Cloned (Comment) | Organism |
---|---|
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 | Homo sapiens |
Crystallization (Comment) | Organism |
---|---|
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 | Homo sapiens |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
N-terminal L-glutaminyl-[protein] + H2O | Homo sapiens | - |
N-terminal L-glutamyl-[protein] + NH3 | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Homo sapiens | Q96HA8 | - |
- |
Purification (Comment) | Organism |
---|---|
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 | Homo sapiens |
Reaction | Comment | Organism | Reaction ID |
---|---|---|---|
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 | Homo sapiens |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
N-terminal L-glutaminyl-[protein] + H2O | - |
Homo sapiens | N-terminal L-glutamyl-[protein] + NH3 | - |
? |
Subunits | Comment | Organism |
---|---|---|
More | 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 | Homo sapiens |
Synonyms | Comment | Organism |
---|---|---|
C8orf32 | - |
Homo sapiens |
hNtaq1 | - |
Homo sapiens |
human N-terminal glutamine amidohydrolase isoform 1 | - |
Homo sapiens |
Ntaq | - |
Homo sapiens |
General Information | Comment | Organism |
---|---|---|
evolution | the catalytic triad, comprising Cys28, His81, and Asp97, is highly conserved among Ntaq proteins, transglutaminases, and cysteine proteases of diverse organisms | Homo sapiens |
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 | Homo sapiens |
physiological function | enzyme Ntaq is an initial component of the N-end rule pathway and converts N-terminal glutamine to glutamate | Homo sapiens |