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Information on EC 6.3.5.5 - carbamoyl-phosphate synthase (glutamine-hydrolysing) and Organism(s) Escherichia coli and UniProt Accession P00968

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EC Tree
IUBMB Comments
The product carbamoyl phosphate is an intermediate in the biosynthesis of arginine and the pyrimidine nucleotides . The enzyme from Escherichia coli has three separate active sites, which are connected by a molecular tunnel that is almost 100 A in length . The amidotransferase domain within the small subunit of the enzyme hydrolyses glutamine to ammonia via a thioester intermediate. The ammonia migrates through the interior of the protein, where it reacts with carboxyphosphate to produce the carbamate intermediate. The carboxyphosphate intermediate is formed by the phosphorylation of hydrogencarbonate by ATP at a site contained within the N-terminal half of the large subunit. The carbamate intermediate is transported through the interior of the protein to a second site within the C-terminal half of the large subunit, where it is phosphorylated by another ATP to yield the final product, carbamoyl phosphate . cf. EC 6.3.4.16, carbamoyl-phosphate synthase (ammonia).
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Escherichia coli
UNIPROT: P00968
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Word Map
The taxonomic range for the selected organisms is: Escherichia coli
The enzyme appears in selected viruses and cellular organisms
Reaction Schemes
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Synonyms
cps iii, cad protein, carbamoyl phosphate synthetase iii, carbamoyl phosphate synthetase ii, cpsii, carbamoyl-phosphate synthetase 2, glutamine-dependent carbamyl phosphate synthetase, carbamoyl phosphate synthetase (glutamine-hydrolyzing), cpsase type ii, carbamylphosphate synthetase - aspartate transcarbamylase, more
SYNONYM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Carbamoyl phosphate synthase (glutamine)
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carbamoyl phosphate synthetase
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Carbamoyl phosphate synthetase (glutamine-hydrolyzing)
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carbamoyl phosphate synthetase 1
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carbamoyl-phosphate synthetase
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Carbamoyl-phosphate synthetase (glutamine-hydrolysing)
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Carbamoylphosphate synthase
Carbamoylphosphate synthetase
Carbamoylphosphate synthetase II
Carbamyl phosphate synthetase (glutamine)
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Carbamyl phosphate sythetase II
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CPSase
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CPSase-A
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CPSase-P
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Glutamine-dependent carbamyl phosphate synthetase
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Synthase, carbamoylphosphate (glutamine)
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Synthetase, carbamoylphosphate (glutamine-hydrolyzing)
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additional information
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carbamoyl phosphate synthetase is a member of the amidotransferase family of enzymes
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
2 ATP + L-glutamine + hydrogencarbonate + H2O = 2 ADP + phosphate + L-glutamate + carbamoyl phosphate
show the reaction diagram
carbamate, the product of the reaction involving ATP, bicarbonate, and ammonia, must be delivered from the site of formation to the site of utilization by traveling nearly 40 A within the enzyme. The tunnel is composed of three continuous water pockets and two narrow connecting parts, near residues A23 and G575. The two narrow parts render two free energy barriers of 6.7 and 8.4 kcal/mol, respectively. Three water pockets are filled with about 21, 9, and 9 waters, respectively, and the corresponding relative free energies of carbamate residing in these free energy minima are 5.8, 0, and 1.6 kcal/mol, respectively. The release of phosphate into solution at the site for the formation of carbamate allows the side chain of R306 to rotate toward E25, E383, and E604. This rotation is virtually prohibited by a barrier of at least 23 kcal/mol when phosphate remains bound. This conformational change not only opens the entrance of the tunnel but also shields the charge-charge repulsion from the three glutamate residues when carbamate passes through the tunnel
2 ATP + L-glutamine + hydrogencarbonate + H2O = 2 ADP + phosphate + L-glutamate + carbamoyl phosphate
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Phosphorylation
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amination
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amide group transfer
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PATHWAY SOURCE
PATHWAYS
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-, -, -, -, -, -, -
SYSTEMATIC NAME
IUBMB Comments
hydrogen-carbonate:L-glutamine amido-ligase (ADP-forming, carbamate-phosphorylating)
The product carbamoyl phosphate is an intermediate in the biosynthesis of arginine and the pyrimidine nucleotides [4]. The enzyme from Escherichia coli has three separate active sites, which are connected by a molecular tunnel that is almost 100 A in length [8]. The amidotransferase domain within the small subunit of the enzyme hydrolyses glutamine to ammonia via a thioester intermediate. The ammonia migrates through the interior of the protein, where it reacts with carboxyphosphate to produce the carbamate intermediate. The carboxyphosphate intermediate is formed by the phosphorylation of hydrogencarbonate by ATP at a site contained within the N-terminal half of the large subunit. The carbamate intermediate is transported through the interior of the protein to a second site within the C-terminal half of the large subunit, where it is phosphorylated by another ATP to yield the final product, carbamoyl phosphate [6]. cf. EC 6.3.4.16, carbamoyl-phosphate synthase (ammonia).
CAS REGISTRY NUMBER
COMMENTARY hide
37233-48-0
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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 + gamma-glutamyl hydrazide + HCO3-
?
show the reaction diagram
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-
-
?
2 ATP + gamma-glutamyl hydroxamate + HCO3-
?
show the reaction diagram
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-
-
?
2 ATP + hydrazine + HCO3-
2 ADP + phosphate + N-amino carbamoyl phosphate
show the reaction diagram
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-
-
?
2 ATP + hydroxylamine + HCO3-
2 ADP + phosphate + N-hydroxy carbamoyl phosphate
show the reaction diagram
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-
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?
2 ATP + L-Gln + HCO3-
2 ADP + phosphate + L-Glu + carbamoyl phosphate
show the reaction diagram
2 ATP + L-Gln + HCO3- + H+ + H2O
2 ADP + phosphate + L-glutamate + carbamoyl phosphate
show the reaction diagram
2 ATP + L-glutamine + HCO3- + H2O
2 ADP + phosphate + L-glutamate + carbamoyl phosphate
show the reaction diagram
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carbamoyl phosphate is utilized in the biosynthesis of arginine and pyrimidine nucleotides
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?
2 ATP + NH4+ + HCO3-
2 ADP + phosphate + carbamoyl phosphate
show the reaction diagram
ATP + L-glutamine + HCO3- + H2O
ADP + phosphate + L-glutamate + carbamoyl phosphate
show the reaction diagram
additional information
?
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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
2 ATP + L-Gln + HCO3-
2 ADP + phosphate + L-Glu + carbamoyl phosphate
show the reaction diagram
2 ATP + L-Gln + HCO3- + H+ + H2O
2 ADP + phosphate + L-glutamate + carbamoyl phosphate
show the reaction diagram
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the product carbamoyl phosphate is utilized in the pyrimidine and arginine biosynthetic pathways
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?
2 ATP + L-glutamine + HCO3- + H2O
2 ADP + phosphate + L-glutamate + carbamoyl phosphate
show the reaction diagram
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carbamoyl phosphate is utilized in the biosynthesis of arginine and pyrimidine nucleotides
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?
ATP + L-glutamine + HCO3- + H2O
ADP + phosphate + L-glutamate + carbamoyl phosphate
show the reaction diagram
additional information
?
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CPS uses the hydrolysis of glutamine as a localized source of ammonia for biosynthetic transformations
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?
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
AMP
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enhances activity
GDP
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enhances activity
GMP
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enhances activity
IDP
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enhances activity
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Mn2+
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maximal activity at concentration of Mn2+ approximately equal to the ATP concentration
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
6-diazo-5-oxonorleucine
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selective inactivation of Gln-dependent activity
Alkyl hydrazines
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inhibits Gln-dependent activity, but not NH4+-dependent activity
azaserine
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selective inactivation of Gln-dependent activity
guanidine hydrochloride
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H2O2
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0.2 mM, inhibits Gln-dependent activity. No effect on the activity with NH4+ in carbamoyl-phosphate synthase reaction
hydroxylamine
L-2-Amino-4-oxo-5-chloropentanoate
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selective inactivation of Gln-dependent activity
NEM
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irreversible inactivation of synthetase activity. Increase of glutaminase activity
P1,P5-di(adenosine 5')-pentaphosphate
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inhibits two partial reactions catalyzed by the enzyme: bicarbonate-dependent ATPase and ATP synthesis from carbamoyl phosphate
potassium cyanate
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inhibits Gln-dependent activity, but not NH4+-dependent activity
UDP
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inhibits to a lesser extent than UMP
additional information
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
ammonia
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GTP
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enhances activity
ITP
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enhances activity
L-ornithine
N-acetylglutamate
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carbamoyl-phosphate synthase II: no requirement for N-acetylglutamate
NEM
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250fold activation of glutaminase activity. Irreversible inactivation of synthetase activity
ornithine
phosphoribosyl 5'-diphosphate
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activation of Escherichia coli and chimeric enzyme, the presence of UTP reduces the PRPP binding
potassium
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thiol
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required for Gln-dependent activity
XMP
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enhances activity
additional information
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allosteric effectors bind at domain D of eCPS
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.022 - 0.53
ADP
111
ammonia
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0.004 - 8.36
ATP
0.11 - 0.15
Gln
0.64 - 32.5
HCO3-
0.1 - 14.9
L-Gln
0.12 - 18.87
L-glutamine
12 - 160
NH4+
additional information
additional information
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.01 - 0.32
ADP
5.1
ammonia
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0.006 - 9.01
ATP
0.1 - 4.2
Gln
0.005 - 3.1
L-Gln
0.53 - 9.4
L-glutamine
0.097 - 5.8
NH4+
additional information
additional information
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Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0018 - 0.005
UMP
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.348
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recombinant mutant P909C/G919C
0.472
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recombinant mutant P909C
0.524
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recombinant mutant G919C
0.855
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recombinant wild-type enzyme
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.6
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mutant enzyme E841K
7.8 - 8.2
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9.3
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wild-type enzyme
9.5
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and a second optimum at pH 4.2, glutaminase activity
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25
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assay at
25 - 37
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assay at
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
large subunit
UniProt
Manually annotated by BRENDA team
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
117700
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1 * 41400 + 1 * 117700, the large subunit catalyses the carbamoyl phosphate synthesis from ammonia in three steps, and binds the effectors in its 15000 domain
118000
120000
130000
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1 * 40000 + 1 * 130000, SDS-PAGE
133000
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1 * 40000, subunit contains the binding site for glutamine, + 1 * 133000, subunit contains the binding sites for NH4+, HCO3-, ATP, and the allosteric effectors. The light and heavy subunits are encoded by the genetically linked car A and car B genes, respectively
163000
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sedimentation equilibrium ultracentrifugation
40000
41400
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1 * 41400 + 1 * 117700, the large subunit catalyses the carbamoyl phosphate synthesis from ammonia in three steps, and binds the effectors in its 15000 domain
42000
58000
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2 * 58000, SDS-PAGE, chimeric protein
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
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x * 40000 + x * 118000
dimer
heterodimer
octamer
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4 * 42000 + 4 * 118000
tetramer
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at high concentrations in presence of ornithine
additional information
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
side-chain modification
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
molecular dynamics simulation. Carbamate, the product of the reaction involving ATP, bicarbonate, and ammonia, must be delivered from the site of formation to the site of utilization by traveling nearly 40 A within the enzyme. The tunnel is composed of three continuous water pockets and two narrow connecting parts, near residues A23 and G575. The two narrow parts render two free energy barriers of 6.7 and 8.4 kcal/mol, respectively. Three water pockets are filled with about 21, 9, and 9 waters, respectively, and the corresponding relative free energies of carbamate residing in these free energy minima are 5.8, 0, and 1.6 kcal/mol, respectively. The release of phosphate into solution at the site for the formation of carbamate allows the side chain of R306 to rotate toward E25, E383, and E604. This rotation is virtually prohibited by a barrier of at least 23 kcal/mol when phosphate remains bound. This conformational change not only opens the entrance of the tunnel but also shields the charge-charge repulsion from the three glutamate residues when carbamate passes through the tunnel
C248D mutant of small subunit, crystals are grown at 4°C by batch from 8% poly(ethylene glycol) 8000, 0.65 M tetraethylammonium chloride, 0.5 mM MnCl2, 100 mM KCl, 1.5 mM ADP, 25 mM GEPES, pH 7.4, 0.5 mM L-Orn. The crystals belong to the space group P2(1)2(1)2(1) with unit cell dimensions of a = 151.1 A, b = 164.2 A, and c = 331.5A and one complete (alphabeta)4-heeterotetramer per asymmetric unit
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in presence of both IMP and ornithine, structural analysis of the nucleotide monophosphate allosteric binding site for enzyme
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the structure of enzyme cocrystallized in the presence of the 5’-adenylylimidodiphosphate, is determined to 2.1 A by X-ray crystallographic analysis, the three active sites of enzyme communicate via domain movements, the first example of such a domain movement is described
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the three active sites within the alpha,beta heterodimer are separated by a linear distance of nearly 100 A and are interconnected by two molecular tunnels
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X-ray crystallographic analysis, the three active sites on the protein are widely separated from one another
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X-ray crystallographic structure
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PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
A23F
mutant designed to block the migration of carbamate through the narrowest parts of the carbamate tunnel. Mutant retains 1.7% of the catalytic activity for the synthesis of carbamoyl phosphate relative to the wild type CPS
G575F
mutant designed to block the migration of carbamate through the narrowest parts of the carbamate tunnel. Mutant retains 3.8% of the catalytic activity for the synthesis of carbamoyl phosphate relative to the wild type CPS
A126M
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mutation in large subunit significantly decreases synthesis of carbamoyl phosphate without completely inactivating the enzyme
A144Q
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mutant enzyme retains ATP specificity
A182V
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reduced apparent affinity for HCO3-, sensitivity toward UMP is unchanched in comparison to wild-type enzyme
A182V/S948F
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mutant is insensitive towards pyrimidine and purine nucleosides, activation by ornithine, although the affinity for this ligand is fivefold reduced in comparison to wild-type enzyme
A23F
migration of carbamate through the narrowest part of the carbamate tunnel is blocked. From the kinetic data the only reaction significantly affected by this mutation is the overall synthesis of carbamoyl phosphate (only 1.7% compared to wild-type)
A23K
A23K mutation decreases the glutamine-dependent ATPase activity by an order of magnitude. While there is a decrease in the rate of carbamoyl phosphate formation, the enzyme utilizes two molecules of ATP for every molecule of carbamoyl phosphate synthesized
A251C
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site-directed mutagenesis in the ammonia tunnel, analysis of secondary structure by circular dichroism measurements
A309C
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kinetic properties are similar to those of the wild-type enzyme
A309C/S35C
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kinetic properties are similar to those of the wild-type enzyme
A309S
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kinetic properties are similar to those of the wild-type enzyme
A311L
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site-directed mutagenesis in the ammonia tunnel, analysis of secondary structure by circular dichroism measurements
A314C
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site-directed mutagenesis in the ammonia tunnel, analysis of secondary structure by circular dichroism measurements
C232G/A251G/A314G
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site-directed mutagenesis in the ammonia tunnel, analysis of secondary structure by circular dichroism measurements
C232V/A251V/A314V
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site-directed mutagenesis in the ammonia tunnel, analysis of secondary structure by circular dichroism measurements
C248D
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partial glutaminase activity of the mutant protein is increased 40fold relative to the wild-type enzyme
C269G
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Cys269Gly and Cys269Ser mutants bind significant amounts of Gln but do not hydrolyze Gln. The mutants are able to catalyze carbamoyl-phosphate formation with NH4+ as nitrogen donor, at a rate equal to that of the wild type enzyme. The mutant enzyme catalyzes ATP synthesis from ADP and carbamoyl phosphate at the usual rates. Substantial increase in bicarbonate-dependent ATPase
C269S
D207A
D207N
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mutant enzyme retains ATP specificity
D334A
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site-directed mutagenesis, the mutation has essentially no effect on the Km for L-glutamine
D362A
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mutation in alpha subunit, 2fold increase in turnover number for NH4+, 5.4fold decrease in KM-value for NH4+, 1.7fold increase in turnover number for Gln, 1.7fold increase in KM-value for Gln
D362A/betaR265A
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mutation S362A in alpha-subunit, mutation R265A in beta-subunit,3fold increase in turnover number for NH4+, 2.2fold decrease in KM-value for NH4+, 1.2fold decrease in turnover number for Gln, 71fold increase in KM-value for Gln
D753A
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mutant enzyme retains ATP specificity
D753N
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mutant enzyme retains ATP specificity
D753X
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residues within the carbamate domain of the large subunit of CPS are selected as targets for mutagenesis. Mutant enzymes E761A, E841Q, N843D, R845Q have diminished ability to synthesize carbamoyl phosphate. Mutant enzymes R715A, Q829A, and R675A show elevated Michaelis constants for MgATP2- in the partial back reaction. The mutant enzymes E761A, N827A, E841Q, N843D, and R845Q show significant increases in the Michaelis constant for either bicarbonate or carbamoyl phosphate. No significant alterations are noted upon mutation of either R571 or D753
DELTA119
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truncation mutant DELTA14, with a removal of 14 amino acids at the carboxy terminus of the large subunit shows a 40fold decrease in Gln-dependent ATPase activity. Similar losses in activity are observed for the DELTA50, DELTA65, DELTA91, and DELTA119 mutant proteins. However formation of carbamoyl phosphate is detected even after the deletion of 119 amino acids from the carboxy-terminal end of the large subunit. No allosteric effects are observed in the presence of Orn even after the removal of the last 119 amino acids from the large subunit of CPS. Mutant enzymes G921V and G921I are unstable and are defective for the synthesis of carbamoyl phosphate. The T977A mutant is not regulated by UMP, but the full allosteric effect is observed with Orn. The R1030A and R1031A mutants enzymes exhibit wild type properties, mutant G921A shows no alteration in any of the allosteric properties. Mutant N1015A cannot be purified
DELTA14
-
truncation mutant DELTA14, with a removal of 14 amino acids at the carboxy terminus of the large subunit shows a 40fold decrease in Gln-dependent ATPase activity. Similar losses in activity are observed for the DELTA50, DELTA65, DELTA91, and DELTA119 mutant proteins. However formation of carbamoyl phosphate is detected even after the deletion of 119 amino acids from the carboxy-terminal end of the large subunit. No allosteric effects are observed in the presence of Orn even after the removal of the last 119 amino acids from the large subunit of CPS. Mutant enzymes G921V and G921I are unstable and are defective for the synthesis of carbamoyl phosphate. The T977A mutant is not regulated by UMP, but the full allosteric effect is observed with Orn. The R1030A and R1031A mutants enzymes exhibit wild type properties, mutant G921A shows no alteration in any of the allosteric properties. Mutant N1015A cannot be purified
DELTA50
-
truncation mutant DELTA14, with a removal of 14 amino acids at the carboxy terminus of the large subunit shows a 40fold decrease in Gln-dependent ATPase activity. Similar losses in activity are observed for the DELTA50, DELTA65, DELTA91, and DELTA119 mutant proteins. However formation of carbamoyl phosphate is detected even after the deletion of 119 amino acids from the carboxy-terminal end of the large subunit. No allosteric effects are observed in the presence of Orn even after the removal of the last 119 amino acids from the large subunit of CPS. Mutant enzymes G921V and G921I are unstable and are defective for the synthesis of carbamoyl phosphate. The T977A mutant is not regulated by UMP, but the full allosteric effect is observed with Orn. The R1030A and R1031A mutants enzymes exhibit wild type properties, mutant G921A shows no alteration in any of the allosteric properties. Mutant N1015A cannot be purified
DELTA65
-
truncation mutant DELTA14, with a removal of 14 amino acids at the carboxy terminus of the large subunit shows a 40fold decrease in Gln-dependent ATPase activity. Similar losses in activity are observed for the DELTA50, DELTA65, DELTA91, and DELTA119 mutant proteins. However formation of carbamoyl phosphate is detected even after the deletion of 119 amino acids from the carboxy-terminal end of the large subunit. No allosteric effects are observed in the presence of Orn even after the removal of the last 119 amino acids from the large subunit of CPS. Mutant enzymes G921V and G921I are unstable and are defective for the synthesis of carbamoyl phosphate. The T977A mutant is not regulated by UMP, but the full allosteric effect is observed with Orn. The R1030A and R1031A mutants enzymes exhibit wild type properties, mutant G921A shows no alteration in any of the allosteric properties. Mutant N1015A cannot be purified
DELTA91
-
truncation mutant DELTA14, with a removal of 14 amino acids at the carboxy terminus of the large subunit shows a 40fold decrease in Gln-dependent ATPase activity. Similar losses in activity are observed for the DELTA50, DELTA65, DELTA91, and DELTA119 mutant proteins. However formation of carbamoyl phosphate is detected even after the deletion of 119 amino acids from the carboxy-terminal end of the large subunit. No allosteric effects are observed in the presence of Orn even after the removal of the last 119 amino acids from the large subunit of CPS. Mutant enzymes G921V and G921I are unstable and are defective for the synthesis of carbamoyl phosphate. The T977A mutant is not regulated by UMP, but the full allosteric effect is observed with Orn. The R1030A and R1031A mutants enzymes exhibit wild type properties, mutant G921A shows no alteration in any of the allosteric properties. Mutant N1015A cannot be purified
E215A
-
mutations of the residues E215A, N283A, E299Q, N301D, and R303Q result in proteins which are unable to synthesize carbamoyl phosphate at a significant rate. The binding of bicarbonate is most affected by the mutagenesis of residues E215A, E299Q, N301D, and R303Q. The Km for ATP is most affected in the mutant enzymes R129A, R169A, Q285A, and N301D. No significant changes in the catalytic constants are observed in the mutants R82A and D207A. All of the mutations, with the exception of the N301D mutant, primarily effect the enzyme by altering the step for the phosphorylation of bicarbonate. Mutation N301D also disrupts the catalytic step involved in the phosphorylation of carbamate
E25Q/E383Q
-
46.7fold decreased turnover number for carbamoyl-phosphate synthesis
E25Q/E383Q/E604Q
-
more than 700fold decreased turnover number for carbamoyl-phosphate synthesis
E299Q
-
mutations of the residues E215A, N283A, E299Q, N301D, and R303Q result in proteins which are unable to synthesize carbamoyl phosphate at a significant rate. The binding of bicarbonate is most affected by the mutagenesis of residues E215A, E299Q, N301D, and R303Q. The Km for ATP is most affected in the mutant enzymes R129A, R169A, Q285A, and N301D. No significant changes in the catalytic constants are observed in the mutants R82A and D207A. All of the mutations, with the exception of the N301D mutant, primarily effect the enzyme by altering the step for the phosphorylation of bicarbonate. Mutation N301D also disrupts the catalytic step involved in the phosphorylation of carbamate
E383Q
-
1.1fold decreased turnover number for carbamoyl-phosphate synthesis
E383Q/E916Q
-
3.9fold decreased turnover number for carbamoyl-phosphate synthesis
E577Q
-
437.5fold decreased turnover number for carbamoyl-phosphate synthesis
E604Q
-
3.7fold decreased turnover number for carbamoyl-phosphate synthesis
E761A
E783A
-
the allosteric activation of enzyme by ornithine is completely suppressed
E783K
-
the allosteric activation of enzyme by ornithine is completely suppressed
E841K
-
comparison of 15N-isotope effects in mutant and wild-type enzyme on the hydrolysis of glutamine, the rate of glutamine hydrolysis in the mutant is not affected by MgATP2 and HCO3-, with the wild-type enzyme in the absence of MgATP2 and HCO3- the isotope effect id reduced
E841Q
-
residues within the carbamate domain of the large subunit of CPS are selected as targets for mutagenesis. Mutant enzymes E761A, E841Q, N843D, R845Q have diminished ability to synthesize carbamoyl phosphate. Mutant enzymes R715A, Q829A, and R675A show elevated Michaelis constants for MgATP2- in the partial back reaction. The mutant enzymes E761A, N827A, E841Q, N843D, and R845Q show significant increases in the Michaelis constant for either bicarbonate or carbamoyl phosphate. No significant alterations are noted upon mutation of either R571 or D753
E892A
-
the allosteric activation of enzyme by ornithine is completely suppressed
E892K
-
the allosteric activation of enzyme by ornithine is completely suppressed
E916Q
-
6.7fold decreased turnover number for carbamoyl-phosphate synthesis
F755A
-
mutant enzyme retains ATP specificity
G1008A
-
mutation abolishs IMP activation and UMP inhibition in comparison to wild-type enzyme
G293A
-
kinetic properties are similar to those of the wild-type enzyme
G293I
-
kinetic properties are similar to those of the wild-type enzyme
G293S
-
kinetic properties are similar to those of the wild-type enzyme
G359F
G359L
-
Km values of L-glutamine are increased
G359S
-
Km values of L-glutamine are increased
G359Y
-
glutaminase and bicarbonate-dependent ATPase reaction are uncoupled from one another, the mutant enzyme is fully functional when external ammonia is utilized as the nitrogen source but is unable to use glutamine for the synthesis of carbamoyl phosphate
G575F
mutation to G575 does not exhibit significant pertubations to the kinetic constants of the partial reactions, G575F mutant has a 50fold reduction in the rate of carbamoyl phosphate formation (attributed to the restricted passage of carbamate through the tunnel). The insertion of a larger phenylalanine side chain is anticipated to create a more efficient blockage of the tunnel. Of the mutants made in the carbamate tunnel, G575F is the most efficacious at blocking the passage of carbamate without disrupting any of the active sites
G575K
mutation to G575 does not exhibit significant pertubations to the kinetic constants of the partial reactions, mutant has similar catalytic properties as the wild-type protein, suggesting that the conformational change of Arg-848 may not be crucial for the transport of carbamate
G824D
-
strongly reduced affinity for ornithine in comparison to wild-type enzyme
G919C
-
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
G921A
-
truncation mutant DELTA14, with a removal of 14 amino acids at the carboxy terminus of the large subunit shows a 40fold decrease in Gln-dependent ATPase activity. Similar losses in activity are observed for the DELTA50, DELTA65, DELTA91, and DELTA119 mutant proteins. However formation of carbamoyl phosphate is detected even after the deletion of 119 amino acids from the carboxy-terminal end of the large subunit. No allosteric effects are observed in the presence of Orn even after the removal of the last 119 amino acids from the large subunit of CPS. Mutant enzymes G921V and G921I are unstable and are defective for the synthesis of carbamoyl phosphate. The T977A mutant is not regulated by UMP, but the full allosteric effect is observed with Orn. The R1030A and R1031A mutants enzymes exhibit wild type properties, mutant G921A shows no alteration in any of the allosteric properties. Mutant N1015A cannot be purified
G921I
-
truncation mutant DELTA14, with a removal of 14 amino acids at the carboxy terminus of the large subunit shows a 40fold decrease in Gln-dependent ATPase activity. Similar losses in activity are observed for the DELTA50, DELTA65, DELTA91, and DELTA119 mutant proteins. However formation of carbamoyl phosphate is detected even after the deletion of 119 amino acids from the carboxy-terminal end of the large subunit. No allosteric effects are observed in the presence of Orn even after the removal of the last 119 amino acids from the large subunit of CPS. Mutant enzymes G921V and G921I are unstable and are defective for the synthesis of carbamoyl phosphate. The T977A mutant is not regulated by UMP, but the full allosteric effect is observed with Orn. The R1030A and R1031A mutants enzymes exhibit wild type properties, mutant G921A shows no alteration in any of the allosteric properties. Mutant N1015A cannot be purified
G921V
-
truncation mutant DELTA14, with a removal of 14 amino acids at the carboxy terminus of the large subunit shows a 40fold decrease in Gln-dependent ATPase activity. Similar losses in activity are observed for the DELTA50, DELTA65, DELTA91, and DELTA119 mutant proteins. However formation of carbamoyl phosphate is detected even after the deletion of 119 amino acids from the carboxy-terminal end of the large subunit. No allosteric effects are observed in the presence of Orn even after the removal of the last 119 amino acids from the large subunit of CPS. Mutant enzymes G921V and G921I are unstable and are defective for the synthesis of carbamoyl phosphate. The T977A mutant is not regulated by UMP, but the full allosteric effect is observed with Orn. The R1030A and R1031A mutants enzymes exhibit wild type properties, mutant G921A shows no alteration in any of the allosteric properties. Mutant N1015A cannot be purified
G997A
-
mutation abolishs IMP activation and UMP inhibition in comparison to wild-type enzyme
H975L
-
mutation abolishes oligomer formation even high enzyme concentrations or in the presence of ornithine
H975L/N987V
-
mutation abolishes oligomer formation even high enzyme concentrations or in the presence of ornithine
H995A
I18W/A23F/C24F
triple mutant I18W/A23F/C24F is made to disrupt the water pocket that may facilitate the passage of carbamate through the carbamate tunnel. This mutant significantly hinders the overall rate of carbamoyl phosphate synthesis and it diminishes all of the other partial reactions
I211S
-
mutant enzyme retains ATP specificity
I352F
-
site-directed mutagenesis in the ammonia tunnel, analysis of secondary structure by circular dichroism measurements
K1061A
-
mutation abolishs IMP activation and UMP inhibition in comparison to wild-type enzyme
K954A
-
mutation has little effect on enzyme activity in comparison to wild-type enzyme
K993A
-
mutation reduces enzyme activity in comparison to wild-type enzyme
K993W
-
mutation has little effect on enzyme activity in comparison to wild-type enzyme
K993W/H995A
-
mutation abolishs IMP activation and UMP inhibition in comparison to wild-type enzyme
L270K
-
ammonia-dependent carbamoyl phosphate synthesis activity is very similar to that of the wild-type enzyme, L-glutamine-dependent carbamoyl phosphate synthesis activity is 5fold decreased in comparison to the wild-type enzyme, the glutamine binding is almost entirely abolished
L421E
L421E/H975L/N987V
-
mutation abolishes oligomer formation even high enzyme concentrations or in the presence of ornithine
L421E/N987D
-
no oligomerization
L648E
mutant shows no detectable rate of carbamoyl phosphate formation. Little effect on the rates of all partial reactions is observed. Thus the reactions at the small subunit and the carboxy phosphate active sites remains unperturbed. The L648E mutant exhibits a 10fold drop in the rate of the glutaminase reaction which is due to the uncoupling between the carboxy phosphate and glutaminase active sites
L720E
the rates for both the glutamine- and HCO3--dependent ATPase reactions are largely unaffected by the mutation. The rate of the partial ATP-synthesis reaction is decreased 4fold. These perturbations may be due to an altered active site environment which diminishes the rate ADP phosphorylation by carbamoyl phosphate. No carbamoyl phosphate formation is detected. Mutant can structurally block the exit of the carbamate tunnel although the presence of the glutamate also weakens the assistance of Arg-848 during the synthesis of carbamoyl phosphate
L990A
M174E
mutant has significant reductions in the rates of the ATPase and carbamoyl phosphate synthesis reactions. Mutation does not affect the rate of the partial ATP synthesis reaction
M174E/M378E
catalytic properties of the double mutant, M174E/M378E, are similar to the single mutants with regard to the various partial reactions. Double mutant is unable to synthesize carbamoyl phosphate
M378E
mutant has significant reductions in the rates of the ATPase and carbamoyl phosphate synthesis reactions. Mutation does not affect the rate of the partial ATP synthesis reaction
M911E
the rate for both the glutamine- and HCO3--dependent ATPase reactions are largely unaffected by the mutation. The rate of the partial ATP-synthesis reaction is virtually undetectable. This perturbation may be due to an altered active site environment which diminishes the rate ADP phosphorylation by carbamoyl phosphate. Only limited carbamoyl phosphate formation is detected with mutant M911E. M911E mutant can structurally block the exit of the carbamate tunnel although the presence of glutamate also weakens the assistance of Arg-848 during the synthesis of carbamoyl phosphate
N1015A
-
truncation mutant DELTA14, with a removal of 14 amino acids at the carboxy terminus of the large subunit shows a 40fold decrease in Gln-dependent ATPase activity. Similar losses in activity are observed for the DELTA50, DELTA65, DELTA91, and DELTA119 mutant proteins. However formation of carbamoyl phosphate is detected even after the deletion of 119 amino acids from the carboxy-terminal end of the large subunit. No allosteric effects are observed in the presence of Orn even after the removal of the last 119 amino acids from the large subunit of CPS. Mutant enzymes G921V and G921I are unstable and are defective for the synthesis of carbamoyl phosphate. The T977A mutant is not regulated by UMP, but the full allosteric effect is observed with Orn. The R1030A and R1031A mutants enzymes exhibit wild type properties, mutant G921A shows no alteration in any of the allosteric properties. Mutant N1015A cannot be purified
N283A
-
mutations of the residues E215A, N283A, E299Q, N301D, and R303Q result in proteins which are unable to synthesize carbamoyl phosphate at a significant rate. The binding of bicarbonate is most affected by the mutagenesis of residues E215A, E299Q, N301D, and R303Q. The Km for ATP is most affected in the mutant enzymes R129A, R169A, Q285A, and N301D. No significant changes in the catalytic constants are observed in the mutants R82A and D207A. All of the mutations, with the exception of the N301D mutant, primarily effect the enzyme by altering the step for the phosphorylation of bicarbonate. Mutation N301D also disrupts the catalytic step involved in the phosphorylation of carbamate
N301D
-
mutations of the residues E215A, N283A, E299Q, N301D, and R303Q result in proteins which are unable to synthesize carbamoyl phosphate at a significant rate. The binding of bicarbonate is most affected by the mutagenesis of residues E215A, E299Q, N301D, and R303Q. The Km for ATP is most affected in the mutant enzymes R129A, R169A, Q285A, and N301D. No significant changes in the catalytic constants are observed in the mutants R82A and D207A. All of the mutations, with the exception of the N301D mutant, primarily effect the enzyme by altering the step for the phosphorylation of bicarbonate. Mutation N301D also disrupts the catalytic step involved in the phosphorylation of carbamate
N301K
-
mutation in large subunit significantly decreases synthesis of carbamoyl phosphate without completely inactivating the enzyme
N311A
-
site-directed mutagenesis, the mutant shows increased Km for L-glutamine compared to the wild-type enzyme
N827A
-
residues within the carbamate domain of the large subunit of CPS are selected as targets for mutagenesis. Mutant enzymes E761A, E841Q, N843D, R845Q have diminished ability to synthesize carbamoyl phosphate. Mutant enzymes R715A, Q829A, and R675A show elevated Michaelis constants for MgATP2- in the partial back reaction. The mutant enzymes E761A, N827A, E841Q, N843D, and R845Q show significant increases in the Michaelis constant for either bicarbonate or carbamoyl phosphate. No significant alterations are noted upon mutation of either R571 or D753
N843Q
-
residues within the carbamate domain of the large subunit of CPS are selected as targets for mutagenesis. Mutant enzymes E761A, E841Q, N843D, R845Q have diminished ability to synthesize carbamoyl phosphate. Mutant enzymes R715A, Q829A, and R675A show elevated Michaelis constants for MgATP2- in the partial back reaction. The mutant enzymes E761A, N827A, E841Q, N843D, and R845Q show significant increases in the Michaelis constant for either bicarbonate or carbamoyl phosphate. No significant alterations are noted upon mutation of either R571 or D753
N987D
-
mutant forms a (alpha/beta)-monomer regardless of the presence of any allosteric effectors
N987V
-
mutation decreases the oligomerisation
N992A
-
mutation abolishes oligomer formation even high enzyme concentrations or in the presence of ornithine
P165S
-
reduced apparent affinity for HCO3-, sensitivity toward UMP is increased in comparison to wild-type enzyme
P170L
-
reduced apparent affinity for HCO3-, sensitivity toward UMP is increased in comparison to wild-type enzyme
P360A/H361A
-
mutation in beta-subunit, turnover number for NH4+ is nearly identical to wild-type value, 1.6fold increase in turnover number for Gln, 3.2fold increase in KM-value for Gln
P360A/H361A/R265A
-
mutations P360A and H361A in alpha-subunit, mutation R265A in beta-subunit, mutant enzyme is unable to utilize glutamine for the synthesis of carbamoyl phosphate1.3fold increase in turnover number for NH4+, 11.8fold decrease in KM-value for NH4+
P360L
P690Q
-
mutant enzyme retains ATP specificity
P909C
-
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
P909C/G919C
-
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
Q262A/R265A
-
mutation in beta-subunit 1.9fold increase in turnover number for NH4+, 5fold decrease in KM-value for NH4+, 1.4fold decrease in turnover number for Gln, 43fold increase in KM-value for Gln
Q262A/R265A/N266A
-
mutation in beta-subunit, 1.6fold decrease in turnover number for Gln, 13.5fold increase in KM-value for Gln
Q262P
-
mutation causes marked enzyme instability, mutation in large subunit significantly decreases synthesis of carbamoyl phosphate without completely inactivating the enzyme
Q273E
-
ammonia-dependent carbamoyl phosphate synthesis activity is very similar to that of the wild-type enzyme, L-glutamine-dependent carbamoyl phosphate synthesis activity is equivalent to the wild-type enzyme, but the mutant is 10fold impaired in its L-glutamine binding ability in comparison to wild-type enzyme
Q273E/L270K
-
ammonia-dependent carbamoyl phosphate synthesis activity is very similar to that of the wild-type enzyme, L-glutamine-dependent carbamoyl phosphate synthesis activity is 25fold decreased in comparison to the wild-type enzyme, the glutamine binding is almost entirely abolished
Q273E/N240S
-
ammonia-dependent carbamoyl phosphate synthesis activity is very similar to that of the wild-type enzyme, L-glutamine-dependent carbamoyl phosphate synthesis activity is equivalent to the wild-type enzyme
Q285A
-
mutations of the residues E215A, N283A, E299Q, N301D, and R303Q result in proteins which are unable to synthesize carbamoyl phosphate at a significant rate. The binding of bicarbonate is most affected by the mutagenesis of residues E215A, E299Q, N301D, and R303Q. The Km for ATP is most affected in the mutant enzymes R129A, R169A, Q285A, and N301D. No significant changes in the catalytic constants are observed in the mutants R82A and D207A. All of the mutations, with the exception of the N301D mutant, primarily effect the enzyme by altering the step for the phosphorylation of bicarbonate. Mutation N301D also disrupts the catalytic step involved in the phosphorylation of carbamate
Q310A
-
site-directed mutagenesis, the mutant shows increased Km for L-glutamine compared to the wild-type enzyme
Q351A
-
site-directed mutagenesis, the mutant shows highly increased Km for L-glutamine compared to the wild-type enzyme
Q829A
-
residues within the carbamate domain of the large subunit of CPS are selected as targets for mutagenesis. Mutant enzymes E761A, E841Q, N843D, R845Q have diminished ability to synthesize carbamoyl phosphate. Mutant enzymes R715A, Q829A, and R675A show elevated Michaelis constants for MgATP2- in the partial back reaction. The mutant enzymes E761A, N827A, E841Q, N843D, and R845Q show significant increases in the Michaelis constant for either bicarbonate or carbamoyl phosphate. No significant alterations are noted upon mutation of either R571 or D753
R1020A
-
mutation has little effect on enzyme activity in comparison to wild-type enzyme
R1021A
-
mutation has little effect on enzyme activity in comparison to wild-type enzyme
R1030A
-
truncation mutant DELTA14, with a removal of 14 amino acids at the carboxy terminus of the large subunit shows a 40fold decrease in Gln-dependent ATPase activity. Similar losses in activity are observed for the DELTA50, DELTA65, DELTA91, and DELTA119 mutant proteins. However formation of carbamoyl phosphate is detected even after the deletion of 119 amino acids from the carboxy-terminal end of the large subunit. No allosteric effects are observed in the presence of Orn even after the removal of the last 119 amino acids from the large subunit of CPS. Mutant enzymes G921V and G921I are unstable and are defective for the synthesis of carbamoyl phosphate. The T977A mutant is not regulated by UMP, but the full allosteric effect is observed with Orn. The R1030A and R1031A mutants enzymes exhibit wild type properties, mutant G921A shows no alteration in any of the allosteric properties. Mutant N1015A cannot be purified
R1031A
-
truncation mutant DELTA14, with a removal of 14 amino acids at the carboxy terminus of the large subunit shows a 40fold decrease in Gln-dependent ATPase activity. Similar losses in activity are observed for the DELTA50, DELTA65, DELTA91, and DELTA119 mutant proteins. However formation of carbamoyl phosphate is detected even after the deletion of 119 amino acids from the carboxy-terminal end of the large subunit. No allosteric effects are observed in the presence of Orn even after the removal of the last 119 amino acids from the large subunit of CPS. Mutant enzymes G921V and G921I are unstable and are defective for the synthesis of carbamoyl phosphate. The T977A mutant is not regulated by UMP, but the full allosteric effect is observed with Orn. The R1030A and R1031A mutants enzymes exhibit wild type properties, mutant G921A shows no alteration in any of the allosteric properties. Mutant N1015A cannot be purified
R129A
-
mutations of the residues E215A, N283A, E299Q, N301D, and R303Q result in proteins which are unable to synthesize carbamoyl phosphate at a significant rate. The binding of bicarbonate is most affected by the mutagenesis of residues E215A, E299Q, N301D, and R303Q. The Km for ATP is most affected in the mutant enzymes R129A, R169A, Q285A, and N301D. No significant changes in the catalytic constants are observed in the mutants R82A and D207A. All of the mutations, with the exception of the N301D mutant, primarily effect the enzyme by altering the step for the phosphorylation of bicarbonate. Mutation N301D also disrupts the catalytic step involved in the phosphorylation of carbamate
R169A
-
mutations of the residues E215A, N283A, E299Q, N301D, and R303Q result in proteins which are unable to synthesize carbamoyl phosphate at a significant rate. The binding of bicarbonate is most affected by the mutagenesis of residues E215A, E299Q, N301D, and R303Q. The Km for ATP is most affected in the mutant enzymes R129A, R169A, Q285A, and N301D. No significant changes in the catalytic constants are observed in the mutants R82A and D207A. All of the mutations, with the exception of the N301D mutant, primarily effect the enzyme by altering the step for the phosphorylation of bicarbonate. Mutation N301D also disrupts the catalytic step involved in the phosphorylation of carbamate
R169H
-
mutation in large subunit significantly decreases synthesis of carbamoyl phosphate without completely inactivating the enzyme
R265A
-
mutation in beta-subunit, 1.3fold increase in turnover number for NH4+, 5fold decrease in KM-value for NH4+, 1.3fold decrease in turnover number for Gln, 69fold increase in KM-value for Gln
R303Q
-
mutations of the residues E215A, N283A, E299Q, N301D, and R303Q result in proteins which are unable to synthesize carbamoyl phosphate at a significant rate. The binding of bicarbonate is most affected by the mutagenesis of residues E215A, E299Q, N301D, and R303Q. The Km for ATP is most affected in the mutant enzymes R129A, R169A, Q285A, and N301D. No significant changes in the catalytic constants are observed in the mutants R82A and D207A. All of the mutations, with the exception of the N301D mutant, primarily effect the enzyme by altering the step for the phosphorylation of bicarbonate. Mutation N301D also disrupts the catalytic step involved in the phosphorylation of carbamate
R306A
-
more than 700fold decreased turnover number for carbamoyl-phosphate synthesis
R571X
-
residues within the carbamate domain of the large subunit of CPS are selected as targets for mutagenesis. Mutant enzymes E761A, E841Q, N843D, R845Q have diminished ability to synthesize carbamoyl phosphate. Mutant enzymes R715A, Q829A, and R675A show elevated Michaelis constants for MgATP2- in the partial back reaction. The mutant enzymes E761A, N827A, E841Q, N843D, and R845Q show significant increases in the Michaelis constant for either bicarbonate or carbamoyl phosphate. No significant alterations are noted upon mutation of either R571 or D753
R675A
-
residues within the carbamate domain of the large subunit of CPS are selected as targets for mutagenesis. Mutant enzymes E761A, E841Q, N843D, R845Q have diminished ability to synthesize carbamoyl phosphate. Mutant enzymes R715A, Q829A, and R675A show elevated Michaelis constants for MgATP2- in the partial back reaction. The mutant enzymes E761A, N827A, E841Q, N843D, and R845Q show significant increases in the Michaelis constant for either bicarbonate or carbamoyl phosphate. No significant alterations are noted upon mutation of either R571 or D753
R675L
-
mutation in large subunit significantly decreases synthesis of carbamoyl phosphate without completely inactivating the enzyme
R715A
-
residues within the carbamate domain of the large subunit of CPS are selected as targets for mutagenesis. Mutant enzymes E761A, E841Q, N843D, R845Q have diminished ability to synthesize carbamoyl phosphate. Mutant enzymes R715A, Q829A, and R675A show elevated Michaelis constants for MgATP2- in the partial back reaction. The mutant enzymes E761A, N827A, E841Q, N843D, and R845Q show significant increases in the Michaelis constant for either bicarbonate or carbamoyl phosphate. No significant alterations are noted upon mutation of either R571 or D753
R82A
-
mutations of the residues E215A, N283A, E299Q, N301D, and R303Q result in proteins which are unable to synthesize carbamoyl phosphate at a significant rate. The binding of bicarbonate is most affected by the mutagenesis of residues E215A, E299Q, N301D, and R303Q. The Km for ATP is most affected in the mutant enzymes R129A, R169A, Q285A, and N301D. No significant changes in the catalytic constants are observed in the mutants R82A and D207A. All of the mutations, with the exception of the N301D mutant, primarily effect the enzyme by altering the step for the phosphorylation of bicarbonate. Mutation N301D also disrupts the catalytic step involved in the phosphorylation of carbamate
R845Q
-
residues within the carbamate domain of the large subunit of CPS are selected as targets for mutagenesis. Mutant enzymes E761A, E841Q, N843D, R845Q have diminished ability to synthesize carbamoyl phosphate. Mutant enzymes R715A, Q829A, and R675A show elevated Michaelis constants for MgATP2- in the partial back reaction. The mutant enzymes E761A, N827A, E841Q, N843D, and R845Q show significant increases in the Michaelis constant for either bicarbonate or carbamoyl phosphate. No significant alterations are noted upon mutation of either R571 or D753
R848A
-
233fold decreased turnover number for carbamoyl-phosphate synthesis
S209A
-
mutant enzyme retains ATP specificity
S35C
-
kinetic properties are similar to those of the wild-type enzyme
S35F
-
kinetic properties are similar to the wild-type enzyme, only KM-value of L-glutamine is 5fold increased
S35Y
-
site-directed mutagenesis in the ammonia tunnel, analysis of secondary structure by circular dichroism measurements
S743N
-
minor modification of kinetic parameters in comparison to wild-type enzyme
S743N/G824D
-
strongly reduced affinity for ornithine in comparison to wild-type enzyme
S789P
-
mutation in large subunit significantly decreases synthesis of carbamoyl phosphate without completely inactivating the enzyme
S948A
-
mutation has little effect on enzyme activity in comparison to wild-type enzyme
S948F
-
mutant enzyme is unsensitive to UMP and IMP, but is still activated by ornithine, although to a reduced extent
T1042I
T1042K
-
the residue is responsible for the binding of ornithine to enzyme
T1043K
-
the allosteric activation of enzyme by ornithine is completely suppressed
T249V
-
site-directed mutagenesis in the ammonia tunnel, analysis of secondary structure by circular dichroism measurements
T800F
-
reduced affinity for ornithine, increased sensitivity for UMP in comparison to wild-type enzyme
T974A
-
mutation abolishs IMP activation and UMP inhibition in comparison to wild-type enzyme
T977A
-
truncation mutant DELTA14, with a removal of 14 amino acids at the carboxy terminus of the large subunit shows a 40fold decrease in Gln-dependent ATPase activity. Similar losses in activity are observed for the DELTA50, DELTA65, DELTA91, and DELTA119 mutant proteins. However formation of carbamoyl phosphate is detected even after the deletion of 119 amino acids from the carboxy-terminal end of the large subunit. No allosteric effects are observed in the presence of Orn even after the removal of the last 119 amino acids from the large subunit of CPS. Mutant enzymes G921V and G921I are unstable and are defective for the synthesis of carbamoyl phosphate. The T977A mutant is not regulated by UMP, but the full allosteric effect is observed with Orn. The R1030A and R1031A mutants enzymes exhibit wild type properties, mutant G921A shows no alteration in any of the allosteric properties. Mutant N1015A cannot be purified
V640R
-
mutation in large subunit significantly decreases synthesis of carbamoyl phosphate without completely inactivating the enzyme
V991A
-
mutation has little effect on enzyme activity in comparison to wild-type enzyme
V994A
-
mutation reduces enzyme activity in comparison to wild-type enzyme
W170T
-
increased Km compared to the wild type enzyme
W175T
-
increased Km compared to the wild type enzyme
W213T
-
increased Km compared to the wild type enzyme
W437T
-
increased Km compared to the wild type enzyme
W461T
-
increased Km compared to the wild type enzyme
W71T
-
increased Km compared to the wild type enzyme
additional information
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
treatment with 1 M potassium thiocyanate results in reversible dissociation into its subunits which retain catalytic activity
-
OXIDATION STABILITY
ORGANISM
UNIPROT
LITERATURE
Gln-dependent function of carbamoyl-phosphate synthase is inactivated by incubating the enzyme in air with low concentrations of dithiothreitol, glutathione, Cys, homocysteine, or 2-mercaptoethanol
-
1731
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
Escherichia coli and chimeric enzyme
-
recombinant mutant enzymes from Escherichia coli strain RC50 to over 95% purity
-
recombinant wild-type and mutant enzymes
-
Sephacryl S-300 gel filtration and Resource-Q column chromatography
-
wild-type and mutant enzyme
-
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
cloning of car B wild-type and mutant enzymes
-
Escherichia coli and chimeric enzyme are expressed in Escherichia coli L673
-
expressed in Escherichia coli
expression in Echerichia coli
-
expression in Escherichia coli
-
expression of mutant enzymes in Escherichia coli strain RC50
-
expression of wild-type and mutant enzymes
-
expression of wild-type and mutant enzymes in Escherichia coli strain L673
-
the plasmids containing the carAB genes are transformed in the RC50 cell line of Escherichia coli for expression of the wild-type and mutant forms of CPS
-
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Meister, A.
Mechanism and regulation of the glutamine-dependent carbamyl phosphate synthetase of Escherichia coli
Adv. Enzymol. Relat. Areas Mol. Biol.
62
315-374
1989
Agaricus bisporus, Escherichia coli, Elasmobranchii, Fresh-water teleost, Mammalia, Squalus acanthias
Manually annotated by BRENDA team
Anderson, P.M.
Carbamoyl-phosphate synthetase: an example of effects on enzyme properties of shifting an equilibrium between active monomer and active oligomer
Biochemistry
25
5576-5582
1986
Escherichia coli
Manually annotated by BRENDA team
Kaseman, D.S.; Meister, A.
carbamyl phosphate synthetase (glutamine-utilizing) from Escherichia coli
Methods Enzymol.
113
305-326
1985
Escherichia coli, Escherichia coli B / ATCC 11303
Manually annotated by BRENDA team
Powers, S.G.; Griffith, O.W.; Meister, A.
Inhibition of carbamyl phosphate synthetase by P1,P5-di(adenosine 5')-pentaphosphate
J. Biol. Chem.
252
3558-3560
1977
Escherichia coli
Manually annotated by BRENDA team
Trotta, P.P; Estis, L.F.; Meister, A.; Haschemeyer, R.H.
Self-association and allosteric properties of glutamine-dependent carbamyl phosphate synthetase
J. Biol. Chem.
249
482-491
1974
Escherichia coli
Manually annotated by BRENDA team
Anderson, P.M.; Carlson, J.D.; Rosenthal, G.A.; Meister, A.
Effect of potassium cyanate on the catalytic activities of carbamyl phosphate synthetase
Biochem. Biophys. Res. Commun.
55
246-252
1973
Escherichia coli
Manually annotated by BRENDA team
Anderson, P.M.; Meister, A.
Evidence for an activated form of carbon dioxide in the reaction catalyzed by Escherichia coli carbamyl phosphate synthetase
Biochemistry
4
2803-2808
1965
Escherichia coli, Escherichia coli B / ATCC 11303
Manually annotated by BRENDA team
Rubio, V.; Llorente, P.
Activation of carbamoyl phosphate synthetase from Escherichia coli by glycerol
Biochem. Biophys. Res. Commun.
107
1400-1405
1982
Escherichia coli
Manually annotated by BRENDA team
Avid-Majd, F.; Stapleton, M.A.; Harmon, M.F.; Hanks, B.A.; Mullins, L.S.; Raushel, F.M.
Comparison of the functional differences for the homologous residues within the carboxy phosphate and carbamate domains of carbamoyl phosphate synthetase
Biochemistry
35
14362-14369
1996
Escherichia coli
Manually annotated by BRENDA team
Stapleton, M.A.; Javid-Majd, F.; Harmon, M.F.; Hanks, B.A.; Grahmann, J.L.; Mullins, L.S.; Raushel, F.M.
Role of conserved residues within the carboxy phosphate domain of carbamoyl phosphate synthetase
Biochemistry
35
14352-14361
1996
Escherichia coli
Manually annotated by BRENDA team
Czerwinski, R.M.; Mareya, S.M.; Raushel, F.M.
Regulatory changes in the control of carbamoyl phosphate synthetase induced by truncation and mutagenesis of the allosteric binding domain
Biochemistry
34
13920-13927
1995
Escherichia coli
Manually annotated by BRENDA team
Herve, G.; Nagy, M.; Le Gouar, M.; Penverne, B.; Ladjimi, M.
The carbamoyl phosphate synthetase-aspartate transcarbamoylase complex of Saccharomyces cerevisiae: molecular and cellular aspects
Biochem. Soc. Trans.
21
195-198
1993
Saccharomyces cerevisiae, Escherichia coli, eukaryota, Mesocricetus auratus
Manually annotated by BRENDA team
Guy, H.I.; Bouvier, A.; Evans, D.R.
The smallest carbamoyl-phosphate synthetase. A single catalytic subdomain catalyzes all three partial reactions
J. Biol. Chem.
14; 272
29255-29262
1997
Escherichia coli
Manually annotated by BRENDA team
Guy, H.I.; Rotgeri, A.; Evans, D.R.
Activation by fusion of the glutaminase and synthetase subunits of Escherichia coli carbamyl-phosphate synthetase
J. Biol. Chem.
272
19913-19918
1997
Escherichia coli
Manually annotated by BRENDA team
Pierrat, O.A.; Javid-Majd, F.; Raushel, F.M.
Dissection of the conduit for allosteric control of carbamoyl phosphate synthetase by ornithine
Arch. Biochem. Biophys.
400
26-33
2002
Escherichia coli
Manually annotated by BRENDA team
Thoden, J.B.; Miran, S.G.; Phillips, J.C.; Howard, A.J.; Raushel, F.M.; Holden, H.M.
Carbamoyl phosphate synthetase: caught in the act of glutamine hydrolysis
Biochemistry
37
8825-8831
1998
Escherichia coli
Manually annotated by BRENDA team
Braxton, B.L.; Mullins, L.S.; Raushel, F.M.; Reinhart, G.D.
Allosteric dominance in carbamoyl phosphate synthetase
Biochemistry
38
1394-1401
1999
Escherichia coli
Manually annotated by BRENDA team
Thoden, J.B.; Huang, X.; Raushel, F.M.; Holden, H.M.
The small subunit of carbamoyl phosphate synthetase: snapshots along the reaction pathway
Biochemistry
38
16158-16166
1999
Escherichia coli
Manually annotated by BRENDA team
Thoden, J.B.; Wesenberg, G.; Raushel, F.M.; Holden, H.M.
Carbamoyl phosphate synthetase: closure of the B-domain as a result of nucleotide binding
Biochemistry
38
2347-2357
1999
Escherichia coli
Manually annotated by BRENDA team
Bueso, J.; Cervera, J.; Fresquet, V.; Marina, A.; Lusty, C.J.; Rubio, V.
Photoaffinity labeling with the activator IMP and Site-Directed mutagenesis of histidine 995 of carbamoyl phosphate synthetase from Escherichia coli demonstrate that the binding site for IMP overlaps with that for the inhibitor UMP
Biochemistry
38
3910-3917
1999
Escherichia coli
Manually annotated by BRENDA team
Huang, X.; Raushel, F.M.
An engineered blockage within the ammonia tunnel of carbamoyl phosphate synthetase prevents the use of glutamine as a substrate but not ammonia
Biochemistry
39
3240-3247
2000
Escherichia coli
Manually annotated by BRENDA team
Miles, B.W.; Raushel, F.M.
Synchronization of the three reaction centers within carbamoyl Phosphate synthetase
Biochemistry
39
5051-5056
2000
Escherichia coli
Manually annotated by BRENDA team
Rishavy, M.A.; Cleland, W.W.; Lusty, C.J.
15N isotope effects in glutamine hydrolysis catalyzed by carbamyl phosphate synthetase: evidence for a tetrahedral intermediate in the mechanism
Biochemistry
39
7309-7315
2000
Escherichia coli
Manually annotated by BRENDA team
Kim, J.; Raushel, F.M.
Allosteric control of the oligomerization of carbamoyl phosphate synthetase from Escherichia coli
Biochemistry
40
11030-11036
2001
Escherichia coli
Manually annotated by BRENDA team
Kim, J.; Howell, S.; Huang, X.; Raushel, F.M.
Structural defects within the carbamate tunnel of carbamoyl phosphate synthetase
Biochemistry
41
12575-12581
2002
Escherichia coli
Manually annotated by BRENDA team
Nara, T.; Gao, G.; Yamasaki, H.; Nakajima-Shimada, J.; Aoki, T.
Carbamoyl-phosphate synthetase II in kinetoplastids
Biochim. Biophys. Acta
1387
462-468
1998
Dictyostelium discoideum, Drosophila melanogaster, Escherichia coli, Leishmania mexicana (O15829), Mesocricetus auratus, Saccharomyces cerevisiae, Trypanosoma cruzi (O15830)
Manually annotated by BRENDA team
Gibson, G.E.; Mullins, L.S.; Raushel, F.M.
Carbamoyl phosphate synthetase from Escherichia coli does not catalyze the dehydration of bicarbonate to carbon dioxide
Bioorg. Chem.
26
255-268
1998
Escherichia coli
-
Manually annotated by BRENDA team
Holden, H.M.; Thoden, J.B.; Raushel, F.M.
Carbamoyl phosphate synthetase: an amazing biochemical odyssey from substrate to product
Cell. Mol. Life Sci.
56
507-522
1999
Escherichia coli
Manually annotated by BRENDA team
Raushel, F.M.; Thoden, J.B.; Reinhart, G.D.; Holden, H.M.
Carbamoyl phosphate synthetase: a crooked path from substrates to products
Curr. Opin. Chem. Biol.
2
624-632
1998
Escherichia coli
Manually annotated by BRENDA team
Holden, H.M.; Thoden, J.B.; Raushel, F.M.
Carbamoyl phosphate synthetase: a tunnel runs through it
Curr. Opin. Struct. Biol.
8
679-685
1998
Escherichia coli
Manually annotated by BRENDA team
Mora, P.; Rubio, V.; Cervera, J.
Mechanism of oligomerization of Escherichia coli carbamoyl phosphate synthetase and modulation by the allosteric effectors. A site-directed mutagenesis study
FEBS Lett.
511
6-10
2002
Escherichia coli
Manually annotated by BRENDA team
Vaishnav, P.; Randev, S.; Jatiani, S.; Aggarwal, S.; Keharia, H.; Vyas, P.R.; Nareshkumar, G.; Archana, G.
Characterization of carbamoyl phosphate synthetase of Streptomyces spp
Indian J. Exp. Biol.
38
931-935
2000
Escherichia coli, Streptomyces coelicolor, Streptomyces lividans, Streptomyces lividans MTCC1
Manually annotated by BRENDA team
Guy, H.I.; Schmidt, B.; Herve, G.; Evans, D.R.
Pressure-induced dissociation of carbamoyl-phosphate synthetase domains. The catalytically active form is dimeric
J. Biol. Chem.
273
14172-14178
1998
Escherichia coli
Manually annotated by BRENDA team
Sahay, N.; Guy, H.I.; Liu, X.; Evans, D.R.
Regulation of an Escherichia coli/mammalian chimeric carbamoyl-phosphate synthetase
J. Biol. Chem.
273
31195-31202
1998
Escherichia coli, Mammalia
Manually annotated by BRENDA team
Thoden, J.B.; Raushel, F.M.; Wesenberg, G.; Holden, H.M.
The binding of inosine monophosphate to Escherichia coli carbamoyl phosphate synthetase
J. Biol. Chem.
274
22502-22507
1999
Escherichia coli
Manually annotated by BRENDA team
Hewagama, A.; Guy, H.I.; Vickrey, J.F.; Evans, D.R.
Functional linkage between the glutaminase and synthetase domains of carbamoyl-phosphate synthetase. Role of serine 44 in carbamoyl-phosphate synthetase-aspartate carbamoyltransferase-dihydroorotase (CAD)
J. Biol. Chem.
274
28240-28245
1999
Escherichia coli, Mammalia
Manually annotated by BRENDA team
Huang, X.; Raushel, F.M.
Restricted passage of reaction intermediates through the ammonia tunnel of carbamoyl phosphate synthetase
J. Biol. Chem.
275
26233-26240
2000
Escherichia coli
Manually annotated by BRENDA team
Ahuja, A.; Purcarea, C.; Guy, H.I.; Evans, D.R.
A novel carbamoyl-phosphate synthetase from Aquifex aeolicus
J. Biol. Chem.
276
45694-45703
2001
Aquifex aeolicus, Bacillus subtilis, Cricetinae, Escherichia coli, Methanocaldococcus jannaschii, Pseudomonas aeruginosa
Manually annotated by BRENDA team
Eroglu, B.; Powers-Lee, S.G.
Unmasking a functional allosteric domain in an allosterically nonresponsive carbamoyl-phosphate synthetase
J. Biol. Chem.
277
45466-45472
2002
Saccharomyces cerevisiae, Escherichia coli
Manually annotated by BRENDA team
Saeed-Kothe, A.; Powers-Lee, S.G.
Specificity determining residues in ammonia- and glutamine-dependent carbamoyl phosphate synthetases
J. Biol. Chem.
277
7231-7238
2002
Escherichia coli
Manually annotated by BRENDA team
Saeed-Kothe, A.; Powers-Lee, S.G.
Gain of glutaminase function in mutants of the ammonia-specific frog carbamoyl phosphate synthetase
J. Biol. Chem.
278
26722-26726
2003
Escherichia coli
Manually annotated by BRENDA team
Delannay, S.; Charlier, D.; Tricot, C.; Villeret, V.; Pierard, A.; Stalon, V.
Serine 948 and threonine 1042 are crucial residues for allosteric regulation of Escherichia coli carbamoylphosphate synthetase and illustrate coupling effects of activation and inhibition pathways
J. Mol. Biol.
286
1217-1228
1999
Escherichia coli
Manually annotated by BRENDA team
Fresquet, V.; Mora, P.; Rochera, L.; Ramon-Maiques, S.; Rubio, V.; Cervera, J.
Site-directed mutagenesis of the regulatory domain of Escherichia coli carbamoyl phosphate synthetase identifies crucial residues for allosteric regulation and for transduction of the regulatory signals
J. Mol. Biol.
299
979-991
2000
Escherichia coli, Escherichia coli L814
Manually annotated by BRENDA team
Kim, J.; Raushel, F.M.
Access to the carbamate tunnel of carbamoyl phosphate synthetase
Arch. Biochem. Biophys.
425
33-41
2004
Escherichia coli
Manually annotated by BRENDA team
Kim, J.; Raushel, F.M.
Perforation of the tunnel wall in carbamoyl phosphate synthetase derails the passage of ammonia between sequential active sites
Biochemistry
43
5334-5340
2004
Escherichia coli
Manually annotated by BRENDA team
Yefimenko, I.; Fresquet, V.; Marco-Marin, C.; Rubio, V.; Cervera, J.
Understanding carbamoyl phosphate synthetase deficiency: impact of clinical mutations on enzyme functionality
J. Mol. Biol.
349
127-141
2005
Escherichia coli
Manually annotated by BRENDA team
Thoden, J.B.; Huang, X.; Kim, J.; Raushel, F.M.; Holden, H.M.
Long-range allosteric transitions in carbamoyl phosphate synthetase
Protein Sci.
13
2398-2405
2004
Escherichia coli
Manually annotated by BRENDA team
Kothe, M.; Powers-Lee, S.G.
Nucleotide recognition in the ATP-grasp protein carbamoyl phosphate synthetase
Protein Sci.
13
466-475
2004
Escherichia coli
Manually annotated by BRENDA team
Johnson, J.L.; West, J.K.; Nelson, A.D.; Reinhart, G.D.
Resolving the fluorescence response of Escherichia coli carbamoyl phosphate synthetase: mapping intra- and intersubunit conformational changes
Biochemistry
46
387-397
2007
Escherichia coli
Manually annotated by BRENDA team
Devroede, N.; Huysveld, N.; Charlier, D.
Mutational analysis of intervening sequences connecting the binding sites for integration host factor, PepA, PurR, and RNA polymerase in the control region of the Escherichia coli carAB operon, encoding carbamoylphosphate synthase
J. Bacteriol.
188
3236-3245
2006
Escherichia coli
Manually annotated by BRENDA team
Fan, Y.; Lund, L.; Yang, L.; Raushel, F.M.; Gao, Y.Q.
Mechanism for the transport of ammonia within carbamoyl phosphate synthetase determined by molecular dynamics simulations
Biochemistry
47
2935-2944
2008
Escherichia coli
Manually annotated by BRENDA team
Hart, E.J.; Powers-Lee, S.G.
Role of Cys1327 and Cys1337 in redox sensitivity and allosteric monitoring in human carbamoyl phosphate synthetase
J. Biol. Chem.
284
5977-5985
2008
Escherichia coli
Manually annotated by BRENDA team
Hart, E.J.; Powers-Lee, S.G.
Mutation analysis of carbamoyl phosphate synthetase: does the structurally conserved glutamine amidotransferase triad act as a functional dyad?
Protein Sci.
17
1120-1128
2008
Escherichia coli
Manually annotated by BRENDA team
Fan, Y.; Lund, L.; Shao, Q.; Gao, Y.Q.; Raushel, F.M.
A combined theoretical and experimental study of the ammonia tunnel in carbamoyl phosphate synthetase
J. Am. Chem. Soc.
131
10211-10219
2009
Escherichia coli
Manually annotated by BRENDA team
Lund, L.; Fan, Y.; Shao, Q.; Gao, Y.; Raushel, F.
Carbamate transport in carbamoyl phosphate synthetase: A theoretical and experimental investigation
J. Am. Chem. Soc.
132
3870-3878
2010
Escherichia coli, Escherichia coli (P00968)
Manually annotated by BRENDA team