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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The expected taxonomic range for this enzyme is: Eukaryota, Bacteria
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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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.
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
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
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
the catalytic triad, comprising Cys28, His81, and Asp97, is highly conserved among Ntaq proteins, transglutaminases, and cysteine proteases of diverse organisms
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
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
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
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
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
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
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
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 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
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
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3.5.1.122 protein N-terminal glutamine amidohydrolase
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