Any feedback?
Please rate this page
(literature.php)
(0/150)

BRENDA support

Literature summary for 3.5.1.122 extracted from

  • 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 (2014), PLoS ONE, 9, e111142.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

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 (Commentary)

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/ Products (Substrates)

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

Organism UniProt Comment Textmining
Homo sapiens Q96HA8
-
-

Purification (Commentary)

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

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 and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
N-terminal L-glutaminyl-[protein] + H2O
-
Homo sapiens N-terminal L-glutamyl-[protein] + NH3
-
?

Subunits

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

Synonyms Comment Organism
C8orf32
-
Homo sapiens
hNtaq1
-
Homo sapiens
human N-terminal glutamine amidohydrolase isoform 1
-
Homo sapiens
Ntaq
-
Homo sapiens

General Information

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