Information on EC 6.3.4.16 - carbamoyl-phosphate synthase (ammonia)

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

EC NUMBER
COMMENTARY hide
6.3.4.16
-
RECOMMENDED NAME
GeneOntology No.
carbamoyl-phosphate synthase (ammonia)
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
2 ATP + NH3 + hydrogencarbonate = 2 ADP + phosphate + carbamoyl phosphate
show the reaction diagram
ATP + carbamate = ADP + carbamoyl phosphate
show the reaction diagram
(1c)
-
-
-
ATP + hydrogencarbonate = ADP + carboxyphosphate
show the reaction diagram
(1a)
-
-
-
NH3 + carboxyphosphate = carbamate + phosphate
show the reaction diagram
(1b)
-
-
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
amination
-
-
-
-
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Alanine, aspartate and glutamate metabolism
-
-
Arginine biosynthesis
-
-
Metabolic pathways
-
-
Microbial metabolism in diverse environments
-
-
NIL
-
-
Nitrogen metabolism
-
-
urea cycle
SYSTEMATIC NAME
IUBMB Comments
carbon-dioxide:ammonia ligase (ADP-forming, carbamate-phosphorylating)
The enzyme catalyses the first committed step in the urea cycle. The reaction proceeds via three separate chemical reactions: phosphorylation of hydrogencarbonate to carboxyphosphate; a nucleophilic attack of ammonia on carboxyphosphate yielding carbamate; and the phosphorylation of carbamate forming carbamoyl phosphate. Two moles of ATP are utilized for the synthesis of one molecule of carbamyl phosphate, making the reaction essentially irreversible. The enzyme requires the allosteric activator N-acetyl-L-glutamate. cf. EC 6.3.5.5, carbamoyl-phosphate synthase (glutamine-hydrolysing).
CAS REGISTRY NUMBER
COMMENTARY hide
9026-23-7
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
Frog
-
-
-
Manually annotated by BRENDA team
Texas tortoise
-
-
Manually annotated by BRENDA team
-
UniProt
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
DSM 861
UniProt
Manually annotated by BRENDA team
Mus musculus C57BL/6
C57BL/6 mice
-
-
Manually annotated by BRENDA team
Argentine tree frog
-
-
Manually annotated by BRENDA team
LPL26
-
-
Manually annotated by BRENDA team
LPL26
-
-
Manually annotated by BRENDA team
-
TrEMBL
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
metabolism
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
2 ATP + NH3 + CO2 + H2O
2 ADP + phosphate + carbamoyl phosphate
show the reaction diagram
2 ATP + NH3 + CO2 + H2O
?
show the reaction diagram
ATP + carbamate
ADP + carbamoyl phosphate
show the reaction diagram
ATP + L-Gln + HCO3-
ADP + phosphate + L-Glu + carbamoyl phosphate
show the reaction diagram
ATP + NH3 + CO2 + H2O
ADP + phosphate + carbamoyl phosphate
show the reaction diagram
ATP + NH3 + CO2 + H2O
ADP + phosphate + carbamoylphosphate
show the reaction diagram
ATP + NH4+ + CO2 + H2O
ADP + phosphate + carbamoyl phosphate
show the reaction diagram
ATP + NH4+ + CO2 + H2O
ADP + phosphate + carbamoylphosphate
show the reaction diagram
ATP + NH4+ + HCO3-
ADP + phosphate + carbamoyl phosphate
show the reaction diagram
ATP + NH4+ + HCO3- + H2O
ADP + phosphate + carbamoyl 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
2 ATP + NH3 + CO2 + H2O
2 ADP + phosphate + carbamoyl phosphate
show the reaction diagram
2 ATP + NH3 + CO2 + H2O
?
show the reaction diagram
ATP + NH3 + CO2 + H2O
ADP + phosphate + carbamoyl phosphate
show the reaction diagram
ATP + NH3 + CO2 + H2O
ADP + phosphate + carbamoylphosphate
show the reaction diagram
ATP + NH4+ + CO2 + H2O
ADP + phosphate + carbamoyl phosphate
show the reaction diagram
H9ZVS7
-
-
-
?
ATP + NH4+ + HCO3-
ADP + phosphate + carbamoyl phosphate
show the reaction diagram
ATP + NH4+ + HCO3- + H2O
ADP + phosphate + carbamoyl phosphate
show the reaction diagram
additional information
?
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Cs+
-
monovalent cation required, ATP hydrolysis is activated in the order of the series: Tl+, Cs+, Rb+, K+, Na+. Half-maximal activation at 1-2 mM
K+
-
monovalent cation required, ATP hydrolysis is activated in the order of the series: Tl+, Cs+, Rb+, K+, Na+. Half-maximal activation at 1-2 mM
Na+
-
monovalent cation required, ATP hydrolysis is activated in the order of the series: Tl+, Cs+, Rb+, K+, Na+
Rb+
-
monovalent cation required, ATP hydrolysis is activated in the order of the series: Tl+, Cs+, Rb+, K+, Na+. Half-maximal activation at 1-2 mM
Tl+
-
monovalent cation required, ATP hydrolysis is activated in the order of the series: Tl+, Cs+, Rb+, K+, Na+. Half-maximal activation at 0.2-0.6 mM
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2-Amino-4-oxo-5-chloropentanoic acid
-
-
2-oxoglutarate
-
-
ADP
-
strong
alpha,beta-methyleneATP
-
-
Carbamoyl phosphate
-
50% inhibition at 13 mM
cardiolipin
-
irreversible, inclusion of Mg2+ or MgATP2- in the incubation mixture prevent inactivation
citrate
-
-
cytidine triphosphate
-
dCTP
-
1 mM, inhibition is more pronounced at 37C compared to 70C
dGTP
-
1 mM, inhibition is more pronounced at 37C compared to 70C
glycerol
-
in the presence of N-acetyl-L-glutamate the enzyme is inhibited by increasing concentrations of glycerol
HEPES
-
competitive with N-acetylglutamate
malate
-
-
Mg2+
-
above 20 mM
N-carbamoylglutamate
-
binding of acetylglutamate to carbamoylphosphate synthetase is inhibited
P1,P5-di-(adenosine-5')pentaphosphate
-
no inhibition by P1,P3-di-(adenosine-5')-pentaphosphate
palmitoyl-CoA
-
irreversible inactivation in a time- and concentration-dependent manner, this fatty acylation is specific for long-chain fatty acyl-CoA
peroxynitrite
CPS1 activity is decreased by treatment with peroxynitrite in a peroxynitrite concentration- and time-dependent manner due to tyrosine nitration (47% decrease in 1 min and 60% decrease in 10 min with 1 mM peroxynitrite)
succinate
-
-
Tris
-
competitive with N-acetylglutamate
TTP
-
0.3 mM, about 50% inhibition, inhibition is more pronounced at 37C compared to 70C
UTP
-
0.3 mM, about 50% inhibition, inhibition is more pronounced at 37C compared to 70C
XTP
-
0.3 mM, about 50% inhibition, inhibition is more pronounced at 37C compared to 70C
additional information
-
the urea cycle enzyme CPS1 is the antigen for Hep Par 1 antibody
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
acetylglutamate
dimethyl sulfoxide
-
activates in absence of the activator acetylglutamate
ethylene glycol
-
activates in absence of the activator acetylglutamate
glycerol
inosine monophosphate
-
weak activator, the IMP binding site is located at the C-terminus, binding structure analysis from crystal structure with bound IMP, PDB ID 1CE8
L-alanine
-
infusion of alanine at 50% of resting energy expenditure for 36 h increases the enzyme activity more than twofold
N-acetyl-L-beta-phenylglutamate
-
lower activation of CPSI compared to N-acetyl-L-glutamate
N-acetyl-L-glutamate
N-acetylglutamate
N-carbamoylglutamate
-
effective activator, Km: 2.0 mM
N-carbamyl-L-glutamate
-
-
ornithine
-
-
Sucrose
-
activates in absence of the activator acetylglutamate
additional information
-
not: UTP
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.39
ammonia
-
wild-type enzyme, comparison to native and mutant enzyme
0.0372 - 1.92
ATP
2.3 - 7.2
CO2
2.2
CoATP2-
1 - 242
HCO3-
0.054 - 1.35
MgATP2-
1
MnATP2-
0.29 - 0.44
N-acetylglutamate
0.038 - 0.7
NH3
0.35 - 15.6
NH4+
additional information
additional information
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1.8
ammonia
Rana catesbeiana
-
wild-type enzyme, comparison to native and mutant enzyme
0.043 - 2.4
ATP
0.044
HCO3-
Methanobrevibacter smithii
A5UK38
at pH 8.0 and 37C
5.54 - 5.73
NH3
0.052
NH4+
Methanobrevibacter smithii
A5UK38
at pH 8.0 and 37C
additional information
additional information
Homo sapiens
-
-
-
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0705
ATP
Methanobrevibacter smithii
A5UK38
at pH 8.0 and 37C
4
0.003
HCO3-
Methanobrevibacter smithii
A5UK38
at pH 8.0 and 37C
195
0.0033
NH4+
Methanobrevibacter smithii
A5UK38
at pH 8.0 and 37C
54
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
13
Carbamoyl phosphate
-
-
0.015 - 0.024
palmitoyl-CoA
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.00452
ATP synthetase reverse reaction rate, at pH 8.0 and 37C
0.014
recombinant enzyme expressed in Schizosaccharomyces pombe
0.077
carbamoyl phosphate synthetase reaction rate, at pH 8.0 and 37C
0.12
deletion mutant lacking the last 13 residues of the carboxyl end
0.3 - 0.46
-
-
0.4
recombinant enzyme expressed in Escherischia coli
0.43
-
purified recombinant tagged mutant C1327A
0.47
-
purified recombinant tagged mutant C1337A
0.86
-
purified recombinant tagged wild-type enzyme
16
-
value at 37C and 60C
additional information
-
-
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.8 - 7.6
-
-
7.6
-
assay at
7.6 - 8.6
-
at 37C
7.6
-
at 60C
7.8
-
-
7.8 - 8
-
at 37C
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.7 - 8.5
-
pH 5.7: about 60% of maximal activit, pH 8.5: about 80% of maximal activity, 70C
6.3 - 8
-
50% of maximal activity at pH 6.3 and 8.0
7 - 8.5
-
50% of maximal activity at pH 7.0 and 8.5
7 - 10
-
pH 7.0: about 60% of maximal activity, pH 10.0: about 60% of maximal activity, 37C
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25 - 30
-
assay at
additional information
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4.6
calculated from amino acid sequence
4.7
-
isoelectric focusing
6.3
-
sequence calculation
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
-
hepatocarcinoma cell line, suppression of CPS1 expression occurs at the transcriptional level
Manually annotated by BRENDA team
-
hepatocarcinoma cell line, suppression of CPS1 expression occurs at the transcriptional level
Manually annotated by BRENDA team
-
hepatocarcinoma cell line, suppression of CPS1 expression occurs at the transcriptional level
Manually annotated by BRENDA team
-
expressed at higher level in adult testis than in fetal testis
Manually annotated by BRENDA team
additional information
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
-
contained completely within the inner mitochondrial membrane
Manually annotated by BRENDA team
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
50700
-
gel filtration
78000 - 80000
-
native PAGE, gel filtration
140000
-
gel filtration
160000 - 251000
-
sedimentation velocity measurement, gel filtration, MW depends on protein concentration and composition of solvent
165000 - 178000
-
gel filtration, density gradient centrifugation
166000
-
mass spectrometry
188000
-
gel filtration
190000
-
gel filtration
192000
-
recombinant enzyme, gel filtration
202000
-
native enzyme, gel filtration
222000
-
glycerol or sucrose density gradient centrifugation
250000
-
gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
homodimer
2 * 41403, calculated from amino acid sequence; 2 * 44000, SDS-PAGE
monomer
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
acetylation
lipoprotein
-
the enzyme becomes spontaneously palmitoylated, in a reversible manner depending on the palmitoyl-CoA concentrations, on an active site cysteine residue regulating the enzyme activity through inhibition, and for membrane attachment
additional information
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
1.5 A resolution structure of recombinant enzyme
-
recombinant enzyme
-
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
23
-
24 h, 20% v/v glycerol, 2 mM DTT, 13% loss of activity; 24 h, 20% v/v glycerol, 2 mM DTT, stable
37
-
4 h, 30% loss of activity
38.5
-
transition temperature
46
-
5 min, 25% loss of activity
55
-
5 min, 80% loss of activity
60
-
extreme thermostable
100
-
1 h, 50% loss of activity
additional information
-
the enzyme is extreme thermostable
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
acetylglutamate ATP accelerates the oxidative inactivation by mixtures of Fe3+, ascorbate, and O2
-
elastase inactivates. Addition of ATP, Mg2+, K+ and N-acetyl-L-glutamate protects entirely
-
elastase inactivates. Addition of ATP, Mg2+, K+ and N-acetyl-L-glutamate protects entirely. Acetylglutamate alone speeds inactivation
-
glycerol and DTT stabilize during purification
-
inactivated by the Fe3+-oxygen-ascorbate model system for mixed-function oxidation. The susceptibility is markedly increased by acetylglutamate, when ATP is absent. ATP, and even more a mixture of ATP and bicarbonate protect from inactivation
-
no inactivation by elastase
Frog
-
stabilty under pressure, progressive inactivation down to 50% of initial activity at 200 MPa, this residual activity is constant at 200 MPa for at least 50 min
-
trypsin, chymotrypsin, and papain also hydrolyze the oxidized enzyme considerybly faster than the native enzyme
-
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
4C, 20% v/v glycerol, 0.1 or 2 mM DTT, 48 h, stable
-
4C, 6 months, stable
-
4C, stable for at least 1 month
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
HisTrap column chromatography
-
Kunitz-type soybean trypsin inhibitor-coupled Sepharose CL-4B column chromatography, Sephacryl S-200 gel filtration, and Superdex 200 FPLC gel filtration
-
native enzyme partially by mitochondria preparation and anion exchange chromatography
-
Ni2+ affinity column chromatography, and gel filtration
recombinant enzyme
-
recombinant His6-tagged wild-type and mutant CPSIs from Spodoptera frugiperda SF9 cells by affinity chromatography
-
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
cloning of N-terminally His6-tagged wild-type and mutant CPSIs in Escherichia coli and expression in Spodopetra frugiperda SF9 cells
-
expressed in Escherichia coli BL21(DE3) cells
expressed in Escherichia coli DH10Bac cells
-
expression in Escherichia coli
-
expression in Escherichia coli and Schizosaccharomyces pombe
expression in Saccharomyces pombe
-
gene CPS1 is located on chromosome 2q35, genotyping and cloning of wild-type and mutant CPSIs in Cos7 and HeLa cells, which show increased activity with the wild-type gene, overview
-
gene CPS1, located on chromosome 2, genotyping, overview
gene CPSI, DNA and amino acid sequence determination and analysis, expression of the N-terminally His6-FLAG-tagged enzyme and mutant T1406D in Schizosaccharomyces pombe from pESP5, expression of mature protein of 1462 amino acid residues with the first 39 amino acids of the precursor protein replaced by a methionyl residue
-
gene CPSI, expression of the N-terminally His6-FLAG-tagged enzyme in Schizosaccharomyces pombe from pESP5
-
EXPRESSION
ORGANISM
UNIPROT
LITERATURE
human hepatocellular carcinoma cells do not express CPS1, whereas cultured human primary hepatocytes express abundant levels. CPS1 is silenced or down-regulated in liver tumor tissues compared with the matched noncancerous tissues. The expression of CPS1 in human hepatocellular carcinoma cells is restored with demethylation agent, 5-azacytidine. Two CpG dinucleotides, located near the transcription start site, and a CpG-rich region in the first intron are hypermethylated in human hepatocellular carcinoma cells
nutrient depletion does not increase CPS1 protein levels in wild-type or KO cells
-
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
A1378T
-
the carbamoyl-phosphate synthetase deficiency-causing mutation greatly decreases enzyme activity
A438P
-
inactive
Al38P
-
the carbamoyl-phosphate synthetase deficiency-causing mutation greatly decreases enzyme activity
C1327A
-
the mutant shows reduced activity compared to the wild-type enzyme, the mutation significantly alters activity at the domain C ATP site, binding of N-acetylglutamate is affected, overview
C1337A
-
the mutant shows reduced activity compared to the wild-type enzyme, the mutation significantly alters activity at the domain C ATP site, binding of N-acetylglutamate is affected, overview
G1376S
-
the mutation has no detectable effect on activity
L1381S
-
the mutation is clearly disease-causing (carbamoyl-phosphate synthetase deficiency), since it induces strong enzyme instability
L390R
-
the mutation is clearly disease-causing (carbamoyl-phosphate synthetase deficiency), since it induces strong enzyme instability
N355D
-
the carbamoyl-phosphate synthetase deficiency-causing mutation greatly decreases enzyme activity
R1262X
-
a premature stop codon mutation naturally occuring in carbamoyl phosphate synthetase 1 deficiency, CPS1D
R803G
-
naturally occuring missense mutation involved in carbamoyl phosphate synthetase 1 deficiency, CPS1D
T1405N
-
significant change in CPSI enzymatic function
T1406D
-
naturally occurring single nucleotide polymorphism, phenotype, overview
T1443A
-
the carbamoyl-phosphate synthetase deficiency-causing mutation greatly decreases enzyme activity
T344A
-
the mutation has no detectable effect on activity
T544M
-
the carbamoyl-phosphate synthetase deficiency-causing mutation greatly decreases enzyme activity
W1410K
-
site-directed mutagenesis, the mutant binds N-acetyl-L-beta-phenylglutamate at the N-acetyl-L-glutamate binding site
Y389C
-
the carbamoyl-phosphate synthetase deficiency-causing mutation greatly decreases enzyme activity
E261Q
-
no synthesis of carbonyl phosphate in presence of L-glutamine
K258L
-
synthesis of carbonyl phosphate in presence of L-glutamine
N1437D
-
site-directed mutagenesis, the mutant enzymes shows reduced activation by N-acetyl-L-glutamate compared to the wild-type enzyme
N1440D
-
site-directed mutagenesis, the mutant enzymes shows reduced activation by N-acetyl-L-glutamate compared to the wild-type enzyme
T1391V
-
site-directed mutagenesis, the mutant enzymes shows reduced activation by N-acetyl-L-glutamate compared to the wild-type enzyme
T1394A
-
site-directed mutagenesis, the mutant enzymes shows reduced activation by N-acetyl-L-glutamate compared to the wild-type enzyme
W1410K
-
site-directed mutagenesis, the mutant enzymes shows reduced activation by N-acetyl-L-glutamate compared to the wild-type enzyme
D265A
-
no carbamoyl phosphate synthesis
D265E
-
about 10% of the specific acitivity of the wild-type enzyme
D265N
-
about 10% of the specific acitivity of the wild-type enzyme
D810A
-
no carbamoyl phosphate synthesis
D810E
-
no carbamoyl phosphate synthesis
D810N
-
no carbamoyl phosphate synthesis
H262N
-
no carbamoyl phosphate synthesis
H807N
-
about 30% of the specific acitivity of the wild-type enzyme
I316G
-
about 10% of the specific acitivity of the wild-type enzyme
I316H
-
no carbamoyl phosphate synthesis
I316S
-
about 30% of the specific acitivity of the wild-type enzyme
L229G
-
about 30% of the specific acitivity of the wild-type enzyme
S228A
-
about 30% of the specific acitivity of the wild-type enzyme
D265A
-
no carbamoyl phosphate synthesis
-
D265N
-
about 10% of the specific acitivity of the wild-type enzyme
-
H262N
-
no carbamoyl phosphate synthesis
-
L229G
-
about 30% of the specific acitivity of the wild-type enzyme
-
S228A
-
about 30% of the specific acitivity of the wild-type enzyme
-
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
biotechnology
-
ammonia elimination as functional marker in hepatocyte cultivation and zonation in a bioreactor, and construction of a bioartificial liver, overview
medicine
human hepatocellular carcinoma cells do not express CPS1, whereas cultured human primary hepatocytes express abundant levels. CPS1 is silenced or down-regulated in liver tumor tissues compared with the matched noncancerous tissues. The expression of CPS1 in human hepatocellular carcinoma cells is restored with demethylation agent, 5-azacytidine. Two CpG dinucleotides, located near the transcription start site, and a CpG-rich region in the first intron are hypermethylated in human hepatocellular carcinoma cells