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Information on EC 6.3.4.2 - CTP synthase (glutamine hydrolysing) and Organism(s) Escherichia coli and UniProt Accession P0A7E5
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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: Escherichia coli
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
cytidine triphosphate synthetase
-
-
-
-
synthetase, cytidine triphosphate
-
-
-
-
uridine triphosphate aminase
-
-
-
-
UTP-ammonia ligase
-
-
-
-
CTPS
-
-
-
-
cytidine 5'-triphosphate synthetase
-
-
-
-
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
ATP + UTP + L-glutamine = ADP + phosphate + CTP + L-glutamate
mechanism proceeds by formation of a phosphorylated pyrimidinone
-
ATP + UTP + L-glutamine = ADP + phosphate + CTP + L-glutamate
mechanism, the initial chemical step catalyzed by CTP synthetase is the phosphorylation of UTP by ATP to form an enzyme-bound intermediate
-
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 + gamma-L-glutamylhydroxamate
ADP + phosphate + N4-hydroxy-CTP
-
-
-
?
ATP + UTP + L-gamma-glutamyl hydrazide
ADP + phosphate + N4-amino-CTP + L-glutamate
-
-
-
?
ATP + UTP + L-glutamine
ADP + phosphate + CTP + L-glutamate
-
-
-
?
ATP + UTP + L-glutamine
ADP + phosphate + CTP + L-glutamate
CTPS catalyses the ATP-dependent formation of CTP from UTP using either ammonia or L-glutamine as the nitrogen source
-
-
?
ATP + UTP + NH3
ADP + phosphate + CTP
CTPS catalyses the ATP-dependent formation of CTP from UTP using either ammonia or L-glutamine as the nitrogen source
-
-
?
ATP + UTP + glutamine
ADP + phosphate + CTP + Glu
-
maximal activity with NH4+ is at least 20% greater than with glutamine
-
-
?
UTP + ATP + NH3
CTP + ADP + phosphate
-
the enzyme is regulated in a complex fashion, overview
-
-
?
UTP + ATP + NH3
CTP + ADP + phosphate
-
NH3 from glutamine deamination or exogenous
-
-
?
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
?
-
UTP- and CTP-binding structures, overview
-
-
?
additional information
?
-
-
UTP- and CTP-binding structures, overview
-
-
?
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
UTP + ATP + NH3
CTP + ADP + phosphate
-
the enzyme is regulated in a complex fashion, overview
-
-
?
ATP + UTP + L-glutamine
ADP + phosphate + CTP + L-glutamate
-
-
-
?
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
-
-
?
COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Mg2+
Mg2+
Mg2+
-
the enzyme requires more Mg2+ for full catalytic activity than required simply to complex the nucleotide substrate. Half-saturation value for Mg2+ is 2.6 mM for the reaction with NH4+ and 2.4 mM for the glutamine-dependent reaction
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
6-thio-GTP
the GTP analogue is capable of inhibiting Gln-dependent CTP formation at over 0.15 mM
6-thioguanosine 5'-triphosphate
no guanosine, kact: 8.5/sec, KA: 0.035 mM, Ki: 0.27 mM
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
S-nitroso-L-cysteine
specific irreversible inhibitor,inhibits the activity by 94%
S-nitroso-L-homocysteine
specific irreversible inhibitor, inhibits the activity by 90%
2',3'-dialdehyde adenosine 5'-triphosphate
irreversible inhibitor of CTPS
Cu2+
-
inhibition is not reversed by EDTA, in presence of dithiothreitol inhibition at concentrations below 0.2 mM
glutamate gamma-semialdehyde
-
potent linear mixed-type inhibitor, competitive with respect to ammonia, no inhibition of the mutant enzyme C379A
L-2-pyrrolidone 5-carboxylate
-
weak competitive inhibition of the reaction with ammonia as substrate, no significant inhibition with glutamine as substrate
Ni2+
-
in presence of dithiothreitol inhibition at concentrations below 0.2 mM
pyrrole-2-carboxylate
-
weak competitive inhibition of the reaction with ammonia as substrate, no significant inhibition with glutamine as substrate
Zn2+
-
inhibition is reversed by EDTA, in presence of dithiothreitol inhibition at concentrations below 0.2 mM
2'-deoxy-GTP
the GTP analogue is capable of inhibiting Gln-dependent CTP formation at over 0.15 mM
GTP
allosteric effector, structural requirements for activation are stringent, but requirements for inhibition are lax. GTP promotes Gln hydrolysis but inhibits Gln-dependent CTP formation at concentrations of over 0.15 mM
guanosine 5'-tetraphosphate
no guanosine, kact: 4/sec, KA: 0.19 mM, Ki: 0.5 mM
guanosine 5'-tetraphosphate
the GTP analogue is capable of inhibiting Gln-dependent CTP formation at over 0.15 mM
ITP
the GTP analogue is capable of inhibiting Gln-dependent CTP formation at over 0.15 mM
GTP
-
inhibition of glutamine-dependent CTP formation above 0.15 mM, inhibition of glutamine-dependent CTP formation in a concentration-dependent manner
GTP
-
GTP acts a positive allosteric effector for Gln-dependent CTP formation. However, at concentrations exceeding 0.15 mM, GTP inhibits Gln-dependent CTP formation. Moreover, GTP is an inhibitor of NH3-dependent CTP formation at all concentrations
additional information
incubation of EcCTPS modified by CysNO and HcyNO with 5 mM DTT for 30 min at 37°C reveals that 88% and 97%, respectively, of the original activity can be recovered
-
additional information
it is shown that in the presence of 1 mM Gln, S-nitroso-L-cysteine reduces the enzymatic activity by 88% and by 32% in the presence of 10 mM Gln. Similar studies with S-nitroso-L-homocysteine result in reduction of the activity by 43% and 19%, respectively. The results suggest that the substrate Gln competitively protects the active site of EcCTPS from the modification with S-nitroso-L-cysteine and S-nitroso-L-homocysteine.
-
additional information
no inhibition by S-nitrosoglutathione presumably due to its inability to enter the actve site of the enzyme
-
additional information
GTP analogues inhibite NH3-and Gln-dependent CTP-formation, often in a cooperative manner, to a similar extent as they activate it with IC50 values of 0.2-0.5 mM, the inhibition appears to be due solely to the purine base, binding structures and kinetics, overview. Inhibitor structure-activity study, overview
-
additional information
-
GTP analogues inhibite NH3-and Gln-dependent CTP-formation, often in a cooperative manner, to a similar extent as they activate it with IC50 values of 0.2-0.5 mM, the inhibition appears to be due solely to the purine base, binding structures and kinetics, overview. Inhibitor structure-activity study, overview
-
additional information
-
enzyme loses activity at ionic strengths higher than 0.4 M
-
additional information
-
inhibition by xanthine and derivatives, no inhibition by allantoin, an intact purine ring with anionic character favors inhibition. In general, methylation of the purine does not significantly affect inhibition
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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
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
2'-deoxy-GTP
the GTP analogue is capable of activating Gln-dependent CTP formation
GTP
allosteric effector, structural requirements for activation are stringent, but requirements for inhibition are lax. GTP promotes Gln hydrolysis but inhibits Gln-dependent CTP formation at concentrations of over 0.15 mM
guanosine 5'-tetraphosphate
0 mM guanosine, kact: 4/sec, KA: 0.19 mM, Ki: 0.5 mM
guanosine 5'-tetraphosphate
the GTP analogue is capable of activating Gln-dependent CTP formation
ITP
the GTP analogue is capable of activating Gln-dependent CTP formation
GTP
-
required as allosteric effector to promote reaction with glutamine as substrate, GTP functions by stabilizing the protein conformation that binds the tetrahedral intermediate(s) formed during glutamine hydrolysis
GTP
-
GTP acts a positive allosteric effector for Gln-dependent CTP formation. However, at concentrations exceeding 0.15 mM, GTP inhibits Gln-dependent CTP formation. Moreover, GTP is an inhibitor of NH3-dependent CTP formation at all concentrations
additional information
activation potency in descending order: GTP = 6-thio-GTP, ITP = guanosine 5'-tetraphosphate, O6-methyl-GTP, 2'-deoxy-GTP, no activation with guanosine, GMP, GDP, 2',3'-dideoxy-GTP, acycloguanosine, and acycloguanosine monophosphate, indicating that the 5'-triphosphate, 2'-OH, and 3'-OH are required for full activation, binding structures and kinetics, overview
-
additional information
-
activation potency in descending order: GTP = 6-thio-GTP, ITP = guanosine 5'-tetraphosphate, O6-methyl-GTP, 2'-deoxy-GTP, no activation with guanosine, GMP, GDP, 2',3'-dideoxy-GTP, acycloguanosine, and acycloguanosine monophosphate, indicating that the 5'-triphosphate, 2'-OH, and 3'-OH are required for full activation, binding structures and kinetics, overview
-
additional information
binding of the substrates ATP and UTP, or the product CTP, promotes oligomerization of CTPS from inactive dimers to active tetramers, Gly142 is critical for nucleotide-dependent oligomerization of CTPS to active tetramers
-
additional information
-
binding of the substrates ATP and UTP, or the product CTP, promotes oligomerization of CTPS from inactive dimers to active tetramers, Gly142 is critical for nucleotide-dependent oligomerization of CTPS to active tetramers
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.206
glutamine
wild-type protein, presence of 3 mM ATP, 2 mM UTP
0.345
glutamine
wild-type protein, presence of 1 mM ATP, 1 mM UTP
additional information
additional information
kinetic mechanism, activation and inhibition kinetics
-
additional information
additional information
-
kinetic mechanism, activation and inhibition kinetics
-
additional information
additional information
kinetics of wild-type and mutants
-
additional information
additional information
-
kinetics of wild-type and mutants
-
additional information
additional information
-
positive cooperativity for ATP and UTP
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
8.5
6-thioguanosine 5'-triphosphate
no guanosine, kact: 8.5/sec, KA: 0.035 mM, Ki: 0.27 mM
0.08
UTP
pH 8.0, 37°C, mutant G142A with L-glutamine, in presence of 0.25 mM GTP
0.67
UTP
pH 8.0, 37°C, mutant G143A with L-glutamine, in presence of 0.25 mM GTP
4.2
UTP
pH 8.0, 37°C, mutant G146A with L-glutamine, in presence of 0.25 mM GTP
5
UTP
pH 8.0, 37°C, wild-type enzyme with L-glutamine, in presence of 0.25 mM GTP
0.0058
2',3'-dialdehyde adenosine 5'-triphosphate
K306A: replacement of lysine 306 by alanine reduces the rate of 2',3'-dialdehyde adenosine 5'-triphosphate-dependent inactivation
0.054
2',3'-dialdehyde adenosine 5'-triphosphate
in the presence of 10 mM UTP
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.33
1-beta-D-ribofuranosyl-2-thiouracil 5'-triphosphate
-
pH 7.2, inhibition of glutamine reaction
0.08
2-Thiocytidine 5'-triphosphate
-
pH 7.2, inhibition of glutamine reaction and ammonia reaction
3.36
2',3'-dialdehyde adenosine 5'-triphosphate
in the presence of 10 mM UTP
3.7
2',3'-dialdehyde adenosine 5'-triphosphate
K306A: replacement of lysine 306 by alanine reduces the rate of 2',3'-dialdehyde adenosine 5'-triphosphate-dependent inactivation
0.1
2-thiouridine 5'-triphosphate
-
pH 7.2, inhibition of glutamine reaction
0.25
2-thiouridine 5'-triphosphate
-
pH 7.2, inhibition of ammonia reaction
additional information
additional information
no saturation kinetic is obtained with S-nitroso-L-homocysteine
-
additional information
additional information
nucleotide and nucleoside analogues of GTP and guanosine, respectively, all inhibit NH3- and Gln-dependent CTP formation to a similar extent (IC50 : 0.20.5 mM). This inhibition appears to be due solely to the purine base and is relatively insensitive to the identity of the purine with the exception of inosine, ITP, and adenosine (IC50 : 412 mM). 8-Oxoguanosine is the best inhibitor identified (IC50 : 0.08 mM).
-
additional information
additional information
-
nucleotide and nucleoside analogues of GTP and guanosine, respectively, all inhibit NH3- and Gln-dependent CTP formation to a similar extent (IC50 : 0.20.5 mM). This inhibition appears to be due solely to the purine base and is relatively insensitive to the identity of the purine with the exception of inosine, ITP, and adenosine (IC50 : 412 mM). 8-Oxoguanosine is the best inhibitor identified (IC50 : 0.08 mM).
-
additional information
additional information
kinetic mechanism, activation and inhibition kinetics
-
additional information
additional information
-
kinetic mechanism, activation and inhibition kinetics
-
additional information
additional information
-
inhibition kinetics. Multisite inhibition of CTPS-catalyzed NH3-dependent CTP formation by caffeine
-
IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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
-
0.33
2'-deoxy-guanosine
Escherichia coli
IC50 for inhibition of Gln-dependent CTP formation
0.45
2'-deoxy-guanosine
Escherichia coli
IC50 for inhibition of NH3-dependent CTP formation
0.11
2,6-diaminopurine riboside
Escherichia coli
IC50 for inhibition of Gln-dependent CTP formation
0.18
2,6-diaminopurine riboside
Escherichia coli
IC50 for inhibition of NH3-dependent CTP formation
0.22
2-aminopurine riboside
Escherichia coli
IC50 for inhibition of Gln-dependent CTP formation
0.26
2-aminopurine riboside
Escherichia coli
IC50 for inhibition of NH3-dependent CTP formation
0.34
3'-deoxy-guanosine
Escherichia coli
IC50 for inhibition of Gln-dependent CTP formation
0.4
3'-deoxy-guanosine
Escherichia coli
IC50 for inhibition of NH3-dependent CTP formation
0.39
6-thioguanine
Escherichia coli
IC50 for inhibition of Gln-dependent CTP formation
0.61
6-thioguanine
Escherichia coli
IC50 for inhibition of NH3-dependent CTP formation
0.23
6-thioguanosine
Escherichia coli
IC50 for inhibition of Gln-dependent CTP formation
0.4
6-thioguanosine
Escherichia coli
IC50 for inhibition of NH3-dependent CTP formation
0.08
8-oxoguanosine
Escherichia coli
IC50 for inhibition of Gln-dependent CTP formation
0.13
8-oxoguanosine
Escherichia coli
IC50 for inhibition of NH3-dependent CTP formation
0.11
8-oxoguanosine 5'-triphosphate
Escherichia coli
IC50 for inhibition of Gln-dependent CTP formation
0.15
8-oxoguanosine 5'-triphosphate
Escherichia coli
IC50 for inhibition of NH3-dependent CTP formation
0.33
acycloguanosine
Escherichia coli
IC50 for inhibition of Gln-dependent CTP formation
0.45
acycloguanosine
Escherichia coli
8.16 mM NH3, IC50 for inhibition of NH3-dependent CTP formation
0.45
acycloguanosine
Escherichia coli
IC50 for inhibition of NH3-dependent CTP formation
0.47
acycloguanosine
Escherichia coli
5.44 mM NH3, IC50 for inhibition of NH3-dependent CTP formation
0.48
acycloguanosine
Escherichia coli
2.72 mM NH3, IC50 for inhibition of NH3-dependent CTP formation
0.49
acycloguanosine
Escherichia coli
1.36 mM NH3, IC50 for inhibition of NH3-dependent CTP formation
0.31
acycloguanosine monophosphate
Escherichia coli
IC50 for inhibition of Gln-dependent CTP formation
0.41
acycloguanosine monophosphate
Escherichia coli
IC50 for inhibition of NH3-dependent CTP formation
0.29
GDP
Escherichia coli
2.72 mM NH3, IC50 for inhibition of NH3-dependent CTP formation
0.29
GDP
Escherichia coli
5.44 mM NH3, IC50 for inhibition of NH3-dependent CTP formation
0.3
GDP
Escherichia coli
1.36 mM NH3, IC50 for inhibition of NH3-dependent CTP formation
0.23
GMP
Escherichia coli
5.44 mM NH3, IC50 for inhibition of NH3-dependent CTP formation
0.25
GMP
Escherichia coli
2.72 mM NH3, IC50 for inhibition of NH3-dependent CTP formation
0.28
GMP
Escherichia coli
1.36 mM NH3, IC50 for inhibition of NH3-dependent CTP formation
0.29
GMP
Escherichia coli
8.16 mM NH3, IC50 for inhibition of NH3-dependent CTP formation
0.29
GTP
Escherichia coli
5.44 mM NH3, IC50 for inhibition of NH3-dependent CTP formation
0.3
GTP
Escherichia coli
2.72 mM NH3, IC50 for inhibition of NH3-dependent CTP formation
0.3
GTP
Escherichia coli
8.16 mM NH3, IC50 for inhibition of NH3-dependent CTP formation
0.31
GTP
Escherichia coli
1.36 mM NH3, IC50 for inhibition of NH3-dependent CTP formation
0.22
guanosine
Escherichia coli
5.44 mM NH3, IC50 for inhibition of NH3-dependent CTP formation
0.26
guanosine
Escherichia coli
1.36 mM NH3, IC50 for inhibition of NH3-dependent CTP formation
0.26
guanosine
Escherichia coli
2.72 mM NH3, IC50 for inhibition of NH3-dependent CTP formation
0.29
guanosine
Escherichia coli
8.16 mM NH3, IC50 for inhibition of NH3-dependent CTP formation
0.3
guanosine
Escherichia coli
0.30 mM Gln, IC50 for inhibition of Gln-dependent CTP formation
0.32
guanosine
Escherichia coli
10 mM Gln, IC50 for inhibition of Gln-dependent CTP formation
0.32
guanosine
Escherichia coli
2.5 mM Gln, IC50 for inhibition of Gln-dependent CTP formation
0.35
guanosine
Escherichia coli
0.75 mM Gln, IC50 for inhibition of Gln-dependent CTP formation
0.33
guanosine 5'-tetraphosphate
Escherichia coli
IC50 for inhibition of Gln-dependent CTP formation
0.42
guanosine 5'-tetraphosphate
Escherichia coli
IC50 for inhibition of NH3-dependent CTP formation
2.9
ITP
Escherichia coli
8.16 mM NH3, IC50 for inhibition of NH3-dependent CTP formation
3
ITP
Escherichia coli
2.72 mM NH3, IC50 for inhibition of NH3-dependent CTP formation
3.1
ITP
Escherichia coli
5.44 mM NH3, IC50 for inhibition of NH3-dependent CTP formation
3.3
ITP
Escherichia coli
1.36 mM NH3, IC50 for inhibition of NH3-dependent CTP formation
0.17
N-methylguanosine
Escherichia coli
IC50 for inhibition of Gln-dependent CTP formation
0.23
N-methylguanosine
Escherichia coli
IC50 for inhibition of NH3-dependent CTP formation
0.15
O-methylguanosine
Escherichia coli
0.30 mM Gln, IC50 for inhibition of Gln-dependent CTP formation
0.16
O-methylguanosine
Escherichia coli
10 mM Gln, IC50 for inhibition of Gln-dependent CTP formation
0.16
O-methylguanosine
Escherichia coli
2.5 mM Gln, IC50 for inhibition of Gln-dependent CTP formation
0.16
O-methylguanosine
Escherichia coli
IC50 for inhibition of Gln-dependent CTP formation
0.17
O-methylguanosine
Escherichia coli
0.75 mM Gln, IC50 for inhibition of Gln-dependent CTP formation
0.25
O-methylguanosine
Escherichia coli
IC50 for inhibition of NH3-dependent CTP formation
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
additional information
a structure-activity study using a variety of GTP and guanosine analogues reveals that only a few GTP analogues are capable of activating Gln-dependent CTP formation to varying degrees: GTP > 6-thio-GTP > ITP> guanosine 5'-tetraphosphate > O-methyl-GTP > 2'-deoxy-GTP. No activation is observed with guanosine, GMP, GDP, 2',3'-dideoxy-GTP, acycloguanosine, and acycloguanosine monophosphate indicating that the 5'-triphosphate, 2'-OH, and 3'-OH are required for full activation. The 2-NH2 group is important in binding recognition while substituents at the 6-position are important in activation.
additional information
-
a structure-activity study using a variety of GTP and guanosine analogues reveals that only a few GTP analogues are capable of activating Gln-dependent CTP formation to varying degrees: GTP > 6-thio-GTP > ITP> guanosine 5'-tetraphosphate > O-methyl-GTP > 2'-deoxy-GTP. No activation is observed with guanosine, GMP, GDP, 2',3'-dideoxy-GTP, acycloguanosine, and acycloguanosine monophosphate indicating that the 5'-triphosphate, 2'-OH, and 3'-OH are required for full activation. The 2-NH2 group is important in binding recognition while substituents at the 6-position are important in activation.
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
10.3 - 10.4
-
ammonia-dependent activity,maximal activity with NH4+ is at least 20% greater than with glutamine
8.7
-
reaction with NH4+, glutamine-dependent reaction activated by Mg2+ or Mn2+
pH RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
37
pI VALUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
physiological function
-
central role of CTP in the biosynthesis of nucleic acids, phospholipids, and sialic acid
PDB
SCOP
CATH
UNIPROT
ORGANISM
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
52000
-
4 * 52000, enzyme exists as tetramer in presence of UTP, Mg2+ and ATP
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
dimer
-
the enzyme is a dimer of 108000 Da, the dimer associates to form a tetramer in the presence of either ATP or UTP
tetramer
additional information
tetramer
-
4 * 52000, enzyme exists as tetramer in presence of UTP, Mg2+ and ATP
additional information
binding of the substrates ATP and UTP, or the product CTP, promotes oligomerization of CTPS from inactive dimers to active tetramers, Gly142 is critical for nucleotide-dependent oligomerization of CTPS to active tetramers. Oligomerization of Gly mutant enzymes, overview
additional information
-
binding of the substrates ATP and UTP, or the product CTP, promotes oligomerization of CTPS from inactive dimers to active tetramers, Gly142 is critical for nucleotide-dependent oligomerization of CTPS to active tetramers. Oligomerization of Gly mutant enzymes, overview
additional information
-
the enzyme can dissociate to an apparently inactive monomer. The dissociation is reversible and the rate of association is slow
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
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
G142A
site-directed mutagenesis, inactive mutant with both ammonia and glutamine
G143A
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
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
L109A
uncoupling of the hydrolysis of gamma-glutamyl hydroxamate and nascent NH2OH production from N4-hydroxy-CTP formation is more pronounced with mutant than with wild-type enzyme
C379A
-
mutant enzyme is fully active with ammonia but has no glutamine-dependent activity, no inhibition by glutamate gamma-semialdehyde
D107A
-
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
-
mutant enzyme exhibits no glutamine-dependent activity and is only partially active with NH3
G110A
-
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
G351A
-
mutation increases lability of the enzyme, mutant enzyme is not overproduced because of apparent instability and proteolytic degradation
G352C
-
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
-
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
L109A
-
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
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
additional information
additional information
it can be suggested that the conformational change associated with binding ATP may be transmitted through the L10-alpha11 structural unit (residues 297-312) and thereby mediate effects on the glutaminase activity of CTPS
additional information
-
it can be suggested that the conformational change associated with binding ATP may be transmitted through the L10-alpha11 structural unit (residues 297-312) and thereby mediate effects on the glutaminase activity of CTPS
additional information
-
Escherichia coli CtpS can replace the enzymatic and morphogenic functions of Caulobacter crescentus CtpS
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
4°C, 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
-
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
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.
the soluble histidine-tagged CTPS is purified using metal ion affinity chromatography and the histidine tag is subsequently removed using thrombin-catalyzed cleavage
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
wild-type and L109A recombinant enzyme are expressed in Escherichia coli BL21(DE3)
cloned in Escherichia coli BL-21 as an N-terminal 6xHis-tag fusion protein
APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
drug development
modifying 2-aminopurine or 2-aminopurine riboside may serve as an effective strategy for developing CTPS inhibitors
drug development
-
CTP is a recognized target for the development of anticancer, antiviral, and antiprotozoal agents
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Iyengar, A.; Bearne, S.L.
Aspartate-107 and leucine-109 facilitate efficient coupling of glutamine hydrolysis to CTP synthesis by Escherichia coli CTP synthase
Biochem. J.
369
497-507
2003
Escherichia coli
Bearne, S.L.; Hekmat, O.; Macdonnell, J.E.
Inhibition of Escherichia coli CTP synthase by glutamate gamma-semialdehyde and the role of the allosteric effector GTP in glutamine hydrolysis
Biochem. J.
356
223-232
2001
Escherichia coli
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)
Weng, M.; Zalkin, H.
Structural role for a conserved region in the CTP synthetase glutamine amide transfer domain
J. Bacteriol.
169
3023-3028
1987
Escherichia coli
Zalkin, H.
CTP synthetase
Methods Enzymol.
113
282-287
1985
Escherichia coli, Escherichia coli B / ATCC 11303
von der Saal, W.; Villafranca, J.J.; Anderson, P.M.
Cytidine-5'-triphosphate synthetase catalyzes the phosphorylation of uridine 5'-triphosphate by adenosine 5'-triphosphate
J. Am. Chem. Soc.
107
703-704
1985
Chlamydia trachomatis, Escherichia coli
-
Anderson, P.M.
CTP synthetase from Escherichia coli: an improved purification procedure and characterization of hysteretic and enzyme concentration effects on kinetic properties
Biochemistry
22
3285-3292
1983
Escherichia coli
Scheit, K.H.; Linke, H.J.
Substrate specificity of CTP synthetase from Escherichia coli
Eur. J. Biochem.
126
57-60
1982
Escherichia coli
Long, C.; Koshland jr., D.E.
Cytidine triphosphate synthetase
Methods Enzymol.
51
79-83
1978
Escherichia coli
Koshland jr., D.E.; Levitzki, A.
CTP synthetase and related enzymes
The Enzymes, 3rd Ed. (Boyer, P. D. , ed. )
10
539-559
1974
Escherichia coli
-
Scheit, K.H.; Linke, H.J.
Substrate specificity of CTP synthetase from E. coli
Nucleic Acids Res.
9
229-233
1981
Escherichia coli
Lewis, D.A.; Villafranca, J.J.
Investigation of the mechanism of CTP synthetase using rapid quench and isotope partitioning methods
Biochemistry
28
8454-8459
1989
Escherichia coli
Wylie, J.L.; Berry, J.D.; McClarty, G.
Chlamydia trachomatis CTP synthetase: molecular characterization and developmental regulation of expression
Mol. Microbiol.
22
631-642
1996
Chlamydia trachomatis, Escherichia coli
Robertson, J.G.; Villafranca, J.J.
Characterization of metal ion activation and inhibition of CTP synthetase
Biochemistry
32
3769-3777
1993
Escherichia coli
Endrizzi, J.A.; Kim, H.; Anderson, P.M.; Baldwin, E.P.
Crystal structure of Escherichia coli cytidine triphosphate synthetase, a nucleotide-regulated glutamine amidotransferase/ATP-dependent amidoligase fusion protein and homologue of anticancer and antiparasitic drug targets
Biochemistry
43
6447-6463
2004
Escherichia coli (P0A7E5), Escherichia coli
MacDonnell, J.E.; Lunn, F.A.; Bearne, S.L.
Inhibition of E. coli CTP synthase by the "positive" allosteric effector GTP
Biochim. Biophys. Acta
1699
213-220
2004
Escherichia coli
Lunn, F.A.; Bearne, S.L.
Alternative substrates for wild-type and L109A E. coli CTP synthases: kinetic evidence for a constricted ammonia tunnel
Eur. J. Biochem.
271
4204-4212
2004
Escherichia coli (P0A7E5), Escherichia coli
MacLeod, T.J.; Lunn, F.A.; Bearne, S.L.
The role of lysine residues 297 and 306 in nucleoside triphosphate regulation of E. coli CTP synthase: inactivation by 2,3-dialdehyde ATP and mutational analyses
Biochim. Biophys. Acta
1764
199-210
2006
Escherichia coli (A0A140N932), Escherichia coli
Lunn, F.A.; Macdonnell, J.E.; Bearne, S.L.
Structural requirements for the activation of Escherichia coli CTP synthase by the allosteric effector GTP are stringent, but requirements for inhibition are Lax
J. Biol. Chem.
283
2010-2020
2007
Escherichia coli (P0A7E5), Escherichia coli
Braun, O.; Knipp, M.; Chesnov, S.; Vasak, M.
Specific reactions of S-nitrosothiols with cysteine hydrolases: A comparative study between dimethylargininase-1 and CTP synthetase
Protein Sci.
16
1522-1534
2007
Escherichia coli (P0A7E5)
Lunn, F.A.; Macleod, T.J.; Bearne, S.L.
Mutational analysis of conserved glycine residues 142, 143 and 146 reveals Gly(142) is critical for tetramerization of CTP synthase from Escherichia coli
Biochem. J.
412
113-121
2008
Escherichia coli (P0A7E5), Escherichia coli
Roy, A.C.; Lunn, F.A.; Bearne, S.L.
Inhibition of CTP synthase from Escherichia coli by xanthines and uric acids
Bioorg. Med. Chem. Lett.
20
141-144
2010
Escherichia coli
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