Information on EC 6.3.4.2 - CTP synthase (glutamine hydrolysing)

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

EC NUMBER
COMMENTARY hide
6.3.4.2
-
RECOMMENDED NAME
GeneOntology No.
CTP synthase (glutamine hydrolysing)
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
ATP + UTP + L-glutamine = ADP + phosphate + CTP + L-glutamate
show the reaction diagram
ATP + UTP + NH3 = ADP + phosphate + CTP
show the reaction diagram
(1b)
-
-
-
L-glutamine + H2O = L-glutamate + NH3
show the reaction diagram
(1a)
-
-
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
amination
-
-
-
-
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Metabolic pathways
-
-
pyrimidine metabolism
-
-
Pyrimidine metabolism
-
-
UTP and CTP de novo biosynthesis
-
-
UTP and CTP dephosphorylation I
-
-
UTP and CTP dephosphorylation II
-
-
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
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
-
-
Manually annotated by BRENDA team
strain L2/CPEC
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
HB8
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
-
mutations disrupting CTP synthase isoform C or isoform A expression results in cytoophidium disassembly
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
ATP + dUTP + NH4+
ADP + phosphate + dCTP
show the reaction diagram
ATP + UTP + CH3NHOH
ADP + phosphate + ?
show the reaction diagram
-
-
-
-
?
ATP + UTP + Gln
ADP + phosphate + CTP + Glu
show the reaction diagram
ATP + UTP + Gln + H2O
ADP + CTP + Glu + phosphate
show the reaction diagram
ATP + UTP + Gln-NH2
ADP + phosphate + CTP + N4-amino CTP
show the reaction diagram
-
-
-
-
?
ATP + UTP + Gln-OH
ADP + phosphate + CTP + N4-hydroxy-CTP
show the reaction diagram
-
-
-
-
?
ATP + UTP + glutamine
ADP + phosphate + CTP + Glu
show the reaction diagram
ATP + UTP + glutamine
ADP + phosphate + CTP + L-glutamate
show the reaction diagram
ATP + UTP + hydroxylamine
ADP + phosphate + N4-hydroxyCTP
show the reaction diagram
-
activity is about 3fold higher than activity with NH4+
-
?
ATP + UTP + hydroxylamine
ADP + phosphate + N4-OH-CTP
show the reaction diagram
-
-
-
-
?
ATP + UTP + L-Gln
ADP + phosphate + CTP + L-Glu
show the reaction diagram
-
-
-
?
ATP + UTP + L-glutamine
ADP + phosphate + CTP + L-glutamate
show the reaction diagram
ATP + UTP + NH2NH2
ADP + phosphate + ?
show the reaction diagram
-
-
-
-
?
ATP + UTP + NH2NH2
ADP + phosphate + CTP + N4-amino CTP
show the reaction diagram
-
-
-
-
?
ATP + UTP + NH2OH
ADP + phosphate + N4-hydroxy-CTP
show the reaction diagram
-
-
-
-
?
ATP + UTP + NH3
ADP + CTP + phosphate
show the reaction diagram
ATP + UTP + NH3
ADP + phosphate + CTP
show the reaction diagram
ATP + UTP + NH4+
ADP + phosphate + CTP
show the reaction diagram
ATP + UTP + O-methylhydroxylamine
ADP + phosphate + N4-methoxyCTP
show the reaction diagram
-
-
-
?
deoxyATP + UTP + NH4+
deoxyADP + phosphate + CTP
show the reaction diagram
-
-
-
-
?
deoxyGTP + UTP + NH4+
deoxyGDP + phosphate + CTP
show the reaction diagram
GTP + UTP + NH4+
GDP + phosphate + CTP
show the reaction diagram
UTP + ATP + NH3
CTP + ADP + phosphate
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
ATP + UTP + glutamine
ADP + phosphate + CTP + L-glutamate
show the reaction diagram
ATP + UTP + L-glutamine
ADP + phosphate + CTP + L-glutamate
show the reaction diagram
ATP + UTP + NH3
ADP + phosphate + CTP
show the reaction diagram
ATP + UTP + NH4+
ADP + phosphate + CTP
show the reaction diagram
Q5SIA8
last step in CTP biosynthesis
-
-
?
UTP + ATP + NH3
CTP + ADP + phosphate
show the reaction diagram
-
the enzyme is regulated in a complex fashion, overview
-
-
?
additional information
?
-
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Co2+
-
stimulates at a lower level than Mg2+
Mn2+
-
stimulates at a lower level than Mg2+
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1,3,7,9-tetramethyluric
-
-
1,3,7-trimethyluric acid
-
-
1,3-dimethyluric acid
-
pH-dependent inhibition, overview
1,7-dimethyluric acid
-
pH-dependent inhibition, overview
1-methyluric acid
-
-
2',3'-dialdehyde adenosine 5'-triphosphate
irreversible inhibitor of CTPS
2'-deoxy-GTP
-
no guanosine, kact: 1.5/sec, KA: 0.21 mM, Ki: 0.36 mM; the GTP analogue is capable of inhibiting Gln-dependent CTP formation at over 0.15 mM
2'-deoxy-guanosine
-
-
2,6-diaminopurine riboside
-
-
2-aminopurine riboside
-
-
2-Thiocytidine 5'-triphosphate
-
-
2-thiouridine 5'-triphosphate
-
-
3'-deoxy-guanosine
-
-
3,7-dimethyluric acid
-
-
3-Deazauridine 5'-triphosphate
-
-
4-thiouridine 5'-triphosphate
-
-
6-diazo-5-oxo-L-norleucine
6-thio-GTP
-
the GTP analogue is capable of inhibiting Gln-dependent CTP formation at over 0.15 mM
6-Thioguanine
-
-
6-thioguanosine
-
-
6-thioguanosine 5'-triphosphate
-
no guanosine, kact: 8.5/sec, KA: 0.035 mM, Ki: 0.27 mM
7-deazaguanosine
-
-
8-oxoguanosine
-
-
8-oxoguanosine 5'-triphosphate
-
-
acivicin
-
irreversible inhibition by the glutamine analogue acivin. The acivicin inhibition of Trypanosoma brucei CTPS is more pronounced when the enzyme is preincubated with the drug in the presence of nucleotide substrates than in the absence of substrates
acycloguanosine
-
-
acycloguanosine monophosphate
-
-
adenine
-
-
adenosine
-
-
adenosine 5'-[beta,gamma-imido]triphosphate
-
poor inhibitor compared to ATPgammaS
adenylyl-iminodiphosphate
-
competitive with ATP
alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid
-
reduces the parasite CTP level even further and inhibits trypanosome proliferation in vitro and in Trypanosoma brucei-infected mice
ATPgammaS
-
-
Caffeine
Co2+
-
above 2 mM
Cu2+
-
inhibition is not reversed by EDTA, in presence of dithiothreitol inhibition at concentrations below 0.2 mM
cyclopentenyl cytosine
cyclopentylcytosine triphosphate
-
-
D,L-2-amino-4-phosphonobutyrate
-
-
dCTP
-
very weak inhibitor
DELTA1-Pyrroline-5-carboxylate
-
weak
dideoxy-GTP
-
-
DL-DELTA1-pyrroline 5-carboxylate
-
0.125 mM, complete inhibition of ammonium chloride-dependent CTP synthesis
Gln
-
inhibition of hydroxylamine-dependent N4-OH CTP synthesis in presence of GTP
glutamate gamma-semialdehyde
-
potent linear mixed-type inhibitor, competitive with respect to ammonia, no inhibition of the mutant enzyme C379A
guanosine
guanosine 5'-tetraphosphate
-
; no guanosine, kact: 4/sec, KA: 0.19 mM, Ki: 0.5 mM; the GTP analogue is capable of inhibiting Gln-dependent CTP formation at over 0.15 mM
Inosine
-
-
ITP
-
no guanosine, kact: 5.2/sec, KA: 2.9 mM, Ki: 4.5 mM; the GTP analogue is capable of inhibiting Gln-dependent CTP formation at over 0.15 mM
L-2-pyrrolidone 5-carboxylate
-
weak competitive inhibition of the reaction with ammonia as substrate, no significant inhibition with glutamine as substrate
Mn2+
-
above 2 mM
N-methylguanosine
-
-
NH4Cl
-
substrate inhibition , a significant part of the inhibition can be shown to be due to the increase in ionic strength with increasing substrate concentrations
Ni2+
-
in presence of dithiothreitol inhibition at concentrations below 0.2 mM
O-methylguanosine
-
-
O-methylguanosine 5'-triphosphate
-
no guanosine, kact: 2.8/sec, KA: 0.13 mM, Ki: 0.29 mM
O6-methyl-GTP
-
the GTP analogue is capable of inhibiting Gln-dependent CTP formation at over 0.15 mM
p-chloromercuribenzenesulfonic acid
-
-
paraxanthine
-
pH-dependent inhibition, overview
PCMB
-
0.01 mM, 50% inhibition
pyrrole-2-carboxylate
-
weak competitive inhibition of the reaction with ammonia as substrate, no significant inhibition with glutamine as substrate
S-nitroso-L-cysteine
-
specific irreversible inhibitor,inhibits the activity by 94%
S-nitroso-L-homocysteine
-
specific irreversible inhibitor, inhibits the activity by 90%
Theobromine
-
-
theophylline
-
pH-dependent inhibition, overview
uracil-4-acetic acid
-
-
Uric acid
uridine
-
-
UTP
-
competitive with ATP
xanthine
-
; pH-dependent inhibition, overview
Xanthosine
-
-
Zn2+
-
inhibition is reversed by EDTA, in presence of dithiothreitol inhibition at concentrations below 0.2 mM
additional information
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2'-deoxy-GTP
-
0 mM guanosine, kact: 1.5/sec, KA: 0.21 mM, Ki: 0.36 mM; the GTP analogue is capable of activating Gln-dependent CTP formation
2-mercaptoethanol
6-thio-GTP
-
the GTP analogue is capable of activating Gln-dependent CTP formation
6-thioguanosine 5'-triphosphate
-
0 mM guanosine, kact: 8.5/sec, KA: 0.035 mM, Ki: 0.27 mM
dithiothreitol
-
required for optimal activity, above 50 mM increase activity to 223% mM
GDP
-
slight activation
GMP
-
slight activation
guanosine 5'-tetraphosphate
-
0 mM guanosine, kact: 4/sec, KA: 0.19 mM, Ki: 0.5 mM; the GTP analogue is capable of activating Gln-dependent CTP formation
ITP
-
0 mM guanosine, kact: 5.2/sec, KA: 2.9 mM, Ki: 4.5 mM; the GTP analogue is capable of activating Gln-dependent CTP formation
O-methylguanosine 5'-triphosphate
-
0 mM guanosine, kact: 2.8/sec, KA: 0.13 mM, Ki: 0.29 mM
O6-methyl-GTP
-
the GTP analogue is capable of activating Gln-dependent CTP formation
additional information
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.002 - 0.097
ATP
0.259 - 0.497
Gln
39.4
Gln-NH2
-
37C, pH 8.0, wild-type enzyme
0.063 - 0.165
Gln-OH
0.1 - 0.424
glutamine
0.07
GTP
0.027 - 0.1
L-Gln
0.26
L-glutamine
-
-
75.3 - 82.8
NH2OH
0.627 - 2.79
NH3
54 - 92
NH4+
0.027 - 1.9
UTP
additional information
additional information
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0058 - 0.054
2',3'-dialdehyde adenosine 5'-triphosphate
1.5
2'-deoxy-GTP
Escherichia coli
-
no guanosine
8.5
6-thioguanosine 5'-triphosphate
Escherichia coli
-
no guanosine, kact: 8.5/sec, KA: 0.035 mM, Ki: 0.27 mM
6.26 - 12.8
ATP
1.03 - 6.1
Gln
1.41
Gln-NH2
Escherichia coli
-
37C, pH 8.0, wild-type enzyme
0.063 - 0.453
Gln-OH
0.233 - 8.1
glutamine
8.2 - 10.6
GTP
4
guanosine 5'-tetraphosphate
Escherichia coli
-
no guanosine
5.2
ITP
Escherichia coli
-
no guanosine
14 - 14.1
NH2OH
0.031 - 12.2
NH3
1.8 - 10.1
NH4+
2.8
O-methylguanosine 5'-triphosphate
Escherichia coli
-
no guanosine
0.08 - 14
UTP
additional information
additional information
Escherichia coli
-
-
-
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.33
1-beta-D-ribofuranosyl-2-thiouracil 5'-triphosphate
-
pH 7.2, inhibition of glutamine reaction
3.36 - 3.7
2',3'-dialdehyde adenosine 5'-triphosphate
0.36
2'-deoxy-GTP
-
-
0.08
2-Thiocytidine 5'-triphosphate
-
pH 7.2, inhibition of glutamine reaction and ammonia reaction
0.1 - 0.25
2-thiouridine 5'-triphosphate
0.18 - 0.53
5-bromoUTP
0.27
6-thioguanosine 5'-triphosphate
-
-
0.0023
acivicin
-
-
1.1
adenylyliminodiphosphate
-
pH 8.6
0.08 - 0.09
CTP
5.6
D,L-2-amino-4-phosphonobutyrate
-
pH 8.0, reaction with ammonia
0.182
Gln
-
30C, pH 8.0, inhibition of hydroxylamine-dependent N4-OH CTP synthesis in presence of GTP
0.39
glutamate gamma-semialdehyde
-
-
0.12 - 0.366
GTP
0.5
guanosine 5'-tetraphosphate
-
-
4.5
ITP
-
-
12.6
L-2-pyrrolidone-5-carboxylate
-
pH 8.0, reaction with ammonia
0.29
O-methylguanosine 5'-triphosphate
-
-
11
pyrrole-2-carboxylate
-
pH 8.0, reaction with ammonia
0.027
S-nitroso-L-cysteine
-
Kinact: 0.48/min
0.51
s4UTP
-
pH 7.2, inhibition of ammonia reaction
additional information
additional information
IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.079 - 0.121
1,3,7,9-tetramethyluric
0.067 - 0.07
1,3,7-trimethyluric acid
0.072 - 0.088
1,3-dimethyluric acid
0.113 - 0.119
1,7-dimethyluric acid
0.096 - 0.101
1-methyluric acid
0.33 - 0.42
2'-deoxy-GTP
0.45
2'-deoxy-guanosine
Escherichia coli
-
IC50 for inhibition of NH3-dependent CTP formation
0.11 - 0.18
2,6-diaminopurine riboside
0.22 - 0.26
2-aminopurine riboside
0.34 - 0.4
3'-deoxy-guanosine
0.063 - 0.09
3,7-dimethyluric acid
0.39 - 0.61
6-Thioguanine
0.23 - 0.4
6-thioguanosine
0.08 - 0.13
8-oxoguanosine
0.11 - 0.15
8-oxoguanosine 5'-triphosphate
0.33 - 0.49
acycloguanosine
0.31 - 0.41
acycloguanosine monophosphate
12.9 - 15.8
adenine
11 - 12
adenosine
0.39 - 0.51
Caffeine
0.04 - 0.32
CTP
0.29 - 0.41
dideoxy-GTP
0.3 - 0.33
GDP
0.23 - 0.33
GMP
0.29 - 0.46
GTP
0.22 - 0.38
guanosine
0.33 - 0.42
guanosine 5'-tetraphosphate
3.5 - 5.2
Inosine
2.9 - 4.1
ITP
0.17 - 0.23
N-methylguanosine
0.15 - 0.25
O-methylguanosine
0.44 - 0.48
paraxanthine
0.42 - 0.58
Theobromine
0.43 - 0.55
theophylline
4.2 - 4.7
Uracil
2.6 - 3.2
uracil-4-acetic acid
0.06 - 0.1
Uric acid
3.1 - 4.6
uridine
0.23 - 0.37
xanthine
0.22 - 0.29
Xanthosine
additional information
additional information
Escherichia coli
-
xanthine and related compounds inhibit CTPS activity with IC50 = 0.16-0.58 mM. The presence of an 8-oxo function enhances the inhibition to IC50 = 0.060-0.121 mM. Raising the pH from 8.0 to 8.5 results in slightly increased inhibition of NH3-dependent CTP formation by the xanthines
-
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.00066
-
crude extract of protein
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
0.06
-
-
0.1119
-
1st Talon eluate
0.2
-
mutant enzyme S354A
0.3
-
mutant enzyme S36A
0.3986
-
TEV-cleaved 2nd Talon flow-through
0.5515
-
concentrated SourceQ flow-through
0.6
-
mutant enzyme S454A
0.66
-
wild-type enzyme
0.83
-
mutant enzyme S330A
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7 - 9
-
broad
7.3
-
assay at
7.5 - 9.3
-
glutamine-dependent activity
8 - 8.5
-
assay at
8.1
assay at
8.6 - 8.9
-
reaction with glutamine
8.6
-
reaction with ammonia
8.7
-
reaction with NH4+, glutamine-dependent reaction activated by Mg2+ or Mn2+
10.3 - 10.4
-
ammonia-dependent activity,maximal activity with NH4+ is at least 20% greater than with glutamine
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7 - 10
-
ammonia-dependent activity rises linearly from pH 7 to pH 10
7.3 - 9.5
-
pH 7.3: about 35% of maximal activity, pH 9.5: about 40% of maximal activity
7.3
-
assay at
7.5 - 9
-
pH 7.5: about 70% of maximal activity, pH 9.0: about 55% of maximal activity, dUTP-dependent activity
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
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
additional information
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
additional information
PDB
SCOP
CATH
ORGANISM
UNIPROT
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv)
Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv)
Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv)
Sulfolobus solfataricus (strain ATCC 35092 / DSM 1617 / JCM 11322 / P2)
Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)
Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)
Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)
Trypanosoma brucei brucei (strain 927/4 GUTat10.1)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
60240
-
mass spectroscopy, processed N-terminal Met1
60380
-
mass spectroscopy, unprocessed N-terminal Met1
105000
-
dimer, gel filtration
118000
-
gel filtration
122000
-
sucrose density gradient centrifugation
128000
-
monomer, gel filtration
138000
-
sucrose density gradient centrifugation in absence of nucleotides
210000
234000
-
gel filtration
263000
-
dimer, gel filtration
280000
-
tetramer, gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
tetramer
additional information
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
phosphoprotein
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
2.3 A resolution crystal structure of apoenzyme using Hg-multiwavelength anomalous dispersion phasing, vapor diffusion method, crystals belong to space group P2(1)2(1)2(1), in which each bifunctional monomer contains a dethiobiotin synthetase-like amidoligase N-terminal domain and a type 1 glutamine amidotransferase C-terminal domain
-
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 structurefunction relationship of CTPS-family members; 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
-
dimeric form of CTP synthase at 2.5 A resolution. A comparison of the dimeric interface with the intermolecular interfaces in the tetrameric structures of Thermus thermophilus CTP synthase and Escherichia coli CTP synthase shows that the dimeric interfaces are almost identical in the three systems. Residues that are involved in the tetramerization of Sulfolobus solfataricus CTP synthase all have large thermal parameters in the dimeric form and undergo substantial movement upon tetramerization
hanging-drop vapour-diffusion technique
-
crystal structures of Thermus thermophilus HB8 CTP synthetase as a tetramer in its native form, CTP synthetase as complex with 3 SO42- and CTP synthetase as a complex with glutamine. Crystallization at 20 C using vapor-diffusion method. The space group is I222 with cell dimensions of a = 90.4 A, b = 117.1 A and c = 142.3 A
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
10.3 - 10.4
-
rapid denaturation at
648981
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
-
reversible cold lability
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
2-mercaptoethanol is required for optimal stabilization of enzyme activity
-
the enzyme is stable in absence of ATP and UTP
-
the tetramer is very stable even at dilute enzyme concentrations
-
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20C, 30% glycerol, 100 mM 2-mercaptoethanol, 40% loss of activity after 4 weeks
-
-20C, purified enzyme is stable for months
-
-20C, stable for several months
-
-80C, 30% glycerol, 100 mM 2-mercaptoethanol, 65% of the original activity is retained after 4 weeks
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-80C, stable for at least 6 months
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4C, 30% glycerol, 100 mM 2-mercaptoethanol, 65% loss of activity after 4 weeks
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4C, enzyme concentration 1-4 mg/ml, 20 mM sodium phosphate buffer, pH 7.2, 2 mM L-glutamine, 1 mM EDTA, 70 mM 2-mercaptoethanol, 20% glycerol, stable for 3 months
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
by using talon metal affinity resin, a glass Econo-Column and a Sephadex G-50 column. Proteolysis is performed at 23C for 40 min. The protein solution is then loaded again on a Talon resin column but this time the flow-through, containing CTPS without His tag, is collected. Finally the protein is loaded onto a 0.2-ml SourceQ15 medium column.
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enzyme is purified by gel filtration on a HiLoad 26/60 Superdex 200 column. Active fractions are pooled and applied to a Mono Q HR 5/5 column. Final purifications step is performed on a HiLoad 26/60 Superdex 75 column. The isolated EcCTPS is found >90% pure as judged by SDS-PAGE and ESI-MS.
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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; 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
partial
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protein is purified by using HisTrap HP and Superdex 75 columns
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recombinant wild-type and mutant enzyme L109A
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the soluble histidine-tagged CTPS is purified using metal ion affinity chromatography and the histidine tag is subsequently removed using thrombin-catalyzed cleavage
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wild-type and mutant enzymes S63A, S330A, S254A and S454A
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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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cloned in Escherichia coli as a histidine-tagged fusion protein
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cloned in Escherichia coli BL-21 as an N-terminal 6xHis-tag fusion protein
expressed in Escherichia coli BL21(DE3) cells
expression in a ura7ura8 double mutant that lacks CTP synthetase activity
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expression in Escherichia coli
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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 the mutant enzyme D149E in Escherichia coli
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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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genes ura7D and ura8D, DNA and amino acid sequence determination and analysis, expression analysis
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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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overexpression as a hexahistidine-tagged form
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protein is expressed as a His6-tagged wild type human CTP synthetase 1 fusion protein in Escherichia coli; protein is expressed as a His6-tagged wild type human CTP synthetase 1 fusion protein in Saccharomyces cerevisiae
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synthetase-His6-TEV protease site-tagged Trypanosoma brucei CTP synthetase is expressed in BL21(DE3) pLysS bacteria
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wild-type and L109A recombinant enzyme are expressed in Escherichia coli BL21(DE3)
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C379A
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mutant enzyme is fully active with ammonia but has no glutamine-dependent activity, no inhibition by glutamate gamma-semialdehyde
D107A
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enzyme exhibits wild-type NH3-dependent activity and affinity for glutamine, but impaired glutamine-dependent CTP formation, affinity of the mutant enzyme for GTP is reduced 2-4fold
E103A
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mutant enzyme exhibits no glutamine-dependent activity and is only partially active with NH3
G110A
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affinity of the mutant enzyme for GTP is reduced 2-4fold, enzyme exhibits wild-type NH3-dependent activity and affinity for glutamine, but impaired glutamine-dependent CTP formation
G142A
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site-directed mutagenesis, inactive mutant with both ammonia and glutamine
G143A
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site-directed mutagenesis, kcat/Km for ammonia-dependent and glutamine-dependent CTP formation by mutant G143A are reduced by 22fold and 16fold, respectively, compared to the wild-type enzyme. The mutant is able to form active tetramers in the presence of ATP and UTP
G146A
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site-directed mutagenesis, kcat/Km for ammonia-dependent and glutamine-dependent CTP formation by mutant G143A are reduced by 1.4fold and 1.8fold, respectively, compared to the wild-type enzyme. The mutant is able to form active tetramers in the presence of ATP and UTP
G351A
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mutation increases lability of the enzyme, mutant enzyme is not overproduced because of apparent instability and proteolytic degradation
G352C
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mutation increases lability of the enzyme, mutation abolishes the capacity to form the covalent glutaminyl-cysteine379 catalytic intermediate, thus preventing glutamine amide transfer function, mutant enzyme is not overproduced because of apparent instability and proteolytic degradation
G352P
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mutation increases lability of the enzyme, mutation abolishes the capacity to form the covalent glutaminyl-cysteine379 catalytic intermediate, thus preventing glutamine amide transfer function, mutant enzyme is not overproduced because of apparent instability and proteolytic degradation
H118A
-
mutant enzyme exhibits no glutamine-dependent activity and is only partially active with NH3
K102A
-
mutant enzyme exhibits wild-type activity with respect to NH3 and glutamine
K297A
replacement of lysine 297 by alanine does not affect NH3-dependent CTP formation, relative to wild-type CTPS, but reduces kcat for the glutaminase activity 78fold
K306A
replacement of lysine 306 by alanine reduces the rate of 2',3'-dialdehyde adenosine 5'-triphosphate-dependent inactivation (Kinact = 0.0058/sec, Ki = 3.7 mM) and reduces the apparent affinity for CTPS for both ATP and UTP by 2fold. The efficiency of K306A-catalyzed glutamine-dependent CTP formation is also reduced 2fold while near wild type activity is observed when NH3 is the substrate. These findings suggest that Lys 206 is not essential for ATP binding, but does play a role in bringing about the conformational changes that mediate interactions between ATP and UTP sites, and between the ATP-binding site and the glutamine amide transfer domain
R104A
-
mutant enzyme exhibits no glutamine-dependent activity and is only partially active with NH3
R105A
-
enzyme exhibits wild-type NH3-dependent activity and affinity for glutamine, but impaired glutamine-dependent CTP formation
V349S
-
mutation increases lability of the enzyme
E579A
HEK 293 cells: no effect on phosphorylation of CTPS1 by glycogen synthase kinase 3
S462A/T455A
-
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
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
T455A
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T455A mutation causes a 78% decrease in protein kinase A phosphorylation
E362Q
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turnover number for NH4Cl-dependent GTP synthesis reaction is 1.6fold higher than wild-type value
G360A
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5fold increase in GTP-dependent activation of uncoupled glutamine hydrolysis compared to wild-type enzyme. Turnover number for NH4Cl-dependent GTP synthesis reaction is 1.2fold higher than wild-type value
G360P
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mutant enzyme shows no GTP activation of the uncoupled glutaminase reaction, about 4fold lower turnover number for NH4Cl-dependent CTP synthesis reaction than wild-type enzyme
R359M
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mutant enzyme shows no GTP activation of the uncoupled glutaminase reaction. Turnover number for NH4Cl-dependent GTP synthesis reaction is 1.1fold higher than wild-type value
R359P
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mutant enzyme shows no GTP activation of the uncoupled glutaminase reaction. Turnover number for NH4Cl-dependent GTP synthesis reaction is 1.2fold lower than wild-type value
E161K
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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
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
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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
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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
S454A
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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
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
analysis
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fast assay allows the processing of a large number of samples
drug development
medicine
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the enzyme is an target for treatment of African sleeping sickness because the trypanosomes, unlike mammalian cells, cannot compensate for the inhibition of CTP synthetase by the salvage of cytidine. The CTP synthetase inhibitors 6-diazo-5-oxo-L-norleucine and alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid reduce the parasite CTP level even further and inhibit trypanosome proliferation in vitro and in Trypanosoma brucei-infected mice
Show AA Sequence (12709 entries)
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