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6.3.5.5: carbamoyl-phosphate synthase (glutamine-hydrolysing)

This is an abbreviated version!
For detailed information about carbamoyl-phosphate synthase (glutamine-hydrolysing), go to the full flat file.

Word Map on EC 6.3.5.5

Reaction

2 ATP +

L-glutamine
+
hydrogencarbonate
 +
H2O
=
2 ADP
 +
phosphate
 +
L-glutamate
 +
Carbamoyl phosphate

Synonyms

CAD, CAD carbamoyl-phosphate synthetase, CAD protein, Carbamoyl phosphate synthase (glutamine), carbamoyl phosphate synthetase, Carbamoyl phosphate synthetase (glutamine-hydrolyzing), carbamoyl phosphate synthetase 1, carbamoyl phosphate synthetase II, carbamoyl phosphate synthetase III, carbamoyl-phosphate synthetase, Carbamoyl-phosphate synthetase (glutamine-hydrolysing), carbamoyl-phosphate synthetase 2, Carbamoylphosphate synthase, Carbamoylphosphate synthetase, Carbamoylphosphate synthetase II, Carbamyl phosphate synthetase (glutamine), Carbamyl phosphate sythetase II, carbamylphosphate synthetase - aspartate transcarbamylase, CPA2, CPS, CPS II (glutamine-dependent), CPS III, CPS III (glutamine- and N-acetyl-L-glutamine-dependent), CPS1, CPSase, CPSase type II, CPSase-A, CPSase-P, CPSII, EC 2.7.2.9, Glutamine-dependent carbamyl phosphate synthetase, glutamine-hydrolyzing CPSase, MGG_04503, More, MtCPSs1, MtCPSs2, Synthase, carbamoylphosphate (glutamine), Synthetase, carbamoylphosphate (glutamine-hydrolyzing)

ECTree

     6 Ligases
         6.3 Forming carbon-nitrogen bonds
             6.3.5 Carbon-nitrogen ligases with glutamine as amido-N-donor
                6.3.5.5 carbamoyl-phosphate synthase (glutamine-hydrolysing)

Engineering

Engineering on EC 6.3.5.5 - carbamoyl-phosphate synthase (glutamine-hydrolysing)

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PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
K986I
-
the substrate kinetics (Km for L-glutamine) increases nearly 1.3fold compared to the wild type enzyme
T941F
-
the substrate kinetics (Km for L-glutamine) increases nearly 1.2fold compared to the wild type enzyme
T941F/K986I
-
the substrate kinetics (Km for L-glutamine) increases nearly 1.96fold compared to the wild type enzyme
T941F/T970A/K986I
-
the substrate kinetics (Km for L-glutamine) increases nearly 2.17fold compared to the wild type enzyme
T970A
-
the substrate kinetics (Km for L-glutamine) increases nearly 1.1fold compared to the wild type enzyme
K986I
-
the substrate kinetics (Km for L-glutamine) increases nearly 1.3fold compared to the wild type enzyme
-
T941F
-
the substrate kinetics (Km for L-glutamine) increases nearly 1.2fold compared to the wild type enzyme
-
T941F/K986I
-
the substrate kinetics (Km for L-glutamine) increases nearly 1.96fold compared to the wild type enzyme
-
T941F/T970A/K986I
-
the substrate kinetics (Km for L-glutamine) increases nearly 2.17fold compared to the wild type enzyme
-
T970A
-
the substrate kinetics (Km for L-glutamine) increases nearly 1.1fold compared to the wild type enzyme
-
K954E
-
enzyme activity in absence of effectors is slightly lower when compared with the enzyme from wild-type. UTP inhibition is reduced
S974A
-
under conditions of saturating Utp (2 mM), mutant enzyme shows similar degree of inhibition as wild-type enzyme. Activation by 5-phosphoribosyl-alpha-pyrophosphate is completely abolished at 0.05 mM, a concentration where near maximal activation is observed with wild-type enzyme
P1850T
mutation affects a conserved residue in the linker region of Cad. Mutant animals display reduced lymphatic vessel development and hyperbranching arteries, reminiscent of Notch pathway mutants. Notch signaling is significantly reduced in the mutants
A126M
-
mutation in large subunit significantly decreases synthesis of carbamoyl phosphate without completely inactivating the enzyme
A144Q
-
mutant enzyme retains ATP specificity
A182V
-
reduced apparent affinity for HCO3-, sensitivity toward UMP is unchanched in comparison to wild-type enzyme
A182V/S948F
-
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
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
-
site-directed mutagenesis in the ammonia tunnel, analysis of secondary structure by circular dichroism measurements
A309C
-
kinetic properties are similar to those of the wild-type enzyme
A309C/S35C
-
kinetic properties are similar to those of the wild-type enzyme
A309S
-
kinetic properties are similar to those of the wild-type enzyme
A311L
-
site-directed mutagenesis in the ammonia tunnel, analysis of secondary structure by circular dichroism measurements
A314C
-
site-directed mutagenesis in the ammonia tunnel, analysis of secondary structure by circular dichroism measurements
C232G/A251G/A314G
-
site-directed mutagenesis in the ammonia tunnel, analysis of secondary structure by circular dichroism measurements
C232V/A251V/A314V
-
site-directed mutagenesis in the ammonia tunnel, analysis of secondary structure by circular dichroism measurements
C248D
-
partial glutaminase activity of the mutant protein is increased 40fold relative to the wild-type enzyme
C269G
-
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
-
mutant enzyme retains ATP specificity
D334A
-
site-directed mutagenesis, the mutation has essentially no effect on the Km for L-glutamine
D362A
-
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
-
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
-
mutant enzyme retains ATP specificity
D753N
-
mutant enzyme retains ATP specificity
D753X
-
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
-
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
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
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
-
S948A
-
mutation has little effect on enzyme activity in comparison to wild-type 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
-
S44A
-
mutant of hybrid enzyme shows km value similar to the wild-type hybrid, the kcat values of glutamine and ATP are 14fold lower in comparison to wild-type hybrid, the functional linkage that coordinates the reactions on the glutaminase and carbamoyl-phosphate synthetase domains is lost as a result of mutation
D1220Y
aspartate transcarbamylase activity of the multifunctional mutant protein carbamylphosphate synthetase/aspartate transcarbamylase is no longer inhibited by UTP, carbamylphosphate synthetase activity retains fully sensitive to this effector
N1094D
aspartate transcarbamylase activity of the multifunctional mutant protein carbamylphosphate synthetase/aspartate transcarbamylase is no longer inhibited by UTP, carbamylphosphate synthetase activity retains fully sensitive to this effector
R1076S
aspartate transcarbamylase activity of the multifunctional mutant protein carbamylphosphate synthetase/aspartate transcarbamylase is no longer inhibited by UTP, carbamylphosphate synthetase activity retains fully sensitive to this effector
Y1096C
aspartate transcarbamylase activity of the multifunctional mutant protein carbamylphosphate synthetase/aspartate transcarbamylase is no longer inhibited by UTP, carbamylphosphate synthetase activity retains fully sensitive to this effector
Y1112L
aspartate transcarbamylase activity of the multifunctional mutant protein carbamylphosphate synthetase/aspartate transcarbamylase is no longer inhibited by UTP, carbamylphosphate synthetase activity retains fully sensitive to this effector
C345A
-
site-directed mutagenesis of an essential catalytic triad residue of the GATase domain, the mutation completely abolishes complementation activity and GATase activity
N348A
-
site-directed mutagenesis, the mutation reduces complementation activity, GATase activity and the initial growth rate, but the N348A mutant exhibits the high virulence phenotype of the parental RH strain in C57Bl/6 mice
N348R
-
site-directed mutagenesis, the mutation completely abolishes complementation activity and GATase activity
additional information