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Information on EC 3.5.1.122 - protein N-terminal glutamine amidohydrolase
for references in articles please use BRENDA:EC3.5.1.122
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EC Tree 3 Hydrolases
3.5 Acting on carbon-nitrogen bonds, other than peptide bonds
3.5.1 In linear amides
3.5.1.122 protein N-terminal glutamine amidohydrolase
IUBMB Comments
This enzyme participates in the eukaryotic ubiquitin-dependent Arg/N-end rule pathway of protein degradation, promoting the turnover of intracellular proteins that initiate with Met-Gln. Following the acetylation and removal of the initiator methionine, the exposed N-terminal glutamine is deaminated, resulting in its conversion to L-glutamate. The latter serves as a substrate for EC 2.3.2.8, arginyltransferase, making the protein susceptible to arginylation, polyubiquitination and degradation as specified by the N-end rule.
The enzyme appears in viruses and cellular organisms
Reaction Schemes
showN-terminal L-glutaminyl-[protein]
+
=
N-terminal L-glutamyl-[protein]
+
Synonyms
ntaq1, c8orf32, hntaq1, wdyhv1, cg8253, gln-specific amino-terminal (nt)-amidase, more
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SYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
glutamine-specific N-terminal amidase
NTAQ1
NtQ-amidase
Wdyhv1
NTAQ1
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REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
N-terminal L-glutaminyl-[protein] + H2O = N-terminal L-glutamyl-[protein] + NH3
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N-terminal L-glutaminyl-[protein] + H2O = N-terminal L-glutamyl-[protein] + NH3
catalytic mechanism determination of hNtaq1 based on the crystal structure of hNtaq1 and docking study. In the first step, nucleophilic sulfhydryl group of Cys28 approaches Cd of the amide group of the N-terminal glutamine and becomes deprotonated by His81. The sulfhydryl group of Cys28 plays a crucial role in the nucleophilic attack on acyl group in the N-terminal glutamine side chain of substrates, which results in formation of a tetrahedral intermediate. Asp97 facilitates the process by forming a hydrogen bond and electrostatic interactions with His81. The ammonia is released upon productive collapse of the tetrahedral intermediate and a water molecule enters the active site cavity and attacks S-acyl intermediate to convert glutamine to a glutamate. As the final step, the glutamate side chain is cleaved from S-acyl of Cys28. His81 first acts as a general base activation water for a nucleophilic attack on the S-acyl intermediate, and then upon collapse of the tetrahedral intermediate acts a general acid to protonate the leaving group, i.e. the thiolate of Cys28. The substrate peptide with newly formed N-terminal glutamate is released from the binding cleft at this stage and the enzyme is ready for another round of catalysis
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PATHWAY SOURCE
PATHWAYS
MetaCyc
Arg/N-end rule pathway (eukaryotic)
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SYSTEMATIC NAME
IUBMB Comments
protein N-terminal glutamine amidohydrolase
This enzyme participates in the eukaryotic ubiquitin-dependent Arg/N-end rule pathway of protein degradation, promoting the turnover of intracellular proteins that initiate with Met-Gln. Following the acetylation and removal of the initiator methionine, the exposed N-terminal glutamine is deaminated, resulting in its conversion to L-glutamate. The latter serves as a substrate for EC 2.3.2.8, arginyltransferase, making the protein susceptible to arginylation, polyubiquitination and degradation as specified by the N-end rule.
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SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
LITERATURE
COMMENTARY
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
N-terminal L-glutaminyl-[DHFRbt] + H2O
N-terminal L-glutamyl-[DHFRbt] + NH3
Substrates: substrate is Escherichia coli dihydrofolate reductase (DHFR) moiety fused to a C-terminal peptide (denoted as bt) that is biotinylated in vivo
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QLGSGAW + H2O
ELGSGAW + NH3
Substrates: about 80% of the activity compared to QKGSGAW
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N-terminal L-glutaminyl-[protein] + H2O
N-terminal L-glutamyl-[protein] + NH3
Substrates: -
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N-terminal L-glutaminyl-[protein] + H2O
N-terminal L-glutamyl-[protein] + NH3
Substrates: -
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N-terminal L-glutaminyl-[protein] + H2O
N-terminal L-glutamyl-[protein] + NH3
Substrates: -
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N-terminal L-glutaminyl-[protein] + H2O
N-terminal L-glutamyl-[protein] + NH3
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Substrates: -
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N-terminal L-glutaminyl-[protein] + H2O
N-terminal L-glutamyl-[protein] + NH3
Substrates: -
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N-terminal L-glutaminyl-[protein] + H2O
N-terminal L-glutamyl-[protein] + NH3
Substrates: -
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additional information
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Substrates: the amidohydrolase activity of the native enzyme and of proteolytic fragments is analyzed using either L-glutamine or gamma-L-glutamic 4-nitroanilide as substrates
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additional information
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Substrates: NTAQ1 primarily interacts with the protein N-terminal backbone, instead of the side chain, for substrate recognition. NTAQ1 recognizes substrates through water-mediated hydrogen-bonding networks and then establishes a specific binding conformation. The ionization properties of the second N-terminal residue in the substrate play an auxiliary role in substrate recognition
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additional information
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Substrates: dual specificity of yeast Nta1 (yNta1), importance of second-position residues in Asn/Gln-bearing N-terminal degradation signals (N-degrons), also cf. EC 3.5.1.121. Specific hydrogen bonds stabilize interactions between N-degron peptides and hydrophobic peripheral regions of the active site pocket, interactions between Nta1 and N-degron peptides, detailed overview. The enzyme shows glutamine-specific enzyme activity with dipeptides Gln-Val and Gln-Gly, Michaelis-Menten kinetics
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additional information
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Substrates: dual specificity of yeast Nta1 (yNta1), importance of second-position residues in Asn/Gln-bearing N-terminal degradation signals (N-degrons), also cf. EC 3.5.1.121. Specific hydrogen bonds stabilize interactions between N-degron peptides and hydrophobic peripheral regions of the active site pocket, interactions between Nta1 and N-degron peptides, detailed overview. The enzyme shows glutamine-specific enzyme activity with dipeptides Gln-Val and Gln-Gly, Michaelis-Menten kinetics
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NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
LITERATURE
COMMENTARY
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
N-terminal L-glutaminyl-[protein] + H2O
N-terminal L-glutamyl-[protein] + NH3
Substrates: -
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N-terminal L-glutaminyl-[protein] + H2O
N-terminal L-glutamyl-[protein] + NH3
Substrates: -
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N-terminal L-glutaminyl-[protein] + H2O
N-terminal L-glutamyl-[protein] + NH3
Substrates: -
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N-terminal L-glutaminyl-[protein] + H2O
N-terminal L-glutamyl-[protein] + NH3
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Substrates: -
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N-terminal L-glutaminyl-[protein] + H2O
N-terminal L-glutamyl-[protein] + NH3
Substrates: -
Products: -
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N-terminal L-glutaminyl-[protein] + H2O
N-terminal L-glutamyl-[protein] + NH3
Substrates: -
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
additional information
additional information
glutamine-specific enzyme activity with dipeptides Gln-Val and Gln-Gly, Michaelis-Menten kinetics, overview
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additional information
additional information
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glutamine-specific enzyme activity with dipeptides Gln-Val and Gln-Gly, Michaelis-Menten kinetics, overview
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
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SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SOURCE
additional information
additional information
varying levels of NtQ-amidase activity in all mouse tissues examined
brenda
additional information
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varying levels of NtQ-amidase activity in all mouse tissues examined
brenda
Highest Expressing Human Cell Lines
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GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
evolution
malfunction
metabolism
physiological function
additional information
evolution
the catalytic triad, comprising Cys28, His81, and Asp97, is highly conserved among Ntaq proteins, transglutaminases, and cysteine proteases of diverse organisms
evolution
Ntaq1 and its crystal structure indicate that the active site and catalytic mechanism of NtQ-amidase are similar to those of transglutaminases
evolution
Ntaq1 and its crystal structure indicate that the active site and catalytic mechanism of NtQ-amidase are similar to those of transglutaminases
evolution
Ntaq1 and its crystal structure indicate that the active site and catalytic mechanism of NtQ-amidase are similar to those of transglutaminases
evolution
Ntaq1 and its crystal structure indicate that the active site and catalytic mechanism of NtQ-amidase are similar to those of transglutaminases
malfunction
a mutant in the Drosophila Cg8253 gene, encoding NtQ-amidase, has defective long-term memory
malfunction
downregulation of Ntaq1 in mouse cells results in a decrease of NtQ-amidase activity
metabolism
the enzyme is involved in the the N-end rule pathway, overview
metabolism
the enzyme is involved in the the N-end rule pathway, overview
metabolism
the enzyme is involved in the the N-end rule pathway, overview
metabolism
the first step of the hierarchically organized Arg/N-end rule pathway of protein degradation is deamidation of the N-terminal glutamine and asparagine residues of substrate proteins to glutamate and aspartate, respectively. These reactions are catalyzed by the N-terminal amidase (Nt-amidase) Nta1 in fungi such as Saccharomyces cerevisiae, and by the glutamine-specific Ntaq1 and asparagine-specific Ntan1 Nt-amidases in mammals. Specific deamidation mechanisms in the first step of the N-end rule pathway, overview
physiological function
enzyme Ntaq is an initial component of the N-end rule pathway and converts N-terminal glutamine to glutamate
physiological function
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the proteolysis of native glucosamine-6-phosphate synthase of molecular weight67 kDa from Escherichia coli using cr-chymotrypsin generates two nonoverlapping polypeptides CT1 and CT2 of molecular weight40 kDa and 27 kDa lacking glucosamine-6-phosphate synthesizing activity. N-terminal and C-terminal sequence analyses shows that cleavage occurs between positions 240 and 241 of the primary sequence without further degradation. The glutamine amidohydrolase activity is located in the CT2 N-terminal polypeptide which is capable of incorporating glutamine site-directed affinity label [2-3H]-iV3-(4-methoxyfumaroyl)-diaminopropionic acid, it bears the amidotransferase function. CT1 displays a higher reactivity than CT2 for fructose 6-phosphate binding contains the ketose/aldose isomerase activity
physiological function
the enzyme controls the expression of specific defence-response genes, activates the synthesis pathway for the phytoalexin camalexin and influences basal resistance to the hemibiotroph pathogen Pseudomonas syringae pv tomato
additional information
substrate binding structure, molecular docking studies of tripeptides with N-terminal glutamine, overview. Upon binding of a substrate with N-terminal glutamine, active site catalytic triad mediates the deamination of the N-terminal residue to glutamate by a mechanism analogous to that of cysteine proteases. Active site structure, ooverview
additional information
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substrate binding structure, molecular docking studies of tripeptides with N-terminal glutamine, overview. Upon binding of a substrate with N-terminal glutamine, active site catalytic triad mediates the deamination of the N-terminal residue to glutamate by a mechanism analogous to that of cysteine proteases. Active site structure, ooverview
additional information
three-dimensional structure, mutational analysis, and mechanism of Ntaq1 NtQ-amidase, comparisons of mouse, human, and bovine enzymes, overview
additional information
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three-dimensional structure, mutational analysis, and mechanism of Ntaq1 NtQ-amidase, comparisons of mouse, human, and bovine enzymes, overview
additional information
three-dimensional structure, mutational analysis, and mechanism of Ntaq1 NtQ-amidase, comparisons of mouse, human, and bovine enzymes, overview
additional information
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three-dimensional structure, mutational analysis, and mechanism of Ntaq1 NtQ-amidase, comparisons of mouse, human, and bovine enzymes, overview
additional information
three-dimensional structure, mutational analysis, and mechanism of Ntaq1 NtQ-amidase, comparisons of mouse, human, and bovine enzymes, overview
additional information
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three-dimensional structure, mutational analysis, and mechanism of Ntaq1 NtQ-amidase, comparisons of mouse, human, and bovine enzymes, overview
additional information
the catalytic triads consists of residues Ser28, His81, and Asp97 on antiparallel beta sheets, surrounded by alpha helices
Show AA Sequence and Transmembrane Helices (2520 entries)
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PDB
SCOP
CATH
UNIPROT
ORGANISM
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MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
monomer
in solution, yNta1 is a monomer containing 14 beta-strands, 11 alpha-helices, and three 310-helices. The core region of the enzyme shows antiparallel and parallel mixed beta-sheets surrounded by helices, and these sixstranded beta-sheets face each other
additional information
?
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x * 40000, CT1 polypeptide fragment of glucosamine-6-phosphate synthase, SDS-PAGE
additional information
the enzyme structure reveals a monomeric globular protein with alpha-beta-alpha three-layer sandwich architecture. The catalytic triad located in the active site, Cys-His-Asp, is highly conserved among Ntaq family and transglutaminases from diverse organisms. Substrate binding mode of hNtaq1 with the N-terminus of a symmetry-related Ntaq1 molecule bound in the substrate binding cleft
additional information
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the enzyme structure reveals a monomeric globular protein with alpha-beta-alpha three-layer sandwich architecture. The catalytic triad located in the active site, Cys-His-Asp, is highly conserved among Ntaq family and transglutaminases from diverse organisms. Substrate binding mode of hNtaq1 with the N-terminus of a symmetry-related Ntaq1 molecule bound in the substrate binding cleft
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CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
purified enzyme Ntaq1 bound with the N-terminus of a symmetry-related Ntaq1 molecule, hanging drop vapor diffusion method, mixing of 10 mg/ml protein in 50 mM sodium chloride, 3 mM sodium azide, 0.3 mM TCEP, and 100 mM Bis-Tris, pH 7.0, with well solution containing 1% ethylene glycol, 1.8 M ammonium sulfate, 100 mM MES, pH 6.0, in 1:1 ratio, at 18°C, X-ray diffraction structure determination and analysis at 1.5 A resolution
structures of N-terminal mutants, space group P21. For the asymmetric unit, each of the NTAQ1 molecules with Glu, Trp, Gln, Ser, His, or Leu at the second position contains a monomer, each of the molecules with Arg, Asp, Ala, Asn, Phe, or Gly at the second position contains a dimer
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PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
native enzyme from a brain extract by ion exchange, hydrophobic chromatography, and gel filtration
recombinant N-terminally His6-MBP-tagged enzyme from Escherichia coli strain B834 as Se-Met labeled protein by nickel affinity chromatography, tag cleavage by TEV protease, gel filtration,and another step of nickel affinity chromatography, followed by desalting gel filtration
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CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
gene C8orf32, the enzyme is expressed as recombinant fusion enzyme containing N-terminally fused His6-maltose binding protein (MBP) and a linker region with the TEV protease site for cleavage of target proteins, expression in Escherichia coli strain B834 as Se-Met labeled protein
gene Cg8253
gene Ntaq1, DNA and amino acid sequence determination and analysis, mouse Hebp2 cDNA and Wdyhv1 cDNA are subcloned into the plasmid p425Met25, the FLAG-tagged mouse Hebp2f and Wdyhv1f are expressed in an nta1DELTA mutant of Saccharomyces cerevisiae that lacks the endogenous Nta1 NtN,Q-amidase activity. Recombinant transient expression of a mouse Ntaq1-EGFP fusion from the PCMV promoter transfected into NIH-3T3 cells. Recombinant expression of C-terminally His6-tagged mouse Ntaq1 in Pichia pastoris
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EXPRESSION
ORGANISM
UNIPROT
LITERATURE
expression in wild-type of NTAQ1 is not strongly affected by infection with bacteria. No age-related differences are found in NTAQ1 expression in wild-type
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REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Denisot, M.; Goffic, F.; Badet, B.
Glucosamine-6-phosphate synthase from Escherichia coli yields two proteins upon limited proteolysis: identification of the glutamine amidohydrolase and 2R ketose/aldose isomerase-bearing domains based on their biochemical properties
Arch. Biochem. Biophys.
288
225-230
1991
Escherichia coli
brenda
Wang, H.; Piatkov, K.I.; Brower, C.S.; Varshavsky, A.
Glutamine-specific N-terminal amidase, a component of the N-end rule pathway
Mol. Cell
34
686-695
2009
Bos taurus (Q3T0D3), Bos taurus, Drosophila melanogaster (Q7K2Y9), Homo sapiens (Q96HA8), Homo sapiens, Mus musculus (Q80WB5), Mus musculus
brenda
Park, M.S.; Bitto, E.; Kim, K.R.; Bingman, C.A.; Miller, M.D.; Kim, H.J.; Han, B.W.; Phillips, G.N.
Crystal structure of human protein N-terminal glutamine amidohydrolase, an initial component of the N-end rule pathway
PLoS ONE
9
e111142
2014
Homo sapiens (Q96HA8), Homo sapiens
brenda
Kim, M.K.; Oh, S.J.; Lee, B.G.; Song, H.K.
Structural basis for dual specificity of yeast N-terminal amidase in the N-end rule pathway
Proc. Natl. Acad. Sci. USA
113
12438-12443
2016
Saccharomyces cerevisiae (P40354), Saccharomyces cerevisiae
brenda
Vicente, J.; Mendiondo, G.M.; Pauwels, J.; Pastor, V.; Izquierdo, Y.; Naumann, C.; Movahedi, M.; Rooney, D.; Gibbs, D.J.; Smart, K.; Bachmair, A.; Gray, J.E.; Dissmeyer, N.; Castresana, C.; Ray, R.V.; Gevaert, K.; Holdsworth, M.J.
Distinct branches of the N-end rule pathway modulate the plant immune response
New Phytol.
221
988-1000
2019
Arabidopsis thaliana (O22944), Arabidopsis thaliana
brenda
Kang, J.M.; Park, J.S.; Lee, J.S.; Jang, J.Y.; Han, B.W.
Structural study for substrate recognition of human N-terminal glutamine amidohydrolase 1 in the arginine N-degron pathway
Protein Sci.
33
e5067
2024
Homo sapiens (Q96HA8)
brenda
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