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2 ATP + gamma-glutamyl hydrazide + HCO3-
?
-
-
-
?
2 ATP + gamma-glutamyl hydroxamate + HCO3-
?
-
-
-
?
2 ATP + hydrazine + HCO3-
2 ADP + phosphate + N-amino carbamoyl phosphate
-
-
-
?
2 ATP + hydroxylamine + HCO3-
2 ADP + phosphate + N-hydroxy carbamoyl phosphate
-
-
-
?
2 ATP + L-Gln + HCO3-
2 ADP + phosphate + L-Glu + carbamoyl phosphate
2 ATP + L-Gln + HCO3- + H+ + H2O
2 ADP + phosphate + L-glutamate + carbamoyl phosphate
2 ATP + L-glutamine + HCO3- + H2O
2 ADP + phosphate + L-glutamate + carbamoyl phosphate
-
-
carbamoyl phosphate is utilized in the biosynthesis of arginine and pyrimidine nucleotides
-
?
2 ATP + NH4+ + HCO3-
2 ADP + phosphate + carbamoyl phosphate
ATP + L-glutamine + HCO3- + H2O
ADP + phosphate + L-glutamate + carbamoyl phosphate
additional information
?
-
2 ATP + L-Gln + HCO3-
2 ADP + phosphate + L-Glu + carbamoyl phosphate
-
-
-
?
2 ATP + L-Gln + HCO3-
2 ADP + phosphate + L-Glu + carbamoyl phosphate
-
-
-
?
2 ATP + L-Gln + HCO3-
2 ADP + phosphate + L-Glu + carbamoyl phosphate
-
-
-
?
2 ATP + L-Gln + HCO3-
2 ADP + phosphate + L-Glu + carbamoyl phosphate
-
-
-
?
2 ATP + L-Gln + HCO3-
2 ADP + phosphate + L-Glu + carbamoyl phosphate
-
-
-
?
2 ATP + L-Gln + HCO3-
2 ADP + phosphate + L-Glu + carbamoyl phosphate
-
-
-
?
2 ATP + L-Gln + HCO3-
2 ADP + phosphate + L-Glu + carbamoyl phosphate
-
-
-
?
2 ATP + L-Gln + HCO3-
2 ADP + phosphate + L-Glu + carbamoyl phosphate
-
-
-
?
2 ATP + L-Gln + HCO3-
2 ADP + phosphate + L-Glu + carbamoyl phosphate
-
-
-
?
2 ATP + L-Gln + HCO3-
2 ADP + phosphate + L-Glu + carbamoyl phosphate
-
-
-
?
2 ATP + L-Gln + HCO3-
2 ADP + phosphate + L-Glu + carbamoyl phosphate
-
-
-
?
2 ATP + L-Gln + HCO3-
2 ADP + phosphate + L-Glu + carbamoyl phosphate
-
-
-
?
2 ATP + L-Gln + HCO3-
2 ADP + phosphate + L-Glu + carbamoyl phosphate
-
-
-
?
2 ATP + L-Gln + HCO3-
2 ADP + phosphate + L-Glu + carbamoyl phosphate
-
-
-
?
2 ATP + L-Gln + HCO3-
2 ADP + phosphate + L-Glu + carbamoyl phosphate
-
-
-
?
2 ATP + L-Gln + HCO3-
2 ADP + phosphate + L-Glu + carbamoyl phosphate
-
-
-
?
2 ATP + L-Gln + HCO3-
2 ADP + phosphate + L-Glu + carbamoyl phosphate
-
-
-
?
2 ATP + L-Gln + HCO3-
2 ADP + phosphate + L-Glu + carbamoyl phosphate
-
-
-
?
2 ATP + L-Gln + HCO3-
2 ADP + phosphate + L-Glu + carbamoyl phosphate
-
-
-
?
2 ATP + L-Gln + HCO3-
2 ADP + phosphate + L-Glu + carbamoyl phosphate
-
-
-
?
2 ATP + L-Gln + HCO3-
2 ADP + phosphate + L-Glu + carbamoyl phosphate
-
-
-
?
2 ATP + L-Gln + HCO3-
2 ADP + phosphate + L-Glu + carbamoyl phosphate
-
-
-
?
2 ATP + L-Gln + HCO3-
2 ADP + phosphate + L-Glu + carbamoyl phosphate
-
-
-
?
2 ATP + L-Gln + HCO3-
2 ADP + phosphate + L-Glu + carbamoyl phosphate
-
-
-
?
2 ATP + L-Gln + HCO3-
2 ADP + phosphate + L-Glu + carbamoyl phosphate
-
-
-
?
2 ATP + L-Gln + HCO3-
2 ADP + phosphate + L-Glu + carbamoyl phosphate
-
-
-
?
2 ATP + L-Gln + HCO3-
2 ADP + phosphate + L-Glu + carbamoyl phosphate
-
-
-
?
2 ATP + L-Gln + HCO3-
2 ADP + phosphate + L-Glu + carbamoyl phosphate
-
-
-
?
2 ATP + L-Gln + HCO3-
2 ADP + phosphate + L-Glu + carbamoyl phosphate
-
-
-
?
2 ATP + L-Gln + HCO3-
2 ADP + phosphate + L-Glu + carbamoyl phosphate
-
-
-
?
2 ATP + L-Gln + HCO3-
2 ADP + phosphate + L-Glu + carbamoyl phosphate
-
-
-
?
2 ATP + L-Gln + HCO3-
2 ADP + phosphate + L-Glu + carbamoyl phosphate
-
-
-
?
2 ATP + L-Gln + HCO3-
2 ADP + phosphate + L-Glu + carbamoyl phosphate
-
-
-
?
2 ATP + L-Gln + HCO3-
2 ADP + phosphate + L-Glu + carbamoyl phosphate
-
-
-
?
2 ATP + L-Gln + HCO3-
2 ADP + phosphate + L-Glu + carbamoyl phosphate
-
-
-
?
2 ATP + L-Gln + HCO3-
2 ADP + phosphate + L-Glu + carbamoyl phosphate
-
-
-
?
2 ATP + L-Gln + HCO3-
2 ADP + phosphate + L-Glu + carbamoyl phosphate
-
-
-
?
2 ATP + L-Gln + HCO3-
2 ADP + phosphate + L-Glu + carbamoyl phosphate
-
-
-
?
2 ATP + L-Gln + HCO3-
2 ADP + phosphate + L-Glu + carbamoyl phosphate
-
-
-
?
2 ATP + L-Gln + HCO3-
2 ADP + phosphate + L-Glu + carbamoyl phosphate
-
-
-
?
2 ATP + L-Gln + HCO3-
2 ADP + phosphate + L-Glu + carbamoyl phosphate
-
-
-
-
?
2 ATP + L-Gln + HCO3-
2 ADP + phosphate + L-Glu + carbamoyl phosphate
-
-
-
ir
2 ATP + L-Gln + HCO3-
2 ADP + phosphate + L-Glu + carbamoyl phosphate
-
-
-
ir
2 ATP + L-Gln + HCO3-
2 ADP + phosphate + L-Glu + carbamoyl phosphate
-
-
-
ir
2 ATP + L-Gln + HCO3-
2 ADP + phosphate + L-Glu + carbamoyl phosphate
-
overall reaction is irreversible
-
?
2 ATP + L-Gln + HCO3-
2 ADP + phosphate + L-Glu + carbamoyl phosphate
-
enzyme is a key enzyme in the pyrimidine nucleotide and arginine biosynthetic pathways
-
?
2 ATP + L-Gln + HCO3-
2 ADP + phosphate + L-Glu + carbamoyl phosphate
-
enzyme is a key enzyme in the pyrimidine nucleotide and arginine biosynthetic pathways
-
?
2 ATP + L-Gln + HCO3-
2 ADP + phosphate + L-Glu + carbamoyl phosphate
-
enzyme is a key enzyme in the pyrimidine nucleotide and arginine biosynthetic pathways
-
?
2 ATP + L-Gln + HCO3-
2 ADP + phosphate + L-Glu + carbamoyl phosphate
-
the enzyme catalyses the entry and rate-limiting step of the urea cycle
-
?
2 ATP + L-Gln + HCO3-
2 ADP + phosphate + L-Glu + carbamoyl phosphate
-
the enzyme catalyses the first step in the synthesis of arginine and pyrimidine
-
?
2 ATP + L-Gln + HCO3-
2 ADP + phosphate + L-Glu + carbamoyl phosphate
-
the product carbamoyl phosphate is an essential precursor of arginine and pyrimidine biosynthesis
-
?
2 ATP + L-Gln + HCO3-
2 ADP + phosphate + L-Glu + carbamoyl phosphate
-
the product carbamoyl phosphate is an essential precursor of arginine and pyrimidine biosynthesis
-
?
2 ATP + L-Gln + HCO3-
2 ADP + phosphate + L-Glu + carbamoyl phosphate
-
the product carbamoyl phosphate is an essential precursor of arginine and pyrimidine biosynthesis
-
?
2 ATP + L-Gln + HCO3-
2 ADP + phosphate + L-Glu + carbamoyl phosphate
-
the product carbamoyl phosphate is utilized for the arginine biosynthesis
-
?
2 ATP + L-Gln + HCO3-
2 ADP + phosphate + L-Glu + carbamoyl phosphate
-
the product carbamoyl phosphate is utilized for the arginine biosynthesis
-
?
2 ATP + L-Gln + HCO3-
2 ADP + phosphate + L-Glu + carbamoyl phosphate
-
the product carbamoyl phosphate plays a critical role in both arginine and pyrimidine biosynthesis
-
?
2 ATP + L-Gln + HCO3-
2 ADP + phosphate + L-Glu + carbamoyl phosphate
-
the product carbamoyl phosphate plays a critical role in both arginine and pyrimidine biosynthesis
-
?
2 ATP + L-Gln + HCO3-
2 ADP + phosphate + L-Glu + carbamoyl phosphate
-
the product carbamoylphosphate is utilized in the synthesis of arginine and pyrimidine nucleotides and in the urea cycle
-
?
2 ATP + L-Gln + HCO3-
2 ADP + phosphate + L-Glu + carbamoyl phosphate
-
carbamoyl phosphate synthetase contains an internal molecular tunnel, which has been proposed to facilitate the translocation of reaction intermediates from one active site to another. Ammonia, the product from the hydrolysis of glutamine in the small subunit, is apparently transported to the next active site in the large subunit of CPS over a distance of about 45 Å. The ammonia tunnel that connects these two active sites provides a direct path for the guided diffusion of ammonia and protection from protonation
-
-
?
2 ATP + L-Gln + HCO3- + H+ + H2O
2 ADP + phosphate + L-glutamate + carbamoyl phosphate
-
-
-
?
2 ATP + L-Gln + HCO3- + H+ + H2O
2 ADP + phosphate + L-glutamate + carbamoyl phosphate
-
the product carbamoyl phosphate is utilized in the pyrimidine and arginine biosynthetic pathways
-
?
2 ATP + NH4+ + HCO3-
2 ADP + phosphate + carbamoyl phosphate
-
-
-
?
2 ATP + NH4+ + HCO3-
2 ADP + phosphate + carbamoyl phosphate
-
-
-
-
?
2 ATP + NH4+ + HCO3-
2 ADP + phosphate + carbamoyl phosphate
-
-
-
?
2 ATP + NH4+ + HCO3-
2 ADP + phosphate + carbamoyl phosphate
-
-
-
?
2 ATP + NH4+ + HCO3-
2 ADP + phosphate + carbamoyl phosphate
-
-
-
?
2 ATP + NH4+ + HCO3-
2 ADP + phosphate + carbamoyl phosphate
-
-
-
?
2 ATP + NH4+ + HCO3-
2 ADP + phosphate + carbamoyl phosphate
-
-
-
?
2 ATP + NH4+ + HCO3-
2 ADP + phosphate + carbamoyl phosphate
-
-
-
?
2 ATP + NH4+ + HCO3-
2 ADP + phosphate + carbamoyl phosphate
-
-
-
?
2 ATP + NH4+ + HCO3-
2 ADP + phosphate + carbamoyl phosphate
-
-
-
?
2 ATP + NH4+ + HCO3-
2 ADP + phosphate + carbamoyl phosphate
-
-
-
?
2 ATP + NH4+ + HCO3-
2 ADP + phosphate + carbamoyl phosphate
-
-
-
?
2 ATP + NH4+ + HCO3-
2 ADP + phosphate + carbamoyl phosphate
-
-
-
?
2 ATP + NH4+ + HCO3-
2 ADP + phosphate + carbamoyl phosphate
-
-
-
?
2 ATP + NH4+ + HCO3-
2 ADP + phosphate + carbamoyl phosphate
-
-
-
?
2 ATP + NH4+ + HCO3-
2 ADP + phosphate + carbamoyl phosphate
-
-
-
?
2 ATP + NH4+ + HCO3-
2 ADP + phosphate + carbamoyl phosphate
-
-
-
?
2 ATP + NH4+ + HCO3-
2 ADP + phosphate + carbamoyl phosphate
-
-
-
?
2 ATP + NH4+ + HCO3-
2 ADP + phosphate + carbamoyl phosphate
-
-
-
-
?
2 ATP + NH4+ + HCO3-
2 ADP + phosphate + carbamoyl phosphate
-
-
-
ir
2 ATP + NH4+ + HCO3-
2 ADP + phosphate + carbamoyl phosphate
-
the enzyme is able to utilize external NH4+ as an alternative nitrogen source when glutamine is absent
-
-
?
ATP + L-glutamine + HCO3- + H2O
ADP + phosphate + L-glutamate + carbamoyl phosphate
-
-
-
-
?
ATP + L-glutamine + HCO3- + H2O
ADP + phosphate + L-glutamate + carbamoyl phosphate
-
CPS1 plays a critical role in the biosynthesis of pyrimidine nucleotides and the detoxification of ammonia
-
-
?
ATP + L-glutamine + HCO3- + H2O
ADP + phosphate + L-glutamate + carbamoyl phosphate
-
evolutionarily conserved triad glutamine amidotransferase, GAT, domains catalyze the cleavage of L-glutamine to yield ammonia and sequester the ammonia in a tunnel until delivery to a variety of acceptor substrates in synthetase domains of variable structure. The Cys269 and His353 catalytic triad residues are essential for L-glutamine hydrolysis, whereas Glu355 is not critical for eCPS activity, Gln351 plays a key role in L-glutamine binding, overview
-
-
?
ATP + L-glutamine + HCO3- + H2O
ADP + phosphate + L-glutamate + carbamoyl phosphate
-
neither residue P909 nor residue G919 is critical for eCPS function due to the absence of vicinal cysteinyl residues in wild-type eCPS
-
-
?
ATP + L-glutamine + HCO3- + H2O
ADP + phosphate + L-glutamate + carbamoyl phosphate
-
the enzyme from Escherichia coli catalyzes the synthesis of carbamoyl phosphate from bicarbonate, glutamine, and two molecules of ATP via a chemical mechanism that involves four separate reactions and three unstable intermediates, e.g. carboxyphosphate and carbamate
-
-
r
additional information
?
-
-
existence of an alternate route to the bicarbonate phosphorylation site when ammonia is provided as an external nitrogen source
-
?
additional information
?
-
-
CPS uses the hydrolysis of glutamine as a localized source of ammonia for biosynthetic transformations
-
-
?
additional information
?
-
-
the smaller subunit contains the binding site for the hydrolysis of glutamine, whereas the large subunit catalyzes the formation of carbamoyl phosphate. Molecular dynamics simulations and free-energy profile for the transfer of ammonia and ammonium through a tunnel in the small subunit of CPS, by use of the enzyme's crystal structure, resulting in five successful trajectories for ammonia transfer, while ammonium is immobilized in a water pocket inside the small subunit of the heterodimeric protein, the carbamate tunnel connects the two active sites within the large subunit to one another and guides the unstable carbamate intermediate to the site of phosphorylation, mechanism, overview
-
-
?
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additional information
additional information
-
0.022
ADP
mutant A23K, ATP-synthesis, pH 7.6, temperature not specified in the publication
0.03
ADP
mutant G575F, ATP-synthesis, pH 7.6, temperature not specified in the publication
0.054
ADP
mutant G575K, ATP-synthesis, pH 7.6, temperature not specified in the publication
0.088
ADP
mutant M174E, ATP-synthesis, pH 7.6, temperature not specified in the publication
0.13
ADP
mutant M378E, ATP-synthesis, pH 7.6, temperature not specified in the publication
0.13
ADP
wild-type, ATP-synthesis, pH 7.6, temperature not specified in the publication
0.17
ADP
mutant M174E/M378E, ATP-synthesis, pH 7.6, temperature not specified in the publication
0.18
ADP
mutant I18W/A23F/C24F, ATP-synthesis, pH 7.6, temperature not specified in the publication
0.34
ADP
mutant L648E, ATP-synthesis, pH 7.6, temperature not specified in the publication
0.42
ADP
mutant L720E, ATP-synthesis, pH 7.6, temperature not specified in the publication
0.53
ADP
mutant A23F, ATP-synthesis, pH 7.6, temperature not specified in the publication
0.004
ATP
-
pH 7.6, 25°C, HCO3(-)-dependent ATPase, mutant F755A
0.005
ATP
-
pH 7.6, 25°C, HCO3(-)-dependent ATPase, wild-type enzyme
0.006
ATP
-
pH 7.6, 25°C, HCO3(-)-dependent ATPase, mutant D207A
0.007
ATP
-
pH 7.6, 25°C, HCO3(-)-dependent ATPase, mutant D753A
0.008
ATP
-
pH 7.6, 25°C, HCO3(-)-dependent ATPase, mutant D753N
0.009
ATP
-
pH 7.6, 25°C, HCO3(-)-dependent ATPase, mutant D207N
0.009
ATP
-
pH 7.6, 25°C, HCO3(-)-dependent ATPase, mutant P690Q
0.013
ATP
mutant G575K, HCO3-dependent ATPase activity, pH 7.6, temperature not specified in the publication
0.014
ATP
mutant G575F, Gln-dependent ATPase activity, pH 7.6, temperature not specified in the publication
0.014
ATP
mutant G575F, HCO3-dependent ATPase activity, pH 7.6, temperature not specified in the publication
0.015
ATP
-
pH 7.6, 25°C, Gln-dependent ATPase, mutant D207A
0.016
ATP
-
pH 7.6, 25°C, HCO3(-)-dependent ATPase, mutant S209A
0.02
ATP
-
pH 7.6, 25°C, Gln-dependent ATPase, mutant D207N
0.02
ATP
-
pH 7.6, 25°C, HCO3(-)-dependent ATPase, mutant I211S
0.021
ATP
-
pH 7.6, 25°C, HCO3(-)-dependent ATPase, mutant A144Q/D207A
0.027
ATP
-
pH 7.6, 25°C, mutant D334A, L-glutamine hydrolysis
0.029
ATP
-
pH 7.6, 25°C, Gln-dependent ATPase, wild-type enzyme
0.03
ATP
mutant M378E, HCO3-dependent ATPase activity, pH 7.6, temperature not specified in the publication
0.031
ATP
-
pH 7.6, 25°C, Gln-dependent ATPase, mutant S209A
0.034
ATP
-
pH 7.6, 25°C, HCO3(-)-dependent ATPase, mutant A144Q
0.038
ATP
-
pH 7.6, wild-type enzyme in presence of ornithine and in comparison to mutant enzymes
0.0383
ATP
-
pH 7.6, 25°C, Gln-dependent ATPase, mutant P690Q
0.044
ATP
-
pH 7.6, 25°C, wild-type enzyme, L-glutamine hydrolysis
0.05
ATP
-
glutamine-dependent ADP formation
0.051
ATP
wild-type, HCO3-dependent ATPase activity, pH 7.6, temperature not specified in the publication
0.052
ATP
-
pH 7.6, 25°C, mutant Q351A, L-glutamine hydrolysis
0.06
ATP
-
ammonia-dependent ADP formation
0.064
ATP
-
pH 7.6, 25°C, Gln-dependent ATPase, mutant D753A
0.067
ATP
-
pH 7.6, 25°C, mutant Q310A, L-glutamine hydrolysis
0.07
ATP
mutant G575K, Gln-dependent ATPase activity, pH 7.6, temperature not specified in the publication
0.072
ATP
-
pH 7.6, 25°C, Gln-dependent ATPase, mutant I211S
0.075
ATP
-
pH 7.6, 25°C, mutant N311A, L-glutamine hydrolysis
0.12
ATP
mutant A23F, HCO3-dependent ATPase activity, pH 7.6, temperature not specified in the publication
0.121
ATP
-
pH 7.6, 25°C, Gln-dependent ATPase, mutant D753N
0.135
ATP
-
pH 7.6, 25°C, Gln-dependent ATPase, mutant F755A
0.154
ATP
-
pH 7.6, 25°C, Gln-dependent ATPase, mutant A144Q/D207A
0.16
ATP
mutant M378E, Gln-dependent ATPase activity, pH 7.6, temperature not specified in the publication
0.21
ATP
mutant I18W/A23F/C24F, HCO3-dependent ATPase activity, pH 7.6, temperature not specified in the publication
0.22
ATP
-
pH 8.0, 37°C, wild-type enzyme
0.22
ATP
-
mutant enzyme E306A, Gln-dependent ATP hydrolysis
0.22
ATP
mutant L720E, HCO3-dependent ATPase activity, pH 7.6, temperature not specified in the publication
0.24
ATP
wild-type, Gln-dependent ATPase activity, pH 7.6, temperature not specified in the publication
0.25
ATP
mutant I18W/A23F/C24F, Gln-dependent ATPase activity, pH 7.6, temperature not specified in the publication
0.26
ATP
mutant M911E, HCO3-dependent ATPase activity, pH 7.6, temperature not specified in the publication
0.28
ATP
mutant M174E/M378E, Gln-dependent ATPase activity, pH 7.6, temperature not specified in the publication
0.285
ATP
-
pH 7.6, 25°C, Gln-dependent ATPase, mutant A144Q
0.29
ATP
mutant M174E/M378E, HCO3-dependent ATPase activity, pH 7.6, temperature not specified in the publication
0.32
ATP
-
pH 8.0, 25°C, wild-type enzyme
0.33
ATP
mutant A23K, HCO3-dependent ATPase activity, pH 7.6, temperature not specified in the publication
0.36
ATP
mutant M174E, HCO3-dependent ATPase activity, pH 7.6, temperature not specified in the publication
0.37
ATP
-
pH 7.6, wild-type enzyme in presence of IMP and in comparison to mutant enzymes
0.38
ATP
mutant A23K, Gln-dependent ATPase activity, pH 7.6, temperature not specified in the publication
0.4
ATP
-
mutant enzyme R848A, Gln-dependent ATP hydrolysis
0.44
ATP
-
wild type enzyme, at 25°C in 50 mM HEPES buffer, pH 7.5
0.45
ATP
-
pH 8.0, 25°C, mutant enzyme Q262P
0.46
ATP
-
chimeric enzyme, in comparison to effect of phosphorylation and presence of effectors
0.47
ATP
-
pH 7.6, wild-type enzyme in absence of effectors and in comparison to mutant enzymes
0.5
ATP
mutant L648E, HCO3-dependent ATPase activity, pH 7.6, temperature not specified in the publication
0.51
ATP
-
mutant enzyme W437T, at 25°C in 50 mM HEPES buffer, pH 7.5
0.53
ATP
-
deletion mutant, in comparison to the presence of effectors
0.69
ATP
mutant M174E, Gln-dependent ATPase activity, pH 7.6, temperature not specified in the publication
0.75
ATP
-
mutant enzyme W213T, at 25°C in 50 mM HEPES buffer, pH 7.5
0.76
ATP
-
pH 8.0, 37°C, mutant enzyme R675L
0.87
ATP
-
pH 8.0, 37°C, mutant enzyme A126M
1
ATP
mutant M911E, Gln-dependent ATPase activity, pH 7.6, temperature not specified in the publication
1.14
ATP
-
pH 8.0, 37°C, mutant enzyme R169H
1.2
ATP
mutant L648E, Gln-dependent ATPase activity, pH 7.6, temperature not specified in the publication
1.28
ATP
-
mutant enzyme W71T, at 25°C in 50 mM HEPES buffer, pH 7.5
1.34
ATP
-
mutant enzyme W461T, at 25°C in 50 mM HEPES buffer, pH 7.5
1.57
ATP
-
mutant enzyme W170T, at 25°C in 50 mM HEPES buffer, pH 7.5
1.6
ATP
-
pH 7.6, wild-type enzyme in presence of UMP and in comparison to mutant enzymes
1.6
ATP
mutant A23F, Gln-dependent ATPase activity, pH 7.6, temperature not specified in the publication
1.7
ATP
mutant L720E, Gln-dependent ATPase activity, pH 7.6, temperature not specified in the publication
1.94
ATP
-
mutant enzyme W175T, at 25°C in 50 mM HEPES buffer, pH 7.5
3.02
ATP
-
pH 8.0, 37°C, mutant enzyme V640R
8.13
ATP
-
pH 8.0, 37°C, mutant enzyme S789P
8.36
ATP
-
pH 8.0, 37°C, mutant enzyme P360L
0.11
Gln
-
mutant enzyme E916Q, Gln-dependent ATP hydrolysis
0.12
Gln
-
wild-type enzyme, Gln-dependent ATP hydrolysis
0.14
Gln
-
mutant enzyme E383QE916Q, Gln-dependent ASTP hydrolysis
0.15
Gln
-
mutant enzyme E383Q,Gln-dependent ATP hydrolysis
0.64
HCO3-
-
pH 8.0, 37°C, mutant enzyme P360L
0.76
HCO3-
-
pH 8.0, 25°C, mutant enzyme Q262P
1.2
HCO3-
-
pH 7.5, 37°C, value of wild-type enzyme in comparison to values of mutant enzymes
1.33
HCO3-
-
pH 8.0, 25°C, wild-type enzyme
1.9
HCO3-
-
pH 8.0, 37°C, wild-type enzyme
3.65
HCO3-
-
pH 8.0, 37°C, mutant enzyme V640R
4.46
HCO3-
-
pH 8.0, 37°C, mutant enzyme R675L
12.6
HCO3-
-
pH 8.0, 37°C, mutant enzyme R169H
23.8
HCO3-
-
pH 8.0, 37°C, mutant enzyme S789P
32.5
HCO3-
-
pH 8.0, 37°C, mutant enzyme A126M
0.1
L-Gln
mutant A23K, glutaminase activity pH 7.6, temperature not specified in the publication
0.11
L-Gln
-
pH 7.6, wild-type enzyme
0.15
L-Gln
wild-type, glutaminase activity pH 7.6, temperature not specified in the publication
0.16
L-Gln
mutant I18W/A23F/C24F, glutaminase activity pH 7.6, temperature not specified in the publication
0.17
L-Gln
mutant G575K, glutaminase activity pH 7.6, temperature not specified in the publication
0.18
L-Gln
mutant L720E, glutaminase activity pH 7.6, temperature not specified in the publication
0.18
L-Gln
mutant M911E, glutaminase activity pH 7.6, temperature not specified in the publication
0.19
L-Gln
-
pH 7.6, mutant alphaD362A
0.19
L-Gln
mutant G575F, glutaminase activity pH 7.6, temperature not specified in the publication
0.21
L-Gln
mutant A23F, glutaminase activity pH 7.6, temperature not specified in the publication
0.23
L-Gln
mutant L648E, glutaminase activity pH 7.6, temperature not specified in the publication
0.35
L-Gln
mutant M378E, glutaminase activity pH 7.6, temperature not specified in the publication
0.36
L-Gln
-
pH 7.6, mutant alphaP360A/alphaH361A
0.39
L-Gln
mutant M174E, glutaminase activity pH 7.6, temperature not specified in the publication
0.47
L-Gln
mutant M174E/M378E, glutaminase activity pH 7.6, temperature not specified in the publication
4.7
L-Gln
-
pH 7.6, mutant betaQ262A/betaR265A
7.6
L-Gln
-
pH 7.6, mutant betaR265A
7.8
L-Gln
-
pH 7.6, mutant alphaD362A/betaR265A
14.9
L-Gln
-
pH 7.6, mutant betaQ262A/betaR265A/betaN266A
0.12
L-glutamine
-
pH 7.5, 37°C, value of wild-type enzyme in comparison to values of mutant enzymes
0.13
L-glutamine
-
pH 7.6, 25°C, wild-type enzyme, L-glutamine hydrolysis
0.15
L-glutamine
-
pH 7.6, 37°C, L-glutamine hydrolysis
0.15
L-glutamine
-
pH 7.6, 25°C, mutant Q351A, L-glutamine hydrolysis
0.361
L-glutamine
-
chimeric enzyme, in presence of ornithine and absence of UMP
0.89
L-glutamine
-
Escherichia coli enzyme, in presence of ornithine and absence of UMP
0.91
L-glutamine
-
Escherichia coli enzyme, in absence of ornithine and UMP
1.075
L-glutamine
-
chimeric enzyme, in absence of ornithine and UMP
1.49
L-glutamine
-
Escherichia coli enzyme, in absence of ornithine and presence of UMP
1.86
L-glutamine
-
chimeric enzyme, in absence of ornithine and presence of UMP
1.99
L-glutamine
-
pH 7.6, 25°C, mutant Q310A, L-glutamine hydrolysis
3.45
L-glutamine
-
pH 7.6, 25°C, mutant N311A, L-glutamine hydrolysis
18.87
L-glutamine
-
pH 7.6, 25°C, mutant D334A, L-glutamine hydrolysis
12
NH4+
-
pH 7.6, mutant alphaP360A/alphaH361A/betaR265A
24
NH4+
-
pH 7.6, mutant alphaD362A
26
NH4+
-
pH 7.6, mutant betaQ262A/betaR265A
26
NH4+
-
pH 7.6, mutant betaR265A
59
NH4+
-
pH 7.6, mutant alphaD362A/betaR265A
110
NH4+
-
mutant enzyme E383Q, NH4Cl-dependent ATP hydrolysis
130
NH4+
-
pH 7.6, wild-type enzyme
130
NH4+
-
wild-type enzyme, NH4Cl-dependent ATP hydrolysis
160
NH4+
-
mutant enzyme E916Q, NH4Cl-dependent ATP hydrolysis
additional information
additional information
-
-
-
additional information
additional information
-
-
-
additional information
additional information
-
effect of Orn or UMP on Km-values of mutant and wild type enzyme
-
additional information
additional information
-
kinetic constants of mutant and wild type enzymes
-
additional information
additional information
-
kinetic constants of mutant and wild type enzymes
-
additional information
additional information
-
comparison of KM of partial reactions of wild-type and mutant enzymes
-
additional information
additional information
-
comparison of Km-values for wild-type and mutant enzymes in absence and presence of ornithine
-
additional information
additional information
-
Km values for the formation of ADP, glutamate and carbonyl phosphate of the wild-type and mutant enzymes, Km values for the hydrolysis of gamma-glutamyl hydroxamate, gamma-glutamyl hydrazide, gamma-glutamyl hydroxamate and hydroxylamine of the wild-type and mutant enzymes
-
additional information
additional information
-
kinetics of wild-type and mutant enzymes in L-glutamine-dependent and bicarbonate-dependent ATPase activities, overview
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additional information
additional information
-
0.01
ADP
mutant M911E, value below 0.01, ATP-synthesis, pH 7.6, temperature not specified in the publication
0.024
ADP
mutant L648E, ATP-synthesis, pH 7.6, temperature not specified in the publication
0.031
ADP
mutant L720E, ATP-synthesis, pH 7.6, temperature not specified in the publication
0.058
ADP
mutant G575F, ATP-synthesis, pH 7.6, temperature not specified in the publication
0.086
ADP
mutant I18W/A23F/C24F, ATP-synthesis, pH 7.6, temperature not specified in the publication
0.1
ADP
mutant M174E, ATP-synthesis, pH 7.6, temperature not specified in the publication
0.13
ADP
wild-type, ATP-synthesis, pH 7.6, temperature not specified in the publication
0.15
ADP
mutant G575K, ATP-synthesis, pH 7.6, temperature not specified in the publication
0.2
ADP
mutant M378E, ATP-synthesis, pH 7.6, temperature not specified in the publication
0.22
ADP
mutant M174E/M378E, ATP-synthesis, pH 7.6, temperature not specified in the publication
0.23
ADP
mutant A23F, ATP-synthesis, pH 7.6, temperature not specified in the publication
0.32
ADP
mutant A23K, ATP-synthesis, pH 7.6, temperature not specified in the publication
0.006
ATP
mutant I18W/A23F/C24F, Gln-dependent ATPase activity, pH 7.6, temperature not specified in the publication
0.01
ATP
mutant I18W/A23F/C24F, value below 0.01, CP-synthesis (co-substrate: L-Gln, HCO3-), pH 7.6, temperature not specified in the publication
0.01
ATP
mutant L648E,value below 0.01, CP-synthesis (co-substrate: L-Gln, HCO3-), pH 7.6, temperature not specified in the publication
0.01
ATP
mutant L720E, value below 0.01, CP-synthesis (co-substrate: L-Gln, HCO3-), pH 7.6, temperature not specified in the publication
0.01
ATP
mutant M174E/M378E, value below 0.01, CP-synthesis (co-substrate: L-Gln, HCO3-), pH 7.6, temperature not specified in the publication
0.012
ATP
mutant M378E, CP-synthesis (co-substrate: L-Gln, HCO3-), pH 7.6, temperature not specified in the publication
0.013
ATP
mutant M378E, HCO3-dependent ATPase activity, pH 7.6, temperature not specified in the publication
0.015
ATP
mutant M911E, value below 0.01, CP-synthesis (co-substrate: L-Gln, HCO3-), pH 7.6, temperature not specified in the publication
0.028
ATP
mutant M174E/M378E, HCO3-dependent ATPase activity, pH 7.6, temperature not specified in the publication
0.033
ATP
mutant A23F, CP-synthesis (co-substrate: L-Gln, HCO3-), pH 7.6, temperature not specified in the publication
0.035
ATP
mutant M378E, Gln-dependent ATPase activity, pH 7.6, temperature not specified in the publication
0.039
ATP
mutant I18W/A23F/C24F, HCO3-dependent ATPase activity, pH 7.6, temperature not specified in the publication
0.043
ATP
mutant M174E/M378E, Gln-dependent ATPase activity, pH 7.6, temperature not specified in the publication
0.053
ATP
mutant M174E, HCO3-dependent ATPase activity, pH 7.6, temperature not specified in the publication
0.06
ATP
-
pH 7.6, 25°C, HCO3(-)-dependent ATPase, mutant D207N
0.07
ATP
-
pH 7.6, 25°C, HCO3(-)-dependent ATPase, mutant F755A
0.072
ATP
mutant G575F, CP-synthesis (co-substrate: L-Gln, HCO3-), pH 7.6, temperature not specified in the publication
0.09
ATP
-
pH 7.6, 25°C, HCO3(-)-dependent ATPase, mutant P690Q
0.09
ATP
-
pH 7.6, 25°C, HCO3(-)-dependent ATPase, wild-type enzyme
0.097
ATP
mutant A23F, HCO3-dependent ATPase activity, pH 7.6, temperature not specified in the publication
0.1
ATP
-
pH 7.6, 25°C, HCO3(-)-dependent ATPase, mutant A144Q
0.11
ATP
-
pH 7.6, 25°C, HCO3(-)-dependent ATPase, mutant D207A
0.11
ATP
mutant M174E, Gln-dependent ATPase activity, pH 7.6, temperature not specified in the publication
0.12
ATP
-
pH 7.6, 25°C, HCO3(-)-dependent ATPase, mutant A144Q/D207A
0.13
ATP
-
pH 7.6, 25°C, HCO3(-)-dependent ATPase, mutant D753N
0.14
ATP
-
pH 7.6, 25°C, HCO3(-)-dependent ATPase, mutant D753A
0.16
ATP
mutant A23K, CP-synthesis (co-substrate: L-Gln, HCO3-), pH 7.6, temperature not specified in the publication
0.17
ATP
mutant A23K, HCO3-dependent ATPase activity, pH 7.6, temperature not specified in the publication
0.17
ATP
wild-type, HCO3-dependent ATPase activity, pH 7.6, temperature not specified in the publication
0.18
ATP
mutant G575K, HCO3-dependent ATPase activity, pH 7.6, temperature not specified in the publication
0.19
ATP
mutant G575F, HCO3-dependent ATPase activity, pH 7.6, temperature not specified in the publication
0.28
ATP
mutant A23K, Gln-dependent ATPase activity, pH 7.6, temperature not specified in the publication
0.29
ATP
-
pH 7.6, 25°C, HCO3(-)-dependent ATPase, mutant I211S
0.33
ATP
mutant M911E, HCO3-dependent ATPase activity, pH 7.6, temperature not specified in the publication
0.35
ATP
-
pH 7.6, 25°C, HCO3(-)-dependent ATPase, mutant S209A
0.39
ATP
mutant L720E, HCO3-dependent ATPase activity, pH 7.6, temperature not specified in the publication
0.5
ATP
mutant M911E, Gln-dependent ATPase activity, pH 7.6, temperature not specified in the publication
0.59
ATP
mutant L648E, HCO3-dependent ATPase activity, pH 7.6, temperature not specified in the publication
1.3
ATP
mutant A23F, Gln-dependent ATPase activity, pH 7.6, temperature not specified in the publication
1.33
ATP
-
pH 7.6, 25°C, mutant D334A, L-glutamine hydrolysis
1.51
ATP
-
pH 7.6, 25°C, Gln-dependent ATPase, mutant D207A
1.65
ATP
-
pH 7.6, 25°C, Gln-dependent ATPase, mutant D207N
1.7
ATP
-
pH 7.6, 25°C, Gln-dependent ATPase, mutant A144Q/D207A
1.71
ATP
-
pH 7.6, 25°C, Gln-dependent ATPase, mutant D753A
1.9
ATP
mutant G575K, CP-synthesis (co-substrate: L-Gln, HCO3-), pH 7.6, temperature not specified in the publication
1.9
ATP
mutant M174E, CP-synthesis (co-substrate: L-Gln, HCO3-), pH 7.6, temperature not specified in the publication
1.9
ATP
wild-type, CP-synthesis (co-substrate: L-Gln, HCO3-), pH 7.6, temperature not specified in the publication
2
ATP
mutant L720E, Gln-dependent ATPase activity, pH 7.6, temperature not specified in the publication
2.13
ATP
-
pH 7.6, 25°C, mutant N311A, L-glutamine hydrolysis
2.2
ATP
-
ammonia-dependent ADP formation
2.43
ATP
-
pH 7.6, 25°C, Gln-dependent ATPase, mutant S209A
3.19
ATP
-
pH 7.6, 25°C, mutant Q351A, L-glutamine hydrolysis
3.2
ATP
mutant L648E, Gln-dependent ATPase activity, pH 7.6, temperature not specified in the publication
3.3
ATP
mutant G575F, Gln-dependent ATPase activity, pH 7.6, temperature not specified in the publication
3.3
ATP
mutant G575K, Gln-dependent ATPase activity, pH 7.6, temperature not specified in the publication
3.35
ATP
-
chimeric enzyme, in comparison to effect of phosphorylation and presence of effectors
3.6
ATP
wild-type, Gln-dependent ATPase activity, pH 7.6, temperature not specified in the publication
3.94
ATP
-
pH 7.6, 25°C, Gln-dependent ATPase, mutant A144Q
4.04
ATP
-
pH 7.6, 25°C, Gln-dependent ATPase, wild-type enzyme
4.77
ATP
-
pH 7.6, 25°C, Gln-dependent ATPase, mutant I211S
4.79
ATP
-
pH 7.6, 25°C, wild-type enzyme, L-glutamine hydrolysis
5.18
ATP
-
pH 7.6, 25°C, Gln-dependent ATPase, mutant F755A
5.3
ATP
-
deletion mutant, in comparison to the presence of effectors
5.6
ATP
-
pH 7.6, 25°C, Gln-dependent ATPase, mutant D753N
5.75
ATP
-
pH 7.6, 25°C, mutant Q310A, L-glutamine hydrolysis
6.8
ATP
-
glutamine-dependent ADP formation
9.01
ATP
-
pH 7.6, 25°C, Gln-dependent ATPase, mutant P690Q
0.1
Gln
-
mutant enzyme E306A, Gln-dependent ATP hydrolysis
0.15
Gln
-
mutant enzyme E25Q/E383Q/E604Q, Gln-dependent ATP hydrolysis
0.17
Gln
-
mutant enzyme E577Q, Gln-dependent ATP hydrolysis
0.32
Gln
-
mutant enzyme E25Q/E383Q, Gln-dependent ATP hydrolysis
0.95
Gln
-
mutant enzyme R848A, Gln-dependent ATP hydrolysis
1.5
Gln
-
mutant enzyme E604Q, Gln-dependent ATP hydrolysis
3.2
Gln
-
mutant enzyme E383Q,Gln-dependent ATP hydrolysis
4
Gln
-
mutant enzyme E383QE916Q, Gln-dependent ATP hydrolysis
4
Gln
-
wild-type enzyme, Gln-dependent ATP hydrolysis
4.2
Gln
-
mutant enzyme E916Q, Gln-dependent ATP hydrolysis
0.005
L-Gln
-
pH 7.6, mutant alphaP360A/alphaH361A/betaR265A
0.1
L-Gln
mutant M174E/M378E, glutaminase activity pH 7.6, temperature not specified in the publication
0.13
L-Gln
mutant I18W/A23F/C24F, glutaminase activity pH 7.6, temperature not specified in the publication
0.13
L-Gln
mutant M378E, glutaminase activity pH 7.6, temperature not specified in the publication
0.14
L-Gln
mutant M911E, glutaminase activity pH 7.6, temperature not specified in the publication
0.15
L-Gln
mutant M174E, glutaminase activity pH 7.6, temperature not specified in the publication
0.21
L-Gln
mutant A23K, glutaminase activity pH 7.6, temperature not specified in the publication
0.84
L-Gln
mutant L720E, glutaminase activity pH 7.6, temperature not specified in the publication
0.88
L-Gln
-
pH 7.6, mutant betaQ262A/betaR265A/betaN266A
1
L-Gln
-
pH 7.6, mutant betaQ262A/betaR265A
1.08
L-Gln
mutant A23F, glutaminase activity pH 7.6, temperature not specified in the publication
1.1
L-Gln
-
pH 7.6, mutant betaR265A
1.2
L-Gln
-
pH 7.6, mutant alphaD362A/betaR265A
1.3
L-Gln
mutant L648E, glutaminase activity pH 7.6, temperature not specified in the publication
1.4
L-Gln
-
pH 7.6, wild-type enzyme
2.2
L-Gln
wild-type, glutaminase activity pH 7.6, temperature not specified in the publication
2.3
L-Gln
-
pH 7.6, mutant alphaP360A/alphaH361A
2.4
L-Gln
-
pH 7.6, mutant alphaD362A
3
L-Gln
mutant G575F, glutaminase activity pH 7.6, temperature not specified in the publication
3.1
L-Gln
mutant G575K, glutaminase activity pH 7.6, temperature not specified in the publication
0.53
L-glutamine
-
pH 7.6, 25°C, mutant Q351A, L-glutamine hydrolysis
1.05
L-glutamine
-
pH 7.6, 25°C, mutant N311A, L-glutamine hydrolysis
1.17
L-glutamine
-
pH 7.6, 25°C, mutant D334A, L-glutamine hydrolysis
1.6
L-glutamine
-
pH 7.6, 25°C, mutant Q310A, L-glutamine hydrolysis
2.13
L-glutamine
-
pH 7.6, 25°C, wild-type enzyme, L-glutamine hydrolysis
0.097
NH4+
-
mutant enzyme E25Q/E383Q/E604Q, NH4Cl-dependent ATP hydrolysis
0.12
NH4+
-
mutant enzyme E577Q, NH4Cl-dependent ATP hydrolysis
0.51
NH4+
-
mutant enzyme E25Q/E383Q, NH4Cl-dependent ATP hydrolysis
1.3
NH4+
-
mutant enzyme E604Q, NH4Cl-depebndent ATP hydrolysis
1.3
NH4+
-
mutant enzyme E916Q, NH4Cl-dependent ATP hydrolysis
1.7
NH4+
-
mutant enzyme E383Q, NH4Cl-dependent ATP hydrolysis
1.9
NH4+
-
pH 7.6, wild-type enzyme
2
NH4+
-
pH 7.6, mutant alphaP360A/alphaH361A
2.5
NH4+
-
pH 7.6, mutant alphaP360A/alphaH361A/betaR265A
2.5
NH4+
-
pH 7.6, mutant betaR265A
3.6
NH4+
-
pH 7.6, mutant betaQ262A/betaR265A
3.8
NH4+
-
pH 7.6, mutant alphaD362A
3.9
NH4+
-
wild-type enzyme, NH4Cl-dependent ATP hydrolysis
5.8
NH4+
-
pH 7.6, mutant alphaD362A/betaR265A
additional information
additional information
-
-
-
additional information
additional information
-
-
-
additional information
additional information
-
comparison of kcat in absence and presence of L-ornithine, UMP and IMP for wild-type and mutant enzymes
-
additional information
additional information
-
comparison of kcat of partial reactions of wild-type and mutant enzymes
-
additional information
additional information
-
comparison of kcat-values for wild-type and mutant enzymes in absence and presence of ornithine
-
additional information
additional information
-
kcat values for the formation of ADP, glutamate and carbonyl phosphate of the wild-type and mutant enzymes, kcat values for the hydrolysis of gamma-glutamyl hydroxamate, gamma-glutamyl hydrazide, gamma-glutamyl hydroxamate and hydroxylamine of the wild-type and mutant enzymes
-
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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
-
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
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
-
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
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
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
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
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/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
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+
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
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
C269S
-
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
-
mutant is unable to hydrolyse glutamine, the mutant is proved to be an adequate mimic for studies of the Michaelis complex
D207A
-
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
D207A
-
mutant enzyme retains ATP specificity
E761A
-
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
E761A
-
the residue is a key in the allosteric signal transduction pathway from ornithine to ATP
G359F
-
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
G359F
-
Km values of L-glutamine are increased
H995A
-
mutant exhibits normal kinetics for the activator ornithine and for the substrates in presence of ornithine, the mutation selectiviely decrease 5.6 and 2.3fold the apparent affinity for UMP and IMP, mutation abolishes the ability to be photoaffinity labeled with IMP
H995A
-
mutation has little effect on enzyme activity in comparison to wild-type enzyme
L421E
-
mutant forms a (alpha/beta)2-dimer in presence of ornithine, IMP and UMP
L421E
-
mutation prevents tetramer but not dimer formation
L990A
-
mutation abolishes oligomer formation even high enzyme concentrations or in the presence of ornithine
L990A
-
mutation abolishs IMP activation and UMP inhibition in comparison to wild-type enzyme
P360L
-
UMP still inhibits the activity of the mutant enzyme, 30fold reduced affinity for ornithine and 20fold reduced affinity for IMP in comparison to wild-type enzyme
P360L
-
mutation in large subunit significantly decreases synthesis of carbamoyl phosphate without completely inactivating the enzyme
T1042I
-
greatly reduced activation by ornithine, the affinities for both UMP and IMP are reduced in comparison to wild-type enzyme in comparison to wild-type enzyme
T1042I
-
mutation reduces activation by ornithine, the mutated enzyme is still sensitive to UMP and IMP
additional information
-
smaller recombinant proteins in which the subdomains A1, A2, A3, B1, B2, or B3 of the synthetase subunit are deleted, show that subdomains A1 and B1 are attenuation domains which suppress the intrinsically high activity and are required for the physical association with the glutaminase subunit
additional information
-
the 120000 MW synthetase subunit and the 40000 MW glutaminase subunit, in which the intergenic region between the contiguous carA and carB genes is deleted and the sequence encoding the carbamoyl-phosphate synthase subunits is fused in frame. The resulting fusion protein is activated 10fold relative to the native protein, it is responsive to the allosteric activator Orn, and cannot use NH4+ as a nitrogen donor. The functional linkage that coordinates the rate of glutamine hydrolysis with the activation of bicarbonate is abolished
additional information
-
a chimeric deletion mutant A1A2B3 lacks 59 of 163 residues from the carboxyl end of the Escherichia coli /mammalian chimera, this mutant is somewhat more active than the full-length chimeric protein but has reduced sensitivity to allosteric effectors
additional information
-
A series of mutants created and probes the direct transport of carbamate through the carbamate tunnel, the partial reactions of enzyme have not been significantly impaired by these mutations, and thus, the catalytic machinery at the individual active site has not been functionally pertured
additional information
-
A series of mutants created within the ammonia tunnel are prepared by placement of a constriction via site-directed mutagenesis. The degree of constriction within the ammonia tunnel of these enzymes is found to correlate to the extent of the uncoupling of the partial reactions, the diminution of carbamoyl phosphate formation, and the percentage of the internally derived ammonia that is channelled through the ammonia tunnel
additional information
-
several mutations increase, reduce, suppresse, or reverse the allosteric effects of ornithine, UMP and IMP, the mutational analysis of ornithine, and potassium binding site strongly supports the proposal for a coupling between the allosteric transduction signal pathways for UMP,IMP and ornithine
additional information
-
the mutants are constructed to mimic the potentially significant substitutions which is observed in the frog enzyme and to allow determination of the effects of these changes on the glutamine binding ability of enzyme
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