Glutathione synthase (prokaryotic)

 

Glutathione synthetase catalyses the ligation of glycine to gamma-L-glutamyl-L-cysteine (gamma-Glu-Cys) with concomitant hydrolysis of ATP. This is the second and final step in the synthesis of glutathione. Glutathione has many important functions in the cell, including protection against oxidative damage, detoxification of harmful substances, and as a coenzyme for several reactions. Glutathione synthetase is present in species ranging from bacteria to mammals. This entry represents the glutathione synthetases found in Gram-negative bacteria. This gene does not appear to be present in genomes of Gram-positive bacteria.

 

Reference Protein and Structure

Sequence
P04425 UniProt (6.3.2.3) IPR006284 (Sequence Homologues) (PDB Homologues)
Biological species
Escherichia coli K-12 (Bacteria) Uniprot
PDB
1gsa - STRUCTURE OF GLUTATHIONE SYNTHETASE COMPLEXED WITH ADP AND GLUTATHIONE (2.0 Å) PDBe PDBsum 1gsa
Catalytic CATH Domains
3.30.1490.20 CATHdb 3.30.470.20 CATHdb (see all for 1gsa)
Cofactors
Magnesium(2+) (2) Metal MACiE
Click To Show Structure

Enzyme Reaction (EC:6.3.2.3)

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

Enzyme Mechanism

Introduction

The reaction proceeds via an enzyme-bound acylphosphate intermediate. In the first step, a phosphate group is transferred from ATP to the carboxyl group of the Cys in gamma-Glu-Cys. Arg 225, Lys 160, and two Mg(II) ions may stabilise negative charge in the transition state of phosphoryl transfer. In the second step, the carbonyl of the acyl phosphate is attacked by the amino group of glycine to form a tetrahedral intermediate that is proposed to be stabilised by the guanidinium group of Arg 210. Collapse of the tetrahedral intermediate with loss of phosphate completes the reaction.

Catalytic Residues Roles

UniProt PDB* (1gsa)
Arg210 Arg210A Provides a positive charge to stabilise the tetrahedral intermediate resulting from attack by glycine on the carbonyl of the acylphosphate intermediate. hydrogen bond donor, electrostatic stabiliser
Arg225, Lys160 Arg225A, Lys160A Proposed to stabilise negative charge in the transition state for phosphoryl transfer. hydrogen bond donor, electrostatic stabiliser
Lys125, Arg233 Lys125A, Arg233A Involved in binding and stabilising the negatively charged substrates, intermediates and transition states. electrostatic stabiliser
Asp273 Asp273A Forms part of the first magnesium binding site. metal ligand
Glu281 Glu281A Acts as a bridging ligand between the two magnesium binding sites. metal ligand
Asn283 Asn283A Forms part of the second magnesium binding site. metal ligand
*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

bimolecular nucleophilic substitution, bimolecular nucleophilic addition, proton transfer, unimolecular elimination by the conjugate base

References

  1. Hara T et al. (1996), Biochemistry, 35, 11967-11974. A Pseudo-Michaelis Quaternary Complex in the Reverse Reaction of a Ligase:  Structure ofEscherichia coliB Glutathione Synthetase Complexed with ADP, Glutathione, and Sulfate at 2.0 Å Resolution†,‡. DOI:10.1021/bi9605245. PMID:8810901.
  2. Tanaka T et al. (1997), Arch Biochem Biophys, 339, 151-156. Nicked Multifunctional Loop of Glutathione Synthetase Still Protects the Catalytic Intermediate. DOI:10.1006/abbi.1996.9821. PMID:9056244.
  3. Matsuda K et al. (1996), Protein Eng, 9, 1083-1092. Crystal structure of glutathione synthetase at optimal pH: domain architecture and structural similarity with other proteins. PMID:9010922.
  4. Tanaka T et al. (1993), Biochemistry, 32, 12398-12404. Flexibility impaired by mutations revealed the multifunctional roles of the loop in glutathione synthetase. PMID:8241129.
  5. 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.
  6. Tanaka T et al. (1992), Biochemistry, 31, 2259-2265. 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. PMID:1540581.
  7. Kato H et al. (1988), J Biol Chem, 263, 11646-11651. Role of cysteine residues in glutathione synthetase from Escherichia coli B. Chemical modification and oligonucleotide site-directed mutagenesis. PMID:3042775.

Step 1. The carboxylate group of gamma-L-glutamyl-L-cysteine acts as a nucleophile and attacks the alpha-phoposphate of ATP in a substitution reaction, liberating ADP. The two Mg(II) ions, Lys160, Arg210 and Arg225 all stabilise the intermediates formed.

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Catalytic Residues Roles

Residue Roles
Lys160A hydrogen bond donor, electrostatic stabiliser
Arg210A hydrogen bond donor, electrostatic stabiliser
Arg225A hydrogen bond donor, electrostatic stabiliser
Glu281A metal ligand
Asp273A metal ligand
Asn283A metal ligand
Arg233A electrostatic stabiliser
Lys125A electrostatic stabiliser

Chemical Components

ingold: bimolecular nucleophilic substitution

Step 2. The amine group of L-glycine acts as a nucleophile and attacks the carbonyl group of the phosphorylated substrate in an addition reaction. The two Mg(II) ions, Lys160, Arg210 and Arg225 all stabilise the intermediates formed.

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Catalytic Residues Roles

Residue Roles
Lys160A hydrogen bond donor, electrostatic stabiliser
Arg210A hydrogen bond donor, electrostatic stabiliser
Arg225A hydrogen bond donor, electrostatic stabiliser
Lys125A electrostatic stabiliser
Arg233A electrostatic stabiliser
Glu281A metal ligand
Asp273A metal ligand
Asn283A metal ligand

Chemical Components

ingold: bimolecular nucleophilic addition

Step 3. The oxyanion formed re-forms the carbonyl group, cleaving the P-O bond in a conjugate base elimination reaction. The leaving phosphate group deprotonates the newly formed secondary amine. The two Mg(II) ions, Lys160, Arg210 and Arg225 all stabilise the intermediates formed.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys160A hydrogen bond donor, electrostatic stabiliser
Arg210A hydrogen bond donor, electrostatic stabiliser
Arg225A hydrogen bond donor, electrostatic stabiliser
Lys125A electrostatic stabiliser
Arg233A electrostatic stabiliser
Glu281A metal ligand
Asp273A metal ligand
Asn283A metal ligand

Chemical Components

proton transfer, ingold: unimolecular elimination by the conjugate base
Download: Image, Marvin File

Step 1. The carboxylate group of gamma-L-glutamyl-L-cysteine acts as a nucleophile and attacks the alpha-phoposphate of ATP in a substitution reaction, liberating ADP. The two Mg(II) ions, Lys160, Arg210 and Arg225 all stabilise the intermediates formed.

Step 2. The amine group of L-glycine acts as a nucleophile and attacks the carbonyl group of the phosphorylated substrate in an addition reaction. The two Mg(II) ions, Lys160, Arg210 and Arg225 all stabilise the intermediates formed.

Step 3. The oxyanion formed re-forms the carbonyl group, cleaving the P-O bond in a conjugate base elimination reaction. The leaving phosphate group deprotonates the newly formed secondary amine. The two Mg(II) ions, Lys160, Arg210 and Arg225 all stabilise the intermediates formed.

Products.

Contributors

Gemma L. Holliday, Daniel E. Almonacid, Steven Smith