EzCatDB: D00300

DB codeD00300
RLCP classification1.13.30110.56
CATH domainDomain 13.60.20.10Catalytic domain
Domain 23.40.50.620Catalytic domain
E.C.6.3.5.4
CSA1ct9
MACiEM0302

CATH domainRelated DB codes (homologues)
3.40.50.620S00314,S00549,S00316,S00317,S00318,S00315,T00085,T00249,M00177,M00178,T00106,T00114
3.60.20.10T00201,M00123,M00174

Enzyme Name
Swiss-protKEGG

P22106
Protein nameAsparagine synthetase B {glutamine-hydrolyzing}asparagine synthase (glutamine-hydrolysing)
asparagine synthetase (glutamine-hydrolysing)
glutamine-dependent asparagine synthetase
asparagine synthetase B
AS
AS-B
SynonymsEC 6.3.5.4

KEGG pathways
MAP codePathways
MAP00252Alanine and aspartate metabolism
MAP00910Nitrogen metabolism

Swiss-prot:Accession NumberP22106
Entry nameASNB_ECOLI
ActivityATP + L-aspartate + L-glutamine + H(2)O = AMP + diphosphate + L-asparagine + L-glutamate.
SubunitHomodimer.
Subcellular location
Cofactor


CofactorsSubstratesProducts
KEGG-idC00305C00002C00049C00064C00001C00020C00013C00152C00025
CompoundMagnesiumATPL-AspartateL-GlutamineH2OAMPPyrophosphateL-AsparagineL-Glutamate
Typedivalent metal (Ca2+, Mg2+)amine group,nucleotideamino acids,carboxyl groupamino acids,amide groupH2Oamine group,nucleotidephosphate group/phosphate ionamino acids,amide groupamino acids,carboxyl group
1ct9A01UnboundUnboundUnboundBound:GLN
UnboundUnboundUnboundUnbound
1ct9B01UnboundUnboundUnboundBound:GLN
UnboundUnboundUnboundUnbound
1ct9C01UnboundUnboundUnboundBound:GLN
UnboundUnboundUnboundUnbound
1ct9D01UnboundUnboundUnboundBound:GLN
UnboundUnboundUnboundUnbound
1ct9A02Analogue:IUM 1102,IUM 1101UnboundUnboundUnbound
Bound:AMPUnboundUnboundUnbound
1ct9B02Analogue:IUM 1109,IUM 1108UnboundUnboundUnbound
Bound:AMPUnboundUnboundUnbound
1ct9C02Analogue:IUM 1116,IUM 1115UnboundUnboundUnbound
Bound:AMPUnboundUnboundUnbound
1ct9D02Analogue:IUM 1123,IUM 1122UnboundUnboundUnbound
Bound:AMPUnboundUnboundUnbound

Active-site residues
resource
literature [3], [6] & [9]
pdbCatalytic residuesCofactor-binding residuesMain-chain involved in catalysiscomment
1ct9A01     ;ASN 74

GLY 75
mutant C1A
1ct9B01     ;ASN 74

GLY 75
mutant C1A
1ct9C01     ;ASN 74

GLY 75
mutant C1A
1ct9D01     ;ASN 74

GLY 75
mutant C1A
1ct9A02ARG 324
GLU 352;TYR 357;ASP 384(Magnesium-1 binding);ASP 238;ASP 351(Magnesium-2 binding)


1ct9B02ARG 324
GLU 352;TYR 357;ASP 384(Magnesium-1 binding);ASP 238;ASP 351(Magnesium-2 binding)


1ct9C02ARG 324
GLU 352;TYR 357;ASP 384(Magnesium-1 binding);ASP 238;ASP 351(Magnesium-2 binding)


1ct9D02ARG 324
GLU 352;TYR 357;ASP 384(Magnesium-1 binding);ASP 238;ASP 351(Magnesium-2 binding)



References for Catalytic Mechanism
ReferencesSectionsNo. of steps in catalysis
[2]Fig.15
[3]Fig.3, Fig.4
[5]Fig.6, p.8076
[6]Fig.4
[9]p.16151

references
[1]
PubMed ID5076775
JournalJ Biol Chem
Year1972
Volume247
Pages6708-19
AuthorsHorowitz B, Meister A
TitleGlutamine-dependent asparagine synthetase from leukemia cells. Chloride dependence, mechanism of action, and inhibition.
[2]
PubMed ID7907328
JournalJ Biol Chem
Year1994
Volume269
Pages7450-7
AuthorsBoehlein SK, Richards NG, Schuster SM
TitleGlutamine-dependent nitrogen transfer in Escherichia coli asparagine synthetase B. Searching for the catalytic triad.
[3]
PubMed ID7929415
JournalJ Biol Chem
Year1994
Volume269
Pages26789-95
AuthorsBoehlein SK, Richards NG, Walworth ES, Schuster SM
TitleArginine 30 and asparagine 74 have functional roles in the glutamine dependent activities of Escherichia coli asparagine synthetase B.
[4]
PubMed ID8691431
JournalJ Med Chem
Year1996
Volume39
Pages2367-78
AuthorsParr IB, Boehlein SK, Dribben AB, Schuster SM, Richards NG
TitleMapping the aspartic acid binding site of Escherichia coli asparagine synthetase B using substrate analogs.
[5]
CommentsX-RAY CRYSTALLOGRAPHY (1.8 ANGSTROMS) OF 1-240
PubMed ID8805567
JournalStructure
Year1996
Volume4
Pages801-10
AuthorsIsupov MN, Obmolova G, Butterworth S, Badet-Denisot MA, Badet B, Polikarpov I, Littlechild JA, Teplyakov A
TitleSubstrate 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.
[6]
PubMed ID9139684
JournalJ Biol Chem
Year1997
Volume272
Pages12384-92
AuthorsBoehlein SK, Walworth ES, Richards NG, Schuster SM
TitleMutagenesis and chemical rescue indicate residues involved in beta-aspartyl-AMP formation by Escherichia coli asparagine synthetase B.
[7]
PubMed ID9437423
JournalNat Struct Biol
Year1998
Volume5
Pages15-9
AuthorsNakatsu T, Kato H, Oda J
TitleCrystal structure of asparagine synthetase reveals a close evolutionary relationship to class II aminoacyl-tRNA synthetase.
[8]
PubMed ID9748330
JournalBiochemistry
Year1998
Volume37
Pages13230-8
AuthorsBoehlein SK, Stewart JD, Walworth ES, Thirumoorthy R, Richards NG, Schuster SM
TitleKinetic mechanism of Escherichia coli asparagine synthetase B.
[9]
CommentsX-RAY CRYSTALLOGRAPHY (2.0 ANGSTROMS)
Medline ID20056034
PubMed ID10587437
JournalBiochemistry
Year1999
Volume38
Pages16146-57
AuthorsLarsen TM, Boehlein SK, Schuster SM, Richards NG, Thoden JB, Holden HM, Rayment I
TitleThree-dimensional structure of Escherichia coli asparagine synthetase B: a short journey from substrate to product.
Related PDB1ct9
Related Swiss-protP22106
[10]
PubMed ID11551215
JournalBiochemistry
Year2001
Volume40
Pages11168-75
AuthorsBoehlein SK, Nakatsu T, Hiratake J, Thirumoorthy R, Stewart JD, Richards NG, Schuster SM
TitleCharacterization of inhibitors acting at the synthetase site of Escherichia coli asparagine synthetase B.
[11]
PubMed ID12706338
JournalArch Biochem Biophys
Year2003
Volume413
Pages23-31
AuthorsTesson AR, Soper TS, Ciustea M, Richards NG
TitleRevisiting the steady state kinetic mechanism of glutamine-dependent asparagine synthetase from Escherichia coli.

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:

createdupdated
2004-03-252009-02-26
Copyright: Nozomi Nagano, JST & CBRC-AIST
Funded by PRESTO/Japan Science and Technology Corporation (JST) (December 2001 - November 2004)
Funded by Grant-in-Aid for Publication of Scientific Research Results/Japan Society for the Promotion of Science (JSPS) (April 2005 - March 2006)
Funded by Grant-in-Aid for Scientific Research (B)/Japan Society for the Promotion of Science (JSPS) (April 2005 - March 2008)
Funded by BIRD/Japan Science and Technology Corporation (JST) (September 2005 - September 2010)
Funded by BIRD/Japan Science and Technology Corporation (JST) (October 2007 - September 2010)
Funded by Grant-in-Aid for Publication of Scientific Research Results/Japan Society for the Promotion of Science (JSPS) (April 2011 - March 2012)

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