B.P.; Elzinga, L.; Van Gennip, A.H.
Kinetic properties of CTP synthetase from HL-60 cells
Adv. Exp. Med. Biol.
431
255-258
1998
Homo sapiens
Manually annotated by BRENDA team
van Kuilenburg, A.B.P.; Meinsma, R.; Vreken, P.; Waterham, H.R.; van Gennip, A.H.
Isoforms of human CTP synthetase
Adv. Exp. Med. Biol.
486
257-261
2000
Homo sapiens
Manually annotated by BRENDA team
Kursula, P.; Flodin, S.; Ehn, M.; Hammarstroem, M.; Schueler, H.; Nordlund, P.; Stenmark, P.
Structure of the synthetase domain of human CTP synthetase, a target for anticancer therapy
Acta Crystallogr. Sect. F
62
613-617
2006
Homo sapiens
Manually annotated by BRENDA team
Chang, Y.F.; Martin, S.S.; Baldwin, E.P.; Carman, G.M.
Phosphorylation of human CTP synthetase 1 by protein kinase C: identification of Ser(462) and Thr(455) as major sites of phosphorylation
J. Biol. Chem.
282
17613-17622
2007
Homo sapiens
Manually annotated by BRENDA team
Higgins, M.J.; Graves, P.R.; Graves, L.M.
Regulation of human cytidine triphosphate synthetase 1 by glycogen synthase kinase 3
J. Biol. Chem.
282
29493-29503
2007
Homo sapiens (Q9NRF8), Homo sapiens
Manually annotated by BRENDA team
Choi, M.G.; Carman, G.M.
Phosphorylation of human CTP synthetase 1 by protein kinase A: identification of Thr455 as a major site of phosphorylation
J. Biol. Chem.
282
5367-5377
2007
Homo sapiens
Manually annotated by BRENDA team
Higgins, M.J.; Loiselle, D.; Haystead, T.A.; Graves, L.M.
Human cytidine triphosphate synthetase 1 interacting proteins
Nucleosides Nucleotides Nucleic Acids
27
850-857
2008
Homo sapiens
Manually annotated by BRENDA team
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
Manually annotated by BRENDA team
Kassel, K.M.; Au, d.a..R.; Higgins, M.J.; Hines, M.; Graves, L.M.
Regulation of human cytidine triphosphate synthetase 2 by phosphorylation
J. Biol. Chem.
285
33727-33736
2010
Homo sapiens, Homo sapiens (Q9NRF8)
Manually annotated by BRENDA team
Gou, K.M.; Chang, C.C.; Shen, Q.J.; Sung, L.Y.; Liu, J.L.
CTP synthase forms cytoophidia in the cytoplasm and nucleus
Exp. Cell Res.
323
242-253
2014
Homo sapiens, Mus musculus
Manually annotated by BRENDA team
Chang, C.C.; Jeng, Y.M.; Peng, M.; Keppeke, G.D.; Sung, L.Y.; Liu, J.L.
CTP synthase forms the cytoophidium in human hepatocellular carcinoma
Exp. Cell Res.
361
292-299
2017
Homo sapiens, Drosophila melanogaster (X2JCY2)
Manually annotated by BRENDA team