EzCatDB: T00107

DB codeT00107
RLCP classification3.113.90030.1133
3.1143.90000.1132
CATH domainDomain 13.40.50.20
Domain 23.30.470.20Catalytic domain
Domain 33.30.1490.20Catalytic domain
E.C.6.3.2.3
CSA1gsa
MACiEM0199

CATH domainRelated DB codes (homologues)
3.30.1490.20T00082,M00035,M00037,T00108
3.30.470.20T00082,D00298,M00035,M00037,M00051,T00108
3.40.50.20T00082,M00037,T00108

Enzyme Name
Swiss-protKEGG

P04425
Protein nameGlutathione synthetaseglutathione synthase
glutathione synthetase
GSH synthetase
SynonymsEC 6.3.2.3
Glutathione synthase
GSH synthetase
GSH-S
GSHase

KEGG pathways
MAP codePathways
MAP00251Glutamate metabolism
MAP00480Glutathione metabolism

Swiss-prot:Accession NumberP04425
Entry nameGSHB_ECOLI
ActivityATP + gamma-L-glutamyl-L-cysteine + glycine = ADP + phosphate + glutathione.
SubunitHomotetramer.
Subcellular location
CofactorBinds 1 magnesium or manganese ion per subunit (By similarity).


CofactorsSubstratesProducts
KEGG-idC00305C00002C00669C00037C00008C00009C00051
CompoundMagnesiumATPgamma-L-Glutamyl-L-cysteineGlycineADPOrthophosphateGlutathione
Typedivalent metal (Ca2+, Mg2+)amine group,nucleotideamino acids,amide group,sulfhydryl groupamino acidsamine group,nucleotidephosphate group/phosphate ionamino acids,carboxyl group,peptide/protein,sulfhydryl group
1gltA01UnboundUnboundUnboundUnboundUnboundUnboundUnbound
1glvA01UnboundUnboundUnboundUnboundUnboundUnboundUnbound
1gsaA01UnboundUnboundUnboundUnboundUnboundUnboundUnbound
1gshA01UnboundUnboundUnboundUnboundUnboundUnboundUnbound
2gltA01UnboundUnboundUnboundUnboundUnboundUnboundUnbound
1gltA02UnboundUnboundUnboundUnboundUnboundUnboundUnbound
1glvA02UnboundUnboundUnboundUnboundUnboundUnboundUnbound
1gsaA02Bound:2x_MGUnboundUnboundUnboundBound:ADPAnalogue:SO4Bound:GTT
1gshA02UnboundUnboundUnboundUnboundUnboundUnboundUnbound
2gltA02UnboundUnboundUnboundUnboundUnboundUnboundUnbound
1gltA03UnboundUnboundUnboundUnboundUnboundUnboundUnbound
1glvA03UnboundUnboundUnboundUnboundUnboundUnboundUnbound
1gsaA03UnboundUnboundUnboundUnboundUnboundUnboundUnbound
1gshA03UnboundUnboundUnboundUnboundUnboundUnboundUnbound
2gltA03UnboundUnboundUnboundUnboundUnboundUnboundUnbound

Active-site residues
resource
literature [14]
pdbCatalytic residuesCofactor-binding residuesMain-chain involved in catalysiscomment
1gltA01



1glvA01



1gsaA01



1gshA01



2gltA01



1gltA02LYS 125;ARG 210;ARG 225
ASP 273(Mg1 binding);GLU 281(Mg1 & Mg2 binding);ASN 283(Mg2 binding)
THR 288
invisible 226-241
1glvA02LYS 125;ARG 210;ARG 225
ASP 273(Mg1 binding);GLU 281(Mg1 & Mg2 binding);ASN 283(Mg2 binding)
THR 288
mutant 226-241 replaced by GGG
1gsaA02LYS 125;ARG 210;ARG 225
ASP 273(Mg1 binding);GLU 281(Mg1 & Mg2 binding);ASN 283(Mg2 binding)
THR 288

1gshA02LYS 125;ARG 210;ARG 225
ASP 273(Mg1 binding);GLU 281(Mg1 & Mg2 binding);ASN 283(Mg2 binding)
THR 288
invisible 226-241
2gltA02LYS 125;ARG 210;ARG 225
ASP 273(Mg1 binding);GLU 281(Mg1 & Mg2 binding);ASN 283(Mg2 binding)
THR 288
invisible 226-241
1gltA03LYS 160

       ;       
invisible 164-167
1glvA03LYS 160

       ;       
invisible 164-167
1gsaA03LYS 160

GLY 166;GLY 167

1gshA03LYS 160

       ;       
invisible 164-167
2gltA03LYS 160

       ;       
invisible 164-167

References for Catalytic Mechanism
ReferencesSectionsNo. of steps in catalysis
[5]p.2263-2264
[8]Scheme I, p.12403-124042
[11]Scheme I3
[14]Scheme 1, p.11973-119743
[17]Fig.6A

references
[1]
PubMed ID3553173
JournalJ Biochem (Tokyo)
Year1987
Volume101
Pages207-15
AuthorsKato H, Chihara M, Nishioka T, Murata K, Kimura A, Oda J
TitleHomology of Escherichia coli B glutathione synthetase with dihydrofolate reductase in amino acid sequence and substrate binding site.
[2]
CommentsMUTAGENESIS OF CYSTEINE RESIDUES.
Medline ID88298832
PubMed ID3042775
JournalJ Biol Chem
Year1988
Volume263
Pages11646-51
AuthorsKato H, Tanaka T, Nishioka T, Kimura A, Oda J
TitleRole of cysteine residues in glutathione synthetase from Escherichia coli B. Chemical modification and oligonucleotide site-directed mutagenesis.
Related Swiss-protP04425
[3]
PubMed ID2685323
JournalJ Mol Biol
Year1989
Volume209
Pages503-4
AuthorsKato H, Yamaguchi H, Hata Y, Nishioka T, Katsube Y, Oda J
TitleCrystallization and preliminary X-ray studies of glutathione synthetase from Escherichia coli B.
[4]
PubMed ID1540152
JournalBiochem Biophys Res Commun
Year1992
Volume182
Pages1040-6
AuthorsPeters JM, Dalrymple BP, Jorgensen WK
TitleSequence of a putative glutathione synthetase II gene and flanking regions from Anaplasma centrale.
[5]
CommentsSEQUENCE OF 234-242, AND MUTAGENESIS OF ARG-233 AND ARG-241.
Medline ID92172846
PubMed ID1540581
JournalBiochemistry
Year1992
Volume31
Pages2259-65
AuthorsTanaka T, Kato H, Nishioka T, Oda J
TitleMutational and proteolytic studies on a flexible loop in glutathione synthetase from Escherichia coli B: the loop and arginine 233 are critical for the catalytic reaction.
Related Swiss-protP04425
[6]
PubMed ID8192897
JournalBiochem Cell Biol
Year1993
Volume71
Pages447-53
AuthorsNakagawa CW, Mutoh N, Hayashi Y
TitleGlutathione synthetase from the fission yeast. Purification and its unique heteromeric subunit structure.
[7]
PubMed ID8431434
JournalBiochemistry
Year1993
Volume32
Pages1548-54
AuthorsHibi T, Kato H, Nishioka T, Oda J, Yamaguchi H, Katsube Y, Tanizawa K, Fukui T
TitleUse of adenosine (5')polyphospho(5')pyridoxals to study the substrate-binding region of glutathione synthetase from Escherichia coli B.
[8]
CommentsMUTAGENESIS OF PRO-227 AND GLY-240.
Medline ID94059950
PubMed ID8241129
JournalBiochemistry
Year1993
Volume32
Pages12398-404
AuthorsTanaka T, Yamaguchi H, Kato H, Nishioka T, Katsube Y, Oda J
TitleFlexibility impaired by mutations revealed the multifunctional roles of the loop in glutathione synthetase.
Related Swiss-protP04425
[9]
CommentsX-RAY CRYSTALLOGRAPHY (2.0 ANGSTROMS).
Medline ID93188002
PubMed ID8445637
JournalJ Mol Biol
Year1993
Volume229
Pages1083-100
AuthorsYamaguchi H, Kato H, Hata Y, Nishioka T, Kimura A, Oda J, Katsube Y
TitleThree-dimensional structure of the glutathione synthetase from Escherichia coli B at 2.0 A resolution.
Related PDB1glt,1glv,1gsh,2glt
Related Swiss-protP04425
[10]
PubMed ID8172874
JournalBiochemistry
Year1994
Volume33
Pages4995-9
AuthorsKato H, Tanaka T, Yamaguchi H, Hara T, Nishioka T, Katsube Y, Oda J
TitleFlexible loop that is novel catalytic machinery in a ligase. Atomic structure and function of the loopless glutathione synthetase.
[11]
PubMed ID7862655
JournalProc Natl Acad Sci U S A
Year1995
Volume92
Pages1172-6
AuthorsFan C, Moews PC, Shi Y, Walsh CT, Knox JR
TitleA common fold for peptide synthetases cleaving ATP to ADP: glutathione synthetase and D-alanine:d-alanine ligase of Escherichia coli.
[12]
PubMed ID8577699
JournalProtein Eng
Year1995
Volume8
Pages711-6
AuthorsHara T, Tanaka T, Kato H, Nishioka T, Oda J
TitleSite-directed mutagenesis of glutathione synthetase from Escherichia coli B: mapping of the gamma-L-glutamyl-L-cysteine-binding site.
[13]
PubMed ID7567920
JournalProtein Eng
Year1995
Volume8
Pages353-62
AuthorsMizuguchi K, Go N
TitleComparison of spatial arrangements of secondary structural elements in proteins.
[14]
CommentsX-ray crystallography
PubMed ID8810901
JournalBiochemistry
Year1996
Volume35
Pages11967-74
AuthorsHara T, Kato H, Katsube Y, Oda J
TitleA pseudo-michaelis quaternary complex in the reverse reaction of a ligase: structure of Escherichia coli B glutathione synthetase complexed with ADP, glutathione, and sulfate at 2.0 A resolution.
Related PDB1gsa
[15]
PubMed ID8564538
JournalNat Struct Biol
Year1996
Volume3
Pages128-32
AuthorsArtymiuk PJ, Poirrette AR, Rice DW, Willett P
TitleBiotin carboxylase comes into the fold.
[16]
PubMed ID8548447
JournalNat Struct Biol
Year1996
Volume3
Pages16-8
AuthorsHibi T, Nishioka T, Kato H, Tanizawa K, Fukui T, Katsube Y, Oda J
TitleStructure of the multifunctional loops in the nonclassical ATP-binding fold of glutathione synthetase.
[17]
CommentsX-RAY CRYSTALLOGRAPHY (2.0 ANGSTROMS).
Medline ID97164199
PubMed ID9010922
JournalProtein Eng
Year1996
Volume9
Pages1083-92
AuthorsMatsuda K, Mizuguchi K, Nishioka T, Kato H, Go N, Oda J
TitleCrystal structure of glutathione synthetase at optimal pH: domain architecture and structural similarity with other proteins.
Related Swiss-protP04425
[18]
PubMed ID9056244
JournalArch Biochem Biophys
Year1997
Volume339
Pages151-6
AuthorsTanaka T, Nishioka T, Oda J
TitleNicked multifunctional loop of glutathione synthetase still protects the catalytic intermediate.
[19]
PubMed ID9078268
JournalBiochem J
Year1997
Volume322
Pages241-4
AuthorsWang CL, Oliver DJ
TitleIdentification of a putative flexible loop in Arabidopsis glutathione synthetase.
[20]
PubMed ID9463376
JournalEMBO J
Year1998
Volume17
Pages977-84
AuthorsEsser L, Wang CR, Hosaka M, Smagula CS, Sudhof TC, Deisenhofer J
TitleSynapsin I is structurally similar to ATP-utilizing enzymes.
[21]
PubMed ID9551557
JournalStructure
Year1998
Volume6
Pages363-76
AuthorsLevdikov VM, Barynin VV, Grebenko AI, Melik-Adamyan WR, Lamzin VS, Wilson KS
TitleThe structure of SAICAR synthase: an enzyme in the de novo pathway of purine nucleotide biosynthesis.
[22]
PubMed ID10438618
JournalJ Mol Biol
Year1999
Volume291
Pages239-47
AuthorsGrishin NV
TitlePhosphatidylinositol phosphate kinase: a link between protein kinase and glutathione synthase folds.
[23]
PubMed ID12467574
JournalStructure (Camb)
Year2002
Volume10
Pages1669-76
AuthorsGogos A, Shapiro L
TitleLarge conformational changes in the catalytic cycle of glutathione synthase.
[24]
PubMed ID14990577
JournalJ Biol Chem
Year2004
Volume279
Pages22412-21
AuthorsDinescu A, Cundari TR, Bhansali VS, Luo JL, Anderson ME
TitleFunction of conserved residues of human glutathione synthetase: implications for the ATP-grasp enzymes.

comments
According to the literature [14], this enzyme catalyzes two successive transfer reactions. Firstly, it transfers the gamma-phosphate group of ATP to the C-terminal carboxylate of the second substrate, gamma-glutamylcysteine, to form an acylphosphate intermediate. Secondly, it transfers the acyl group from the intermediate to the amine group of the third substrate, glycine, to form a tetrahedral carbon inermediate, which dissociates into the product GSH, releasing inorganic phosphate and ADP.
The catalytic mechanism seems to be similar to that of the counterpart from human (M00051 in EzCatDB), although the domain strucrures are quite different.
(A) Phosphoryl transfer
(A1) The acceptor group, the C-terminal carboxylate oxygen of gamma-glutamylcysteine (the first substrate), makes a nucleophilic attack on the transferred group, gamma-phosphate of ATP (the second substrate), leading to the formation of the pentacovalent phosphate transition state.
(A2) The mainchain amide of Gly166, and the sidechains of Arg225 and Arg210 stabilize the transferred group, gamma-phosphate, together with two magnesium ions bound to Asp273, Glu281 and Asn283, during the transition state. Meanwhile, Lys125 and Lys160 stabilize the negative charge of the leaving group, alpha- and beta-phosphate groups of ATP (the second substrate), together with the two magnesium ions.
(A3) The leaving group, ADP, dissociates, forming an acylphosphate intermediate.
(B) Acyl transfer
(B1) The acceptor group, the amine of glycine, makes a nucleophilic attack on the transferred group, the carbonyl carbon of the acylphosphate intermediate, forming the tetrahedral intermediate.
(B2) The mainchain amide of Thr288, and the sidechain of Arg210 stabilize the charge on the tetrahedral intermediate. Meanwhile, Arg210, Arg225, and the mainchain amide of Gly166 stabilize the negative charge of the leaving gamma-phosphate, together with the two magnesium ions.
(B3) Finally, the tetrahedral carbon intermediate dissociates to form the product GSH, releasing the inorganic phosphate.

createdupdated
2004-08-012009-02-26


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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