Information on EC 2.7.2.8 - acetylglutamate kinase

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

EC NUMBER
COMMENTARY hide
2.7.2.8
-
RECOMMENDED NAME
GeneOntology No.
acetylglutamate kinase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
ATP + N-acetyl-L-glutamate = ADP + N-acetyl-L-glutamyl 5-phosphate
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
phospho group transfer
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Arginine biosynthesis
-
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arginine metabolism
-
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Biosynthesis of antibiotics
-
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Biosynthesis of secondary metabolites
-
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L-arginine biosynthesis II (acetyl cycle)
-
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L-arginine biosynthesis III (via N-acetyl-L-citrulline)
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L-arginine biosynthesis IV (archaebacteria)
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L-ornithine biosynthesis I
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Metabolic pathways
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SYSTEMATIC NAME
IUBMB Comments
ATP:N-acetyl-L-glutamate 5-phosphotransferase
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CAS REGISTRY NUMBER
COMMENTARY hide
9027-58-1
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
strain W2D, ATCC 25542, derepressed mutant
-
-
Manually annotated by BRENDA team
strain Wc2
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-
Manually annotated by BRENDA team
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-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
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-
-
Manually annotated by BRENDA team
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-
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Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
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argB gene coding the N-acetyl-L-glutamate kinase is first overexpressed in the strain SYPA5-5, whereas the L-arginine production is narrowly increased by 15.4%
metabolism
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Sulfolobus solfataricus lacks ornithine acetyltransferase and thus forms N-acetylglutamate exclusively via the energetically less favourable reaction catalysed by N-acetylglutamate synthase, investing 1 mol of acetyl CoA per mol of N-acetyl intermediate synthesized
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
acetyl-CoA + L-glutamate
coenzyme A + N-acetyl-L-glutamate
show the reaction diagram
ATP + N-acetyl-L-glutamate
ADP + N-acetyl-L-glutamate 5-phosphate
show the reaction diagram
ATP + N-acetyl-L-glutamate
ADP + N-acetyl-L-glutamyl 5-phosphate
show the reaction diagram
ATP + N-carbamoyl-L-glutamate
ADP + N-carbamoyl-L-glutamate 5-phosphate
show the reaction diagram
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at 33% of the activity with N-acetyl-L-glutamate
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-
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ATP + N-formyl-L-glutamate
ADP + N-formyl-L-glutamate 5-phosphate
show the reaction diagram
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at 20% of the activity with N-acetyl-L-glutamate
-
-
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dATP + N-acetyl-L-glutamate
dADP + N-acetyl-L-glutamate 5-phosphate
show the reaction diagram
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as effective as ATP
-
-
-
additional information
?
-
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
ATP + N-acetyl-L-glutamate
ADP + N-acetyl-L-glutamate 5-phosphate
show the reaction diagram
ATP + N-acetyl-L-glutamate
ADP + N-acetyl-L-glutamyl 5-phosphate
show the reaction diagram
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NAGK catalyzes the second step of arginine biosynthesis. In Pseudomonas aeruginosa, this step is rate limiting, and feedback regulated and sigmoidally inhibited by arginine
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-
?
additional information
?
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Ca2+
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Mn2+, Zn2+, Co2+ and Ca2+ in this order can partially replace Mg2+
Zn2+
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Mn2+, Zn2+, Co2+ and Ca2+ in this order can partially replace Mg2+
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2-oxoglutarate
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complete inhibition at 0.5 mM
arginine
L-arginine
L-arginine methyl ester
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1 mM, 80% inhibition
L-citrulline
additional information
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not inhibitory: D-arginine, agmatine, citrulline, L-canavanine, L-lysine, L-ornithine, guanidinium ions, urea at 1 mM, 37C
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2-Ketoglutarate
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only if bound to the PII protein ATP complex
ATP
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maximum activity at 10 mM
L-arginine
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slight activation is observed at 1 mM L-arginine
PII protein
additional information
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2-ketoglutarate shows no additive effect to the activation of PII protein
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1.3
AcCoA
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only for N-acetylglutamate synthase activity
0.29 - 20.1
ATP
2.8
L-glutamate
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only for N-acetylglutamate synthase activity
0.2 - 898
N-acetyl-L-glutamate
additional information
additional information
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
7.7 - 13
ATP
2 - 187.5
N-acetyl-L-glutamate
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1.3 - 220
N-acetyl-L-glutamate
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
30.4 - 41.5
ATP
400
L-arginine
DELTA357-513 (truncated mutant lacking C-terminal 150 amino acids), pH not specified, 37C
IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.02 - 784
L-arginine
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.0008
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pH 8.0, 70C, activity in cell culture in growth medium with 0.2% yeast extract
0.003
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pH 8.0, 70C, activity in cell culture in minimal growth medium with 20 mM glucose and 5 mM NH4+
0.54
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pH 7.4, 37C
3.3
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pH 7.5, free enzyme
12
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without PII protein
13.3
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pH 7.5, complex of enzyme and PII protein
20.1
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pH 7.4, 37C
24
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with PII protein
45
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37C, pH 7.5
130
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37C, pH 7.5
677
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80C, pH 7.5
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.4
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assay at
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4.6 - 7
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pH 4.6: about 75% of maximum activity, pH 7: about 60% of maximum activity
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30
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assay at
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
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sheath and blade, coordinate expression of enzyme and PII-like protein GlnB during the life span
Manually annotated by BRENDA team
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Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
PDB
SCOP
CATH
ORGANISM
UNIPROT
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv)
Pseudomonas aeruginosa (strain ATCC 15692 / PAO1 / 1C / PRS 101 / LMG 12228)
Saccharomyces cerevisiae (strain ATCC 204508 / S288c)
Saccharomyces cerevisiae (strain ATCC 204508 / S288c)
Saccharomyces cerevisiae (strain ATCC 204508 / S288c)
Saccharomyces cerevisiae (strain ATCC 204508 / S288c)
Streptococcus mutans serotype c (strain ATCC 700610 / UA159)
Synechococcus elongatus (strain PCC 7942)
Synechococcus elongatus (strain PCC 7942)
Thermotoga maritima (strain ATCC 43589 / MSB8 / DSM 3109 / JCM 10099)
Xylella fastidiosa (strain Temecula1 / ATCC 700964)
Xylella fastidiosa (strain Temecula1 / ATCC 700964)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30000
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recombinant protein, SDS-PAGE
33000
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mass spectrometry
93000
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gel filtration
180000
190000
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gel filtration in presence of N-acetylglutamate
198000
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calculated from amino acid sequence
230000
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gel filtration
400000
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gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hexamer
tetramer
additional information
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
side-chain modification
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methylation of surface lysine residues
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
as complex with PII protein, NAGK binds Mg2+, ADP, arginine and N-acetylglutamate; hanging drop vapor diffusion method at room temperature, crystal structure of a complex formed between two homotrimers of PII and a single hexamer of enzyme bound to the metabolites N-acetylglutamate, ADP, ATP, and arginine
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crystal structures of EcNAGK free from substrates or complexed with the product N-acetyl-L-glutamyl-5-phosphate (NAGP) and with sulfate are determined at 2 A resolution. Structures reveal a novel, very open NAGK conformation to which substrates associate and from which roducts dissociate. In this conformation, the C-domain, which hosts most of the nucleotide site, rotates 24-28 away from the N-domain, which hosts the acetylglutamate site, whereas the empty ATP site also exhibits some changes
in complex with MgADP-, N-acetyl-glutamte, AlF4-, with MgADP-, N-acetyl-glutamte, with ADp and SO42-
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crystal structure of Maricaulis maris NAGS/K (mmNAGS/K) at 2.7 A resolution shows that it is a tetramer
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without arginine
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crystal structures of both the DUF619 domain-lacking yNAGK, ligand-free as well as complexed with acetylglutamate or acetylglutamate and arginine, and of complete mature yNAGK are determined. yNAGK has as central structure a flat tetramer formed by two dimers of amino acid kinase domains
hanging-drop vapor diffusion method
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crystal structure of the complex between acetylglutamate kinase and PII of Synechococcus elongatus, at 2.75 A resolution
in complex with arginine
of the recombinant wild type protein, the selenomethionine substituted enzyme, the mutants and the methylated enzyme, cocrystallization with ATP, ADP, acetyl-CoA, CoA, N-acetyl-L-glutamate, adenylylimidodiphosphate, arginine
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
64
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10 min, 75% loss of activity without stabilizer, about 40% loss of activity with 75 mM N-acetyl-L-glutamate, 10 min, 10% loss of activity with 1 mM L-arginine
80
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1 h, 80% residual activity
85
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1 h, 60% residual activity
additional information
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GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
L-arginine protects against inactivating effects of high concentrations of urea
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L-arginine protects against the inactivating effects of heat
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loss of activity on repeated freezing and thawing
urea, 4.0 M, complete loss of activity after 120 min
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20C, stable over extended periods
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4C, 0.1 M phosphate buffer, stable
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
affinity chromatography and gel filtration by binding to the PII protein, Ni-nitrilotriacetic acid agarose to purify the recombinant protein
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His-Select nickel affinity gel column chromatography
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of the recombinant protein
recombinant enzyme
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using Ni-NTA chromatography
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli
expressed in Escherichia coli as a His-tagged fusion protein
expressed in Escherichia coli BL21(DE3) cells
expression in Escherichia coli
expression in Escherichia coli, selenomethionine substituted enzyme, site directed mutagenesis
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EXPRESSION
ORGANISM
UNIPROT
LITERATURE
threefold repression of enzyme formation by arginine
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
E19R
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Km and kcat (N-acetyl-L-glutamate) similar to wild-type, IC50 (L-arginine) approximately 55fold increased compared to wild-type
H268N
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Km and kcat (N-acetyl-L-glutamate) similar to wild-type, IC50 (L-arginine) approximately 55fold increased compared to wild-type
H268N/H26E
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Km and kcat (N-acetyl-L-glutamate) similar to wild-type, IC50 (L-arginine) 875fold increased compared to wild-type
H268N/H26E/E19R
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Km and kcat (N-acetyl-L-glutamate) similar to wild-type, IC50 (L-arginine) 1960fold increased compared to wild-type
H26E
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Km and kcat (N-acetyl-L-glutamate) similar to wild-type, IC50 (L-arginine) approximately 55fold increased compared to wild-type
DELTA2-15
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Km (N-acetyl-L-glutamate) increased compared to wild-type, kcat decreased compared to wild-type, IC50 (L-arginine) 71fold increased compared to wild-type
DELTA2-19
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Km (N-acetyl-L-glutamate) increased compared to wild-type, kcat decreased compared to wild-type, IC50 (L-arginine) 148fold increased compared to wild-type
DELTA2-29
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Km (N-acetyl-L-glutamate) increased compared to wild-type, kcat decreased compared to wild-type, IC50 (L-arginine) 163fold increased compared to wild-type
DELTA25
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Km (N-acetyl-L-glutamate) highly increased compared to wild-type, kcat highly decreased compared to wild-type, IC50 (L-arginine) 17fold increased compared to wild-type
DELTA8
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Km (N-acetyl-L-glutamate) increased compared to wild-type, kcat highly decreased compared to wild-type, IC50 (L-arginine) 16fold increased compared to wild-type
E19A
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Km (N-acetyl-L-glutamate) similar to wild-type, kcat similar to wild-type, IC50 (L-arginine) 40fold increased compared to wild-type
E19R
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Km (N-acetyl-L-glutamate) similar to wild-type, kcat similar to wild-type, IC50 (L-arginine) 61fold increased compared to wild-type
E281A
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Km (N-acetyl-L-glutamate) highly increased compared to wild-type, kcat similar to wild-type, IC50 (L-arginine) 21fold increased compared to wild-type
G287A
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Km (N-acetyl-L-glutamate) similar to wild-type, kcat decreased compared to wild-type, IC50 (L-arginine) 37fold increased compared to wild-type
G287D
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Km (N-acetyl-L-glutamate) similar to wild-type, kcat similar to wild-type, IC50 (L-arginine) 40fold increased compared to wild-type
H268A
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Km (N-acetyl-L-glutamate) similar to wild-type, kcat similar to wild-type, IC50 (L-arginine) 39fold increased compared to wild-type
H268N
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Km (N-acetyl-L-glutamate) similar to wild-type, kcat similar to wild-type, IC50 (L-arginine) 58fold increased compared to wild-type
H26A
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Km (N-acetyl-L-glutamate) similar to wild-type, kcat similar to wild-type, IC50 (L-arginine) 54fold increased compared to wild-type
R209A
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Km (N-acetyl-L-glutamate) similar to wild-type, kcat similar to wild-type, IC50 (L-arginine) 46fold increased compared to wild-type
R209K
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Km (N-acetyl-L-glutamate) similar to wild-type, kcat similar to wild-type, IC50 (L-arginine) 37fold increased compared to wild-type
W23A
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Km (N-acetyl-L-glutamate) similar to wild-type, kcat similar to wild-type, IC50 (L-arginine) 4.5fold increased compared to wild-type
D162E
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about 0.1% of wild-type activity
G11A
G11A does not hamper recombinant enzyme expression or purification. Mutant shows a 10fold decrease in Vmax values and it selectively increases 8fold the Km for ATP, affecting much less (3fold increase) the apparent Km for N-acetyl-L-glutamate
K8R
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substantial although diminished activity
N158K
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substantial although diminished activity
R66K
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substantial although diminished activity
I106M
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to increase phasing power, three additional amino acids codons are mutated to methionine (I106M, I294M and L367M). Crystals from this mutant protein are diffracted to 2.7 A
I294M
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to increase phasing power, three additional amino acids codons are mutated to methionine (I106M, I294M and L367M). Crystals from this mutant protein are diffracted to 2.7 A
L367M
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to increase phasing power, three additional amino acids codons are mutated to methionine (I106M, I294M and L367M). Crystals from this mutant protein are diffracted to 2.7 A
E17A
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site-directed mutagenesis, the mutant shows reduced Vmax, the mutation results in decreased affinity of NAGK for arginine
E17D
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site-directed mutagenesis, the mutant shows reduced Vmax, the mutation results in decreased affinity of NAGK for arginine
E17Q
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site-directed mutagenesis, the mutant shows reduced Vmax, the mutation results in decreased affinity of NAGK for arginine
E284D
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site-directed mutagenesis, the mutant shows reduced Vmax and altered Km for the substrates
G290A
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site-directed mutagenesis, the mutant shows reduced Vmax and altered Km for the substrates
H271N
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site-directed mutagenesis, the mutant shows reduced Vmax and altered Km for the substrates
K213
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site-directed mutagenesis, the mutant shows reduced Vmax and altered Km for the substrates
Q10A
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site-directed mutagenesis, the mutant shows reduced Vmax and altered Km for the substrates
R24E
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site-directed mutagenesis, the mutant shows reduced Vmax , the mutation results in increased affinity of NAGK for arginine
Y21A
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site-directed mutagenesis, the mutant shows reduced Vmax and altered Km for the substrates
DELTA357-513
truncated mutant lacking C-terminal 150 amino acids (spanning residues 38-356), belonging to the DUF619 domain family, shows that is it stabilizes yNAGK, slows catalysis and modulates feed-back inhibition by arginine. Truncated yNAGK shows doubled kcat compared to wild-type, Km is almost not affected. IC50 (L-arginine) is lowered compared to wild-type. Truncated mutand shows lower thermal stability compared to wild-type
R233A
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mutant does not interact in vitro with PII protein of wild-type sequence in yeast two-hybrid analysis. Mutant interacts with PII variants containing I86N or I86T mutation
E186A/E387A
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to improve resolution in crystallization
E416A/K417A
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to improve resolution in crystallization
E94A/K95A
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to improve resolution in crystallization
K26A/E27A
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to improve resolution in crystallization
K279A/E280A
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to improve resolution in crystallization
K419A
-
to improve resolution in crystallization
additional information
-
N-helix N-terminal deletions spanning 16 residues dissociate NAGK to active dimers, those of 20 residues decrease the apparent affinity for arginine, and complete N-helix deletion of 26 residues abolishes arginine inhibition, overview
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