DB code: D00300

RLCP classification 1.13.30110.56 : Hydrolysis
CATH domain 3.60.20.10 : Glutamine Phosphoribosylpyrophosphate, subunit 1, domain 1 Catalytic domain
3.40.50.620 : Rossmann fold Catalytic domain
E.C. 6.3.5.4
CSA 1ct9
M-CSA 1ct9
MACiE M0302

CATH domain Related DB codes (homologues)
3.40.50.620 : Rossmann fold S00314 S00549 S00316 S00317 S00318 S00315 T00085 T00249 M00177 M00178 T00106 T00114
3.60.20.10 : Glutamine Phosphoribosylpyrophosphate, subunit 1, domain 1 T00201 M00123 M00174

Uniprot Enzyme Name
UniprotKB Protein name Synonyms RefSeq MEROPS Pfam
P22106 Asparagine synthetase B {glutamine-hydrolyzing}
EC 6.3.5.4
NP_415200.1 (Protein)
NC_000913.2 (DNA/RNA sequence)
YP_488954.1 (Protein)
NC_007779.1 (DNA/RNA sequence)
C44.976 (Cysteine)
PF00733 (Asn_synthase)
PF13537 (GATase_7)
[Graphical View]

KEGG enzyme name
asparagine synthase (glutamine-hydrolysing)
asparagine synthetase (glutamine-hydrolysing)
glutamine-dependent asparagine synthetase
asparagine synthetase B
AS
AS-B

UniprotKB: Accession Number Entry name Activity Subunit Subcellular location Cofactor
P22106 ASNB_ECOLI ATP + L-aspartate + L-glutamine + H(2)O = AMP + diphosphate + L-asparagine + L-glutamate. Homodimer.

KEGG Pathways
Map code Pathways E.C.
MAP00252 Alanine and aspartate metabolism
MAP00910 Nitrogen metabolism

Compound table
Cofactors Substrates Products Intermediates
KEGG-id C00305 C00002 C00049 C00064 C00001 C00020 C00013 C00152 C00025
E.C.
Compound Magnesium ATP L-Aspartate L-Glutamine H2O AMP Pyrophosphate L-Asparagine L-Glutamate
Type divalent metal (Ca2+, Mg2+) amine group,nucleotide amino acids,carboxyl group amino acids,amide group H2O amine group,nucleotide phosphate group/phosphate ion amino acids,amide group amino acids,carboxyl group
ChEBI 18420
15422
17053
18050
58359
15377
16027
29888
17196
58048
16015
PubChem 888
5957
44367445
5960
5961
6992086
22247451
962
6083
1023
21961011
6267
6992089
33032
44272391
88747398
1ct9A01 Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Unbound Unbound Unbound Bound:GLN Unbound Unbound Unbound Unbound
1ct9B01 Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Unbound Unbound Unbound Bound:GLN Unbound Unbound Unbound Unbound
1ct9C01 Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Unbound Unbound Unbound Bound:GLN Unbound Unbound Unbound Unbound
1ct9D01 Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Unbound Unbound Unbound Bound:GLN Unbound Unbound Unbound Unbound
1ct9A02 Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Analogue:IUM_1102, IUM_1101 Unbound Unbound Unbound Bound:AMP Unbound Unbound Unbound
1ct9B02 Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Analogue:IUM_1109, IUM_1108 Unbound Unbound Unbound Bound:AMP Unbound Unbound Unbound
1ct9C02 Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Analogue:IUM_1116, IUM_1115 Unbound Unbound Unbound Bound:AMP Unbound Unbound Unbound
1ct9D02 Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Analogue:IUM_1123, IUM_1122 Unbound Unbound Unbound Bound:AMP Unbound Unbound Unbound

Reference for Active-site residues
resource references E.C.
literature [3], [6] & [9]

Active-site residues
PDB Catalytic residues Cofactor-binding residues Modified residues Main-chain involved in catalysis Comment
1ct9A01 Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain ;ASN 74 GLY 75 mutant C1A
1ct9B01 Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain ;ASN 74 GLY 75 mutant C1A
1ct9C01 Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain ;ASN 74 GLY 75 mutant C1A
1ct9D01 Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain ;ASN 74 GLY 75 mutant C1A
1ct9A02 Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain ARG 324 GLU 352;TYR 357;ASP 384(Magnesium-1 binding);ASP 238;ASP 351(Magnesium-2 binding)
1ct9B02 Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain ARG 324 GLU 352;TYR 357;ASP 384(Magnesium-1 binding);ASP 238;ASP 351(Magnesium-2 binding)
1ct9C02 Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain ARG 324 GLU 352;TYR 357;ASP 384(Magnesium-1 binding);ASP 238;ASP 351(Magnesium-2 binding)
1ct9D02 Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain ARG 324 GLU 352;TYR 357;ASP 384(Magnesium-1 binding);ASP 238;ASP 351(Magnesium-2 binding)

References for Catalytic Mechanism
References Sections No. of steps in catalysis
[2]
Fig.1 5
[3]
Fig.3, Fig.4
[5]
Fig.6, p.807 6
[6]
Fig.4
[9]
p.16151

References
[1]
Resource
Comments
Medline ID
PubMed ID 5076775
Journal J Biol Chem
Year 1972
Volume 247
Pages 6708-19
Authors Horowitz B, Meister A
Title Glutamine-dependent asparagine synthetase from leukemia cells. Chloride dependence, mechanism of action, and inhibition.
Related PDB
Related UniProtKB
[2]
Resource
Comments
Medline ID
PubMed ID 7907328
Journal J Biol Chem
Year 1994
Volume 269
Pages 7450-7
Authors Boehlein SK, Richards NG, Schuster SM
Title Glutamine-dependent nitrogen transfer in Escherichia coli asparagine synthetase B. Searching for the catalytic triad.
Related PDB
Related UniProtKB
[3]
Resource
Comments
Medline ID
PubMed ID 7929415
Journal J Biol Chem
Year 1994
Volume 269
Pages 26789-95
Authors Boehlein SK, Richards NG, Walworth ES, Schuster SM
Title Arginine 30 and asparagine 74 have functional roles in the glutamine dependent activities of Escherichia coli asparagine synthetase B.
Related PDB
Related UniProtKB
[4]
Resource
Comments
Medline ID
PubMed ID 8691431
Journal J Med Chem
Year 1996
Volume 39
Pages 2367-78
Authors Parr IB, Boehlein SK, Dribben AB, Schuster SM, Richards NG
Title Mapping the aspartic acid binding site of Escherichia coli asparagine synthetase B using substrate analogs.
Related PDB
Related UniProtKB
[5]
Resource
Comments X-RAY CRYSTALLOGRAPHY (1.8 ANGSTROMS) OF 1-240
Medline ID
PubMed ID 8805567
Journal Structure
Year 1996
Volume 4
Pages 801-10
Authors Isupov MN, Obmolova G, Butterworth S, Badet-Denisot MA, Badet B, Polikarpov I, Littlechild JA, Teplyakov A
Title Substrate binding is required for assembly of the active conformation of the catalytic site in Ntn amidotransferases: evidence from the 1.8 A crystal structure of the glutaminase domain of glucosamine 6-phosphate synthase.
Related PDB
Related UniProtKB
[6]
Resource
Comments
Medline ID
PubMed ID 9139684
Journal J Biol Chem
Year 1997
Volume 272
Pages 12384-92
Authors Boehlein SK, Walworth ES, Richards NG, Schuster SM
Title Mutagenesis and chemical rescue indicate residues involved in beta-aspartyl-AMP formation by Escherichia coli asparagine synthetase B.
Related PDB
Related UniProtKB
[7]
Resource
Comments
Medline ID
PubMed ID 9437423
Journal Nat Struct Biol
Year 1998
Volume 5
Pages 15-9
Authors Nakatsu T, Kato H, Oda J
Title Crystal structure of asparagine synthetase reveals a close evolutionary relationship to class II aminoacyl-tRNA synthetase.
Related PDB
Related UniProtKB
[8]
Resource
Comments
Medline ID
PubMed ID 9748330
Journal Biochemistry
Year 1998
Volume 37
Pages 13230-8
Authors Boehlein SK, Stewart JD, Walworth ES, Thirumoorthy R, Richards NG, Schuster SM
Title Kinetic mechanism of Escherichia coli asparagine synthetase B.
Related PDB
Related UniProtKB
[9]
Resource
Comments X-RAY CRYSTALLOGRAPHY (2.0 ANGSTROMS)
Medline ID 20056034
PubMed ID 10587437
Journal Biochemistry
Year 1999
Volume 38
Pages 16146-57
Authors Larsen TM, Boehlein SK, Schuster SM, Richards NG, Thoden JB, Holden HM, Rayment I
Title Three-dimensional structure of Escherichia coli asparagine synthetase B: a short journey from substrate to product.
Related PDB 1ct9
Related UniProtKB P22106
[10]
Resource
Comments
Medline ID
PubMed ID 11551215
Journal Biochemistry
Year 2001
Volume 40
Pages 11168-75
Authors Boehlein SK, Nakatsu T, Hiratake J, Thirumoorthy R, Stewart JD, Richards NG, Schuster SM
Title Characterization of inhibitors acting at the synthetase site of Escherichia coli asparagine synthetase B.
Related PDB
Related UniProtKB
[11]
Resource
Comments
Medline ID
PubMed ID 12706338
Journal Arch Biochem Biophys
Year 2003
Volume 413
Pages 23-31
Authors Tesson AR, Soper TS, Ciustea M, Richards NG
Title Revisiting the steady state kinetic mechanism of glutamine-dependent asparagine synthetase from Escherichia coli.
Related PDB
Related UniProtKB

Comments
This enzyme is composed of two domains, N-terminal glutaminase domain, and C-terminal ligase domain. These domains catalyzes the following reactions:
(A) Hydrolysis of glutamine amide (by the N-terminal glutaminase domain) (see [9]):
(A#) This domain is homologous to the N-terminal glutaminase domain of Glutamine-fructose-6-phosphate transaminase (T00201 in EzCatDB), and has a similar reaction mechanism to its mechanism.
(A1) Nucleophile, Cys1 can be either in an active conformation with the thiol group close to the substrate amide, or in an inactive one with the group pointing away from the substrate. During the catalysis, its conformation must be active.
(A2) The N-terminal alpha-amino group acts as a general base, which activates the thiol group of the N-terminal Cys1, through a water molecule.
(A3) The activated thiol group attacks the amide carbon of a substrate, glutamine, to form a tetrahedral intermediate. The intermediate is stabilized by an oxyanion hole, made up by mainchain amide groups of Asn74 and Gly75.
(A4) The intermediate collapses to form a gamma-glutamylthioester and to release ammonia which might be protonated by the water bound to the alpha-amino group of Cys1.
(A5) Another water (distinct from the above bound water) is activated through the bound water by the alpha-amino group.
(A6) The activated water makes a nucleophilic attack on the thiolester carbon, to form a tetrahedral intermediate. This intermediate is also stabilized by the oxyanion hole.
(A7) The intermediate collapses to release the product, glutamate, from Cys1, and the thiol group is protonated through the bound water.
(B) Transfer of adenylate (AMP) to carboxylate oxygen of Aspartate:
(C) Transfer of acyl group of Asp-AMP intermediate to ammonia:

Created Updated
2004-03-25 2009-02-26