Glutathione synthase (eukaryotic)

 

Glutathione synthase is a homodimeric enzyme that catalyses the conversion of gamma-L-glutamyl-L-cysteine and glycine to phosphate and glutathione in the presence of ATP. This is the second step in glutathione biosynthesis and in humans, defects in this enzyme are inherited in an autosomal recessive way and are the cause of severe metabolic acidosis, 5-oxoprolinuria, and increased rate of haemolysis and defective function of the central nervous system.

This entry represents the eukaryotic glutathione synthase, which includes human, yeast, plant and trypanosoma proteins.

 

Reference Protein and Structure

Sequence
P48637 UniProt (6.3.2.3) IPR005615 (Sequence Homologues) (PDB Homologues)
Biological species
Homo sapiens (Human) Uniprot
PDB
2hgs - HUMAN GLUTATHIONE SYNTHETASE (2.1 Å) PDBe PDBsum 2hgs
Catalytic CATH Domains
3.30.1490.50 CATHdb 3.30.470.20 CATHdb 1.10.1080.10 CATHdb (see all for 2hgs)
Cofactors
Magnesium(2+) (2)
Click To Show Structure

Enzyme Reaction (EC:6.3.2.3)

ATP(4-)
CHEBI:30616ChEBI
+
glycine zwitterion
CHEBI:57305ChEBI
+
L-gamma-glutamyl-L-cysteinate(1-)
CHEBI:58173ChEBI
→
hydrogenphosphate
CHEBI:43474ChEBI
+
hydron
CHEBI:15378ChEBI
+
ADP(3-)
CHEBI:456216ChEBI
+
glutathionate(1-)
CHEBI:57925ChEBI
Alternative enzyme names: GSH synthetase, Glutathione synthetase,

Enzyme Mechanism

Introduction

Although this protein shares very little similarity to the prokaryotic enzyme (MACiE 199), the reaction is thought to proceed in a similar manner with 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.

Catalytic Residues Roles

UniProt PDB* (2hgs)
Ser151 (main-N) Ser151A (main-N) Acts to stabilise the build up of negative charge on the tetrahedral intermediate in the second transfer reaction. electrostatic stabiliser
Arg125 Arg125A Acts to stabilise the build up of negative charge on the tetrahedral intermediate in both the first and second transfer steps. electrostatic stabiliser
Glu144, Asn146, Glu368 Glu144A, Asn146A, Glu368A Form part of the magnesium binding site. metal ligand
Gly369 (main-N), Arg450, Lys305, Lys364 Gly369A (main-N), Arg450A, Lys305A, Lys364A Act to stabilise the negative charge on the phosphate and leaving groups during the course of the reaction. electrostatic stabiliser
*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

Chemical Components

References

  1. Dinescu A et al. (2004), J Biol Chem, 279, 22412-22421. Function of Conserved Residues of Human Glutathione Synthetase. DOI:10.1074/jbc.m401334200. PMID:14990577.
  2. De Jesus MC et al. (2014), Protein J, 33, 403-409. The Role of Strong Electrostatic Interactions at the Dimer Interface of Human Glutathione Synthetase. DOI:10.1007/s10930-014-9573-y. PMID:25070563.
  3. Dworeck T et al. (2012), PLoS One, 7, e46580-. Site Directed Mutagenesis of Schizosaccharomyces pombe Glutathione Synthetase Produces an Enzyme with Homoglutathione Synthetase Activity. DOI:10.1371/journal.pone.0046580. PMID:23091597.
  4. Slavens KD et al. (2011), Biochem Biophys Res Commun, 410, 597-601. Valine 44 and valine 45 of human glutathione synthetase are key for subunit stability and negative cooperativity. DOI:10.1016/j.bbrc.2011.06.034. PMID:21683691.
  5. Brown TR et al. (2011), Biochem Biophys Res Commun, 411, 536-542. Aspartate 458 of human glutathione synthetase is important for cooperativity and active site structure. DOI:10.1016/j.bbrc.2011.06.166. PMID:21771585.
  6. Dinescu A et al. (2010), Biochem Biophys Res Commun, 400, 511-516. The role of the glycine triad in human glutathione synthetase. DOI:10.1016/j.bbrc.2010.08.081. PMID:20800579.
  7. Herrera K et al. (2007), J Biol Chem, 282, 17157-17165. Reaction Mechanism of Glutathione Synthetase from Arabidopsis thaliana: SITE-DIRECTED MUTAGENESIS OF ACTIVE SITE RESIDUES. DOI:10.1074/jbc.m700804200. PMID:17452339.
  8. Jez JM et al. (2004), J Biol Chem, 279, 42726-42731. Kinetic Mechanism of Glutathione Synthetase from Arabidopsis thaliana. DOI:10.1074/jbc.m407961200. PMID:15302873.
  9. Gogos A et al. (2002), Structure, 10, 1669-1676. Large conformational changes in the catalytic cycle of glutathione synthase. PMID:12467574.
  10. Polekhina G et al. (1999), EMBO J, 18, 3204-3213. Molecular basis of glutathione synthetase deficiency and a rare gene permutation event. DOI:10.1093/emboj/18.12.3204. PMID:10369661.
  11. Gali RR et al. (1997), Biochem J, 321 ( Pt 1), 207-210. Identification of an essential cysteine residue in human glutathione synthase. PMID:9003420.
  12. Shi ZZ et al. (1996), Nat Genet, 14, 361-365. Mutations in the glutathione synthetase gene cause 5–oxoprolinuria. DOI:10.1038/ng1196-361. PMID:8896573.
  13. Yamaguchi H et al. (1993), J Mol Biol, 229, 1083-1100. Three-dimensional Structure of the Glutathione Synthetase from Escherichia coli B at 2·0 Å Resolution. DOI:10.1006/jmbi.1993.1106. PMID:8445637.

Step 1.

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Catalytic Residues Roles

Residue Roles
Arg125A electrostatic stabiliser
Ser151A (main-N) electrostatic stabiliser
Gly369A (main-N) electrostatic stabiliser
Arg450A electrostatic stabiliser
Glu144A metal ligand
Asn146A metal ligand
Glu368A metal ligand
Lys305A electrostatic stabiliser
Lys364A electrostatic stabiliser

Chemical Components

Step 1.

Contributors

Craig Porter, Gemma L. Holliday