DB code: T00107

RLCP classification 3.113.90030.1133 : Transfer
3.1143.90000.1132 : Transfer
CATH domain 3.40.50.20 : Rossmann fold
3.30.470.20 : D-amino Acid Aminotransferase; Chain A, domain 1 Catalytic domain
3.30.1490.20 : Dna Ligase; domain 1 Catalytic domain
E.C. 6.3.2.3
CSA 1gsa
M-CSA 1gsa
MACiE M0199

CATH domain Related DB codes (homologues)
3.30.1490.20 : Dna Ligase; domain 1 T00082 M00035 M00037 T00108
3.30.470.20 : D-amino Acid Aminotransferase; Chain A, domain 1 T00082 D00298 M00035 M00037 M00051 T00108
3.40.50.20 : Rossmann fold T00082 M00037 T00108

Uniprot Enzyme Name
UniprotKB Protein name Synonyms RefSeq Pfam
P04425 Glutathione synthetase
EC 6.3.2.3
Glutathione synthase
GSH synthetase
GSH-S
GSHase
NP_417422.1 (Protein)
NC_000913.2 (DNA/RNA sequence)
YP_491146.1 (Protein)
NC_007779.1 (DNA/RNA sequence)
PF02955 (GSH-S_ATP)
PF02951 (GSH-S_N)
[Graphical View]

KEGG enzyme name
glutathione synthase
glutathione synthetase
GSH synthetase

UniprotKB: Accession Number Entry name Activity Subunit Subcellular location Cofactor
P04425 GSHB_ECOLI ATP + gamma-L-glutamyl-L-cysteine + glycine = ADP + phosphate + glutathione. Homotetramer. Binds 1 magnesium or manganese ion per subunit (By similarity).

KEGG Pathways
Map code Pathways E.C.
MAP00251 Glutamate metabolism
MAP00480 Glutathione metabolism

Compound table
Cofactors Substrates Products Intermediates
KEGG-id C00305 C00002 C00669 C00037 C00008 C00009 C00051
E.C.
Compound Magnesium ATP gamma-L-Glutamyl-L-cysteine Glycine ADP Orthophosphate Glutathione
Type divalent metal (Ca2+, Mg2+) amine group,nucleotide amino acids,amide group,sulfhydryl group amino acids amine group,nucleotide phosphate group/phosphate ion amino acids,carboxyl group,peptide/protein,sulfhydryl group
ChEBI 18420
 18420
 		15422
 15422
 		17515
 17515
 		15428
 57305
 15428
 57305
 		16761
 16761
 		26078
 26078
 		16856
 16856
PubChem 888
 888
 		5957
 5957
 		123938
 123938
 		5257127
 750
 5257127
 750
 		6022
 6022
 		1004
 22486802
 1004
 22486802
 		124886
 25246407
 124886
 25246407
1gltA01 Unbound Unbound Unbound Unbound Unbound Unbound Unbound
1glvA01 Unbound Unbound Unbound Unbound Unbound Unbound Unbound
1gsaA01 Unbound Unbound Unbound Unbound Unbound Unbound Unbound
1gshA01 Unbound Unbound Unbound Unbound Unbound Unbound Unbound
2gltA01 Unbound Unbound Unbound Unbound Unbound Unbound Unbound
1gltA02 Unbound Unbound Unbound Unbound Unbound Unbound Unbound
1glvA02 Unbound Unbound Unbound Unbound Unbound Unbound Unbound
1gsaA02 Bound:2x_MG Unbound Unbound Unbound Bound:ADP Analogue:SO4 Bound:GTT
1gshA02 Unbound Unbound Unbound Unbound Unbound Unbound Unbound
2gltA02 Unbound Unbound Unbound Unbound Unbound Unbound Unbound
1gltA03 Unbound Unbound Unbound Unbound Unbound Unbound Unbound
1glvA03 Unbound Unbound Unbound Unbound Unbound Unbound Unbound
1gsaA03 Unbound Unbound Unbound Unbound Unbound Unbound Unbound
1gshA03 Unbound Unbound Unbound Unbound Unbound Unbound Unbound
2gltA03 Unbound Unbound Unbound Unbound Unbound Unbound Unbound

Reference for Active-site residues
resource references E.C.
literature [14]

Active-site residues
PDB Catalytic residues Cofactor-binding residues Modified residues Main-chain involved in catalysis Comment
1gltA01
1glvA01
1gsaA01
1gshA01
2gltA01
1gltA02 LYS 125;ARG 210;ARG 225 ASP 273(Mg1 binding);GLU 281(Mg1 & Mg2 binding);ASN 283(Mg2 binding) THR 288 invisible 226-241
1glvA02 LYS 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
1gsaA02 LYS 125;ARG 210;ARG 225 ASP 273(Mg1 binding);GLU 281(Mg1 & Mg2 binding);ASN 283(Mg2 binding) THR 288
1gshA02 LYS 125;ARG 210;ARG 225 ASP 273(Mg1 binding);GLU 281(Mg1 & Mg2 binding);ASN 283(Mg2 binding) THR 288 invisible 226-241
2gltA02 LYS 125;ARG 210;ARG 225 ASP 273(Mg1 binding);GLU 281(Mg1 & Mg2 binding);ASN 283(Mg2 binding) THR 288 invisible 226-241
1gltA03 LYS 160 ; invisible 164-167
1glvA03 LYS 160 ; invisible 164-167
1gsaA03 LYS 160 GLY 166;GLY 167
1gshA03 LYS 160 ; invisible 164-167
2gltA03 LYS 160 ; invisible 164-167

References for Catalytic Mechanism
References Sections No. of steps in catalysis
[5]
p.2263-2264
[8]
Scheme I, p.12403-12404 2
[11]
Scheme I 3
[14]
Scheme 1, p.11973-11974 3
[17]
Fig.6A

References
[1]
Resource
Comments
Medline ID
PubMed ID 3553173
Journal J Biochem (Tokyo)
Year 1987
Volume 101
Pages 207-15
Authors Kato H, Chihara M, Nishioka T, Murata K, Kimura A, Oda J
Title Homology of Escherichia coli B glutathione synthetase with dihydrofolate reductase in amino acid sequence and substrate binding site.
Related PDB
Related UniProtKB
[2]
Resource
Comments MUTAGENESIS OF CYSTEINE RESIDUES.
Medline ID 88298832
PubMed ID 3042775
Journal J Biol Chem
Year 1988
Volume 263
Pages 11646-51
Authors Kato H, Tanaka T, Nishioka T, Kimura A, Oda J
Title Role of cysteine residues in glutathione synthetase from Escherichia coli B. Chemical modification and oligonucleotide site-directed mutagenesis.
Related PDB
Related UniProtKB P04425
[3]
Resource
Comments
Medline ID
PubMed ID 2685323
Journal J Mol Biol
Year 1989
Volume 209
Pages 503-4
Authors Kato H, Yamaguchi H, Hata Y, Nishioka T, Katsube Y, Oda J
Title Crystallization and preliminary X-ray studies of glutathione synthetase from Escherichia coli B.
Related PDB
Related UniProtKB
[4]
Resource
Comments
Medline ID
PubMed ID 1540152
Journal Biochem Biophys Res Commun
Year 1992
Volume 182
Pages 1040-6
Authors Peters JM, Dalrymple BP, Jorgensen WK
Title Sequence of a putative glutathione synthetase II gene and flanking regions from Anaplasma centrale.
Related PDB
Related UniProtKB
[5]
Resource
Comments SEQUENCE OF 234-242, AND MUTAGENESIS OF ARG-233 AND ARG-241.
Medline ID 92172846
PubMed ID 1540581
Journal Biochemistry
Year 1992
Volume 31
Pages 2259-65
Authors Tanaka T, Kato H, Nishioka T, Oda J
Title Mutational 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 PDB
Related UniProtKB P04425
[6]
Resource
Comments
Medline ID
PubMed ID 8192897
Journal Biochem Cell Biol
Year 1993
Volume 71
Pages 447-53
Authors Nakagawa CW, Mutoh N, Hayashi Y
Title Glutathione synthetase from the fission yeast. Purification and its unique heteromeric subunit structure.
Related PDB
Related UniProtKB
[7]
Resource
Comments
Medline ID
PubMed ID 8431434
Journal Biochemistry
Year 1993
Volume 32
Pages 1548-54
Authors Hibi T, Kato H, Nishioka T, Oda J, Yamaguchi H, Katsube Y, Tanizawa K, Fukui T
Title Use of adenosine (5')polyphospho(5')pyridoxals to study the substrate-binding region of glutathione synthetase from Escherichia coli B.
Related PDB
Related UniProtKB
[8]
Resource
Comments MUTAGENESIS OF PRO-227 AND GLY-240.
Medline ID 94059950
PubMed ID 8241129
Journal Biochemistry
Year 1993
Volume 32
Pages 12398-404
Authors Tanaka T, Yamaguchi H, Kato H, Nishioka T, Katsube Y, Oda J
Title Flexibility impaired by mutations revealed the multifunctional roles of the loop in glutathione synthetase.
Related PDB
Related UniProtKB P04425
[9]
Resource
Comments X-RAY CRYSTALLOGRAPHY (2.0 ANGSTROMS).
Medline ID 93188002
PubMed ID 8445637
Journal J Mol Biol
Year 1993
Volume 229
Pages 1083-100
Authors Yamaguchi H, Kato H, Hata Y, Nishioka T, Kimura A, Oda J, Katsube Y
Title Three-dimensional structure of the glutathione synthetase from Escherichia coli B at 2.0 A resolution.
Related PDB 1glt 1glv 1gsh 2glt
Related UniProtKB P04425
[10]
Resource
Comments
Medline ID
PubMed ID 8172874
Journal Biochemistry
Year 1994
Volume 33
Pages 4995-9
Authors Kato H, Tanaka T, Yamaguchi H, Hara T, Nishioka T, Katsube Y, Oda J
Title Flexible loop that is novel catalytic machinery in a ligase. Atomic structure and function of the loopless glutathione synthetase.
Related PDB
Related UniProtKB
[11]
Resource
Comments
Medline ID
PubMed ID 7862655
Journal Proc Natl Acad Sci U S A
Year 1995
Volume 92
Pages 1172-6
Authors Fan C, Moews PC, Shi Y, Walsh CT, Knox JR
Title A common fold for peptide synthetases cleaving ATP to ADP: glutathione synthetase and D-alanine:d-alanine ligase of Escherichia coli.
Related PDB
Related UniProtKB
[12]
Resource
Comments
Medline ID
PubMed ID 8577699
Journal Protein Eng
Year 1995
Volume 8
Pages 711-6
Authors Hara T, Tanaka T, Kato H, Nishioka T, Oda J
Title Site-directed mutagenesis of glutathione synthetase from Escherichia coli B: mapping of the gamma-L-glutamyl-L-cysteine-binding site.
Related PDB
Related UniProtKB
[13]
Resource
Comments
Medline ID
PubMed ID 7567920
Journal Protein Eng
Year 1995
Volume 8
Pages 353-62
Authors Mizuguchi K, Go N
Title Comparison of spatial arrangements of secondary structural elements in proteins.
Related PDB
Related UniProtKB
[14]
Resource
Comments X-ray crystallography
Medline ID
PubMed ID 8810901
Journal Biochemistry
Year 1996
Volume 35
Pages 11967-74
Authors Hara T, Kato H, Katsube Y, Oda J
Title A 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 PDB 1gsa
Related UniProtKB
[15]
Resource
Comments
Medline ID
PubMed ID 8564538
Journal Nat Struct Biol
Year 1996
Volume 3
Pages 128-32
Authors Artymiuk PJ, Poirrette AR, Rice DW, Willett P
Title Biotin carboxylase comes into the fold.
Related PDB
Related UniProtKB
[16]
Resource
Comments
Medline ID
PubMed ID 8548447
Journal Nat Struct Biol
Year 1996
Volume 3
Pages 16-8
Authors Hibi T, Nishioka T, Kato H, Tanizawa K, Fukui T, Katsube Y, Oda J
Title Structure of the multifunctional loops in the nonclassical ATP-binding fold of glutathione synthetase.
Related PDB
Related UniProtKB
[17]
Resource
Comments X-RAY CRYSTALLOGRAPHY (2.0 ANGSTROMS).
Medline ID 97164199
PubMed ID 9010922
Journal Protein Eng
Year 1996
Volume 9
Pages 1083-92
Authors Matsuda K, Mizuguchi K, Nishioka T, Kato H, Go N, Oda J
Title Crystal structure of glutathione synthetase at optimal pH: domain architecture and structural similarity with other proteins.
Related PDB
Related UniProtKB P04425
[18]
Resource
Comments
Medline ID
PubMed ID 9056244
Journal Arch Biochem Biophys
Year 1997
Volume 339
Pages 151-6
Authors Tanaka T, Nishioka T, Oda J
Title Nicked multifunctional loop of glutathione synthetase still protects the catalytic intermediate.
Related PDB
Related UniProtKB
[19]
Resource
Comments
Medline ID
PubMed ID 9078268
Journal Biochem J
Year 1997
Volume 322
Pages 241-4
Authors Wang CL, Oliver DJ
Title Identification of a putative flexible loop in Arabidopsis glutathione synthetase.
Related PDB
Related UniProtKB
[20]
Resource
Comments
Medline ID
PubMed ID 9463376
Journal EMBO J
Year 1998
Volume 17
Pages 977-84
Authors Esser L, Wang CR, Hosaka M, Smagula CS, Sudhof TC, Deisenhofer J
Title Synapsin I is structurally similar to ATP-utilizing enzymes.
Related PDB
Related UniProtKB
[21]
Resource
Comments
Medline ID
PubMed ID 9551557
Journal Structure
Year 1998
Volume 6
Pages 363-76
Authors Levdikov VM, Barynin VV, Grebenko AI, Melik-Adamyan WR, Lamzin VS, Wilson KS
Title The structure of SAICAR synthase: an enzyme in the de novo pathway of purine nucleotide biosynthesis.
Related PDB
Related UniProtKB
[22]
Resource
Comments
Medline ID
PubMed ID 10438618
Journal J Mol Biol
Year 1999
Volume 291
Pages 239-47
Authors Grishin NV
Title Phosphatidylinositol phosphate kinase: a link between protein kinase and glutathione synthase folds.
Related PDB
Related UniProtKB
[23]
Resource
Comments
Medline ID
PubMed ID 12467574
Journal Structure (Camb)
Year 2002
Volume 10
Pages 1669-76
Authors Gogos A, Shapiro L
Title Large conformational changes in the catalytic cycle of glutathione synthase.
Related PDB
Related UniProtKB
[24]
Resource
Comments
Medline ID
PubMed ID 14990577
Journal J Biol Chem
Year 2004
Volume 279
Pages 22412-21
Authors Dinescu A, Cundari TR, Bhansali VS, Luo JL, Anderson ME
Title Function of conserved residues of human glutathione synthetase: implications for the ATP-grasp enzymes.
Related PDB
Related UniProtKB

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.

Created Updated
2004-08-01 2009-02-26