Information on EC 6.3.5.4 - asparagine synthase (glutamine-hydrolysing) and Organism(s) Homo sapiens and UniProt Accession P08243

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Homo sapiens
UNIPROT: P08243
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The expected taxonomic range for this enzyme is: Eukaryota, Bacteria


The taxonomic range for the selected organisms is: Homo sapiens

EC NUMBER
COMMENTARY hide
6.3.5.4
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RECOMMENDED NAME
GeneOntology No.
asparagine synthase (glutamine-hydrolysing)
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
ATP + L-aspartate + L-glutamine + H2O = AMP + diphosphate + L-asparagine + L-glutamate
show the reaction diagram
reaction mechanism, structure-function relationship
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
L-asparagine biosynthesis I
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aspartate and asparagine metabolism
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Alanine, aspartate and glutamate metabolism
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Metabolic pathways
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Biosynthesis of secondary metabolites
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SYSTEMATIC NAME
IUBMB Comments
L-aspartate:L-glutamine amido-ligase (AMP-forming)
The enzyme from Escherichia coli has two active sites [4] that are connected by an intramolecular ammonia tunnel [5,6]. The enzyme catalyses three distinct chemical reactions: glutamine hydrolysis to yield ammonia takes place in the N-terminal domain. The C-terminal active site mediates both the synthesis of a beta-aspartyl-AMP intermediate and its subsequent reaction with ammonia. The ammonia released is channeled to the other active site to yield asparagine [6].
CAS REGISTRY NUMBER
COMMENTARY hide
37318-72-2
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
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asparagine synthetase deficiency, ASD, is a neurological disorder having severe impacts on psychomotor development and mortality at an early age. Children with mutations in the ASNS gene exhibit developmental delays, intellectual disability, microcephaly, intractable seizures, and progressive brain atrophy. Mutations in the ASNS gene have been clinically associated with asparagine synthetase deficiency (ASD), phenotype. Neurologic disorder associated with asparagine synthetase deficiency (ASD). The transcription factor ATF4 binds to an enhancer element within the proximal promoter of the ASNS gene and activates transcription. Role of ATF4 in tumor cell survival and proliferation, ATF4 knockdown causes reduced survival in HT-1080 fibrosarcoma and DLD-1 colorectal adenocarcinoma cells in the absence of nonessential amino acids. Reduced proliferative capacity and increased apoptosis correlate with lower ASNS expression in the ATF4-deficient cells. Supplementation of the tumor cells with asparagine, but not other amino acids, leads to increased cell survival. Role of ASNS activity in modulating tumor growth
physiological function
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asparagine synthetase (ASNS) catalyzes the synthesis of asparagine and glutamate from aspartate and glutamine in an ATP-dependent amidotransferase reaction. Elevated ASNS protein expression is associated with resistance to asparaginase therapy in childhood acute lymphoblastic leukemia. Regulation of ASNS expression, overview. transcription factor ATF4 binds to an enhancer element within the proximal promoter of the ASNS gene and activates transcription. Asparagine depletion activates the amino acid response, AAR, whereas endoplasmic reticulum stress activates the unfolded protein response, UPR
malfunction
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knockdown of asparagine synthetase (ASNS) leads to cell death even in the presence of glutamine, which can be reversed by addition of exogenous asparagine. Asparagine plays a critical role in regulating cellular adaptation to glutamine depletion. ASNS knockdown leads to profound apoptosis even in the presence of glutamine. Addition of extracellular asparagine completely restored cell survival and proliferation. Clinically, the expression of ASNS correlates with the progression of disease and poor prognosis of glioma and neuroblastoma patients. In neuroblastoma with unfavourable prognosis, ASNS expression is significantly higher. Asparagine-induced suppression of apoptosis: asparagine addition to glutamine-deprived cells alters the transcriptional response, suppressing the induction of the reported UPR effectors CHOP and XBP1 while maintaining the transcriptional induction of adaptive components of the UPR-response such as ASNS and HERPUD1. At the protein level, exogenous addition of asparagine suppresses CHOP induction without altering ATF4 accumulation or upstream eIF2alpha phosphorylation
physiological function
additional information
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
ATP + L-aspartate + L-glutamine + H2O
AMP + diphosphate + L-asparagine + L-glutamate
show the reaction diagram
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-
-
-
?
ATP + L-aspartate + NH3
AMP + diphosphate + L-asparagine
show the reaction diagram
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-
-
-
?
L-glutamine + H2O
L-glutamate + NH3
show the reaction diagram
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-
-
-
?
ATP + L-Asp + L-Gln
AMP + diphosphate + Asn + Glu
show the reaction diagram
ATP + L-Asp + NH3
AMP + diphosphate + Asn
show the reaction diagram
ATP + L-aspartate + L-glutamine
AMP + diphosphate + L-asparagine + L-glutamate
show the reaction diagram
ATP + L-aspartate + L-glutamine + H2O
AMP + diphosphate + L-asparagine + L-glutamate
show the reaction diagram
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-
-
-
?
additional information
?
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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 + L-aspartate + L-glutamine + H2O
AMP + diphosphate + L-asparagine + L-glutamate
show the reaction diagram
-
-
-
-
?
ATP + L-aspartate + NH3
AMP + diphosphate + L-asparagine
show the reaction diagram
-
-
-
-
?
L-glutamine + H2O
L-glutamate + NH3
show the reaction diagram
-
-
-
-
?
ATP + L-Asp + L-Gln
AMP + diphosphate + Asn + Glu
show the reaction diagram
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the basic region leucine zipper protein ATF5, a transcriptional activator, stimulates asparagine promoter/reporter gene transcription via the nutrient-sensing response unit
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-
?
ATP + L-aspartate + L-glutamine
AMP + diphosphate + L-asparagine + L-glutamate
show the reaction diagram
ATP + L-aspartate + L-glutamine + H2O
AMP + diphosphate + L-asparagine + L-glutamate
show the reaction diagram
-
-
-
-
?
additional information
?
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upregulation of asparagine synthetase fails to avert cell cycle arrest induced by L-asparaginase in TEL/AML1-positive leukaemic cells
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Mg2+
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required
Mg2+
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required
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
6-diazo-5-oxo-L-norleucine
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loss of Gln-dependent reactions, but no effect on ATP binding as measured during amminoa-dependent Asn synthesis
8-N3ATP
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loss of NH4+-dependent Asn synthesis, but no effect on the glutaminase activity
beta-asparaginyladenylate
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Gln
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0.4-2.0 mM, inhibits the ammonia-dependent production of Asn
mupirocin
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phosmidosine
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sulfoximine adenylate
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most potent inhibitor
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Phytohemagglutinin
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.38
aspartic acid
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pH 8, reaction with glutamine, C-terminally tagged recombinant enzyme
0.08 - 0.11
ATP
1.3
L-aspartic acid
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pH 8, reaction with NH3, C-terminally tagged recombinant enzyme
1.9
L-glutamine
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pH 8, C-terminally tagged recombinant enzyme
1.7
NH3
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pH 8, C-terminally tagged recombinant enzyme
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1.6 - 1.7
ATP
1.7
glutamine
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pH 8, C-terminally tagged recombinant enzyme
1.3 - 1.7
L-aspartic acid
1.8
NH3
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pH 8, C-terminally tagged recombinant enzyme
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.000285
sulfoximine adenylate
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SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
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Manually annotated by BRENDA team
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Manually annotated by BRENDA team
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high expression in T-lineage and low expression in B-lineage
Manually annotated by BRENDA team
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higher expression than in lymphoblastic leukemia cells
Manually annotated by BRENDA team
additional information
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
64000
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SDS-PAGE
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
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the enzyme contains two functional domains, the N-terminal domain (residues 1-208) consists of a two-layer, antiparallel beta-sheet core surrounded by four alpha-helices, this domain harbors the glutamine-binding pocket, consisting of residues Arg49, Asn75, Glu77, and Asp97. The C-terminal domain (residues 209-561) is composed primarily of alpha-helices, but also encompasses a five-stranded, parallel beta-sheet that contains the ATP-binding site: residues Leu256, Val288, Asp295, Ser363, Gly364, Glu365, and Asp401
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
ATP protects from inactivation by UV irradiation in the presence of 8-N3ATP
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-80°C, recombinant C-terminally tagged enzyme is stable on prolonged storage
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
one-step immunoaffinity purification
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
gene ASNS, a single copy gene located on chromosome 7
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C-terminally tagged enzyme, baculovirus-based expression system, the recombinant enzyme is correctly processed, exhibits high activity and is stable on prolonged storage at -80°C
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cloned into a 2 mü plasmid, pBS24.1GAS, suitable for replication in a Saccharomyces cerevisiae ciro strain AB116
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expression of AS-GFP fusion protein in MOLT-4 cells
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expression of several mutant enymes in Saccharomyces cerevisiae
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mutant enzyme in which the N-terminal Cys is replaced by Ala is expressed in Saccharomyces
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EXPRESSION
ORGANISM
UNIPROT
LITERATURE
ASNS activity is highly responsive to cellular stress, primarily by increased transcription from the single gene located on chromosome 7. The transcription factor ATF4 binds to an enhancer element within the proximal promoter of the ASNS gene and activates transcription
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human enzyme activity is highly regulated in response to cell stress, primarily by increased transcription from a single gene located on chromosome 7. Protein limitation or an imbalanced dietary amino acid composition activate the ASNS gene through the amino acid response, a process that is replicated in cell culture through limitation for any single essential amino acid. Endoplasmic reticulum stress also increases ASNS transcription through the PERK-eIF2-ATF4 arm of the unfolded protein response. Both the amino acid response and unfolded protein response lead to increased synthesis of ATF4, which binds to the C/EBP-ATF response element and induces ASNS transcription
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transcription factor ATF4 induces asparagine synthetase which results in glutamine-dependent asparagine synthesis from aspartate, in turn asparagine accumulation then suppresses GCN2 and reduces ATF4
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
A380S
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naturally occuring mutation, homozygous mutation, mutation of a polar residue in the hydrophobic region
A6E
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naturally occuring mutation, compound heterozygous, mutation of a charged amino acid in hydrophobic region, causing steric clash with Phe8
F362V
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naturally occuring mutation, homozygous mutation, causes a decrease in van der Waals interactions
G289A
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naturally occuring mutation, compound heterozygous, mutation proximal to the ATP-binding site, causing steric hindrance with Ser293
L145S
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naturally occuring mutation, compound heterozygous, mutation of a polar side chain in hydrophobic region
L247W
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naturally occuring mutation, causes a decrease in van der Waals interactions
R340H
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naturally occuring mutation, homozygous mutation, causes a loss of hydrogen bonds, and a steric clash with Phe482
R49Q
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naturally occuring mutation, homozygous mutation of the glutamine-binding site, causes loss of hydrogen bonding
R550C
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naturally occuring mutation, causes a decrease in side chain length likely to result in loss of interactions; naturally occuring mutation, homozygous mutation, causes a decrease in side chain length likely to result in loss of interactions
S480F
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naturally occuring mutation, compound heterozygous, mutation of a nonpolar residue on protein surface that may decrease solubility
T337I
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naturally occuring mutation, Proximal to ATP-binding site, causes a hydrophobic patch on protein that may decrease solubility
V489D
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naturally occuring mutation, compound heterozygous, inserts a charged amino acid in hydrophobic region
Y398C
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naturally occuring mutation, homozygous mutation, causes a decrease in van der Waals interactions, solvent-accessible thiol group
C1A
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altering Cys-1 to either Ala or Ser eliminated the Gln-dependent activity, while only minimally affecting the kinetic properties of the NH4+-dependent reaction
C1S
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altering Cys-1 to either Ala or Ser eliminated the Gln-dependent activity, while only minimally affecting the kinetic properties of the NH4+-dependent reaction
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
analysis
mass spectrometry-based procedure for the direct quantification of asparagine synthetase protein concentration in complex sample mixtures. Assay is able to distinguish samples from transformed cell lines that express the enzyme over a wide dynamic range of concentration. The method directly detects asparagine synthetase protein, use in blast samples from patients with acute lymphoblastic leukemia
medicine