Glutathione S-transferase class-μ

 

Glutathione S-transferases (GSTs) are promiscuous enzymes that catalyze several reactions with various substrates. Their essential function is detoxification of xenobiotic compounds that are conjugated to glutathione (GSH) and then excreted; other functions, not associated with detoxification, include repair of macromolecules oxidised by reactive oxygen species, regeneration of S-thiolated proteins, and biosynthesis of physiologically important metabolites. There are two proposed mechanisms regarding GSH activation depending on the conformation of Tyr10 (in and out). Both appear to be viable where evidence supports both conformations.

 

Reference Protein and Structure

Sequence
P30114 UniProt (2.5.1.18) IPR003081 (Sequence Homologues) (PDB Homologues)
Biological species
Schistosoma haematobium (Blood fluke) Uniprot
PDB
1oe8 - 28kDa glutathione S-transferase from Schistosoma haematobium (glutathione saturated) (1.65 Å) PDBe PDBsum 1oe8
Catalytic CATH Domains
1.20.1050.10 CATHdb 3.40.30.10 CATHdb (see all for 1oe8)
Click To Show Structure

Enzyme Reaction (EC:2.5.1.18)

organic halide
CHEBI:17792ChEBI
+
glutathionate(1-)
CHEBI:57925ChEBI
→
halide anion
CHEBI:16042ChEBI
+
S-substitued glutathione(1-)
CHEBI:90779ChEBI
+
hydron
CHEBI:15378ChEBI
Alternative enzyme names: S-(hydroxyalkyl)glutathione lyase, Glutathione S-alkyl transferase, Glutathione S-alkyltransferase, Glutathione S-aralkyltransferase, Glutathione S-aryltransferase, Glutathione S-transferase,

Enzyme Mechanism

Introduction

GSTs catalyse the nucleophilic attack of the activated thiolate of GSH to electrophilic substrates, and an important step of the catalytic cycle is the activation of GSH to GS−. The second mechanism is thought to proceed by the Tyr10-out conformer acting as a general base (facilitated by Arg21) to activate GSH nucleophilic attack when GSH binds.

Catalytic Residues Roles

UniProt PDB* (1oe8)
Tyr10 Tyr10A Activates glutathione (GSH) to GS(-) by acting as a general base to the thiol group of GSH. proton acceptor, proton donor
Arg21 Arg21A Arg21 lowers the pKa of the catalytic Tyr10 via π-cation interactions, as required for catalysis.
*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, proton transfer, overall reactant used, overall product formed, native state of enzyme regenerated

References

  1. Angelucci F et al. (2005), Structure, 13, 1241-1246. Insights into the catalytic mechanism of glutathione S-transferase: the lesson from Schistosoma haematobium. DOI:10.1016/j.str.2005.06.007. PMID:16154081.
  2. Flanagan JU et al. (2011), Drug Metab Rev, 43, 194-214. Sigma-class glutathione transferases. DOI:10.3109/03602532.2011.560157. PMID:21425928.
  3. Baiocco P et al. (2006), J Mol Biol, 360, 678-689. Probing the mechanism of GSH activation in Schistosoma haematobium glutathione-S-transferase by site-directed mutagenesis and X-ray crystallography. DOI:10.1016/j.jmb.2006.05.040. PMID:16777141.
  4. Johnson KA et al. (2003), Biochemistry, 42, 10084-10094. Crystal Structure of the 28 kDa GlutathioneS-Transferase fromSchistosoma haematobium†. DOI:10.1021/bi034449r. PMID:12939136.
  5. Armstrong RN (1997), Chem Res Toxicol, 10, 2-18. Structure, Catalytic Mechanism, and Evolution of the Glutathione Transferases. DOI:10.1021/tx960072x. PMID:9074797.

Step 1. Tyr10-out acts a base to deprotonate glutathione's thiol group, activiating it for nucleophilic attack on GST's electrophilic substrate.

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Catalytic Residues Roles

Residue Roles
Tyr10A proton acceptor

Chemical Components

ingold: bimolecular nucleophilic substitution, proton transfer, overall reactant used, overall product formed

Step 2. Tyr-10 is deprotonated by a water molecule or the leaving group (denoted X-).

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Tyr10A proton donor

Chemical Components

proton transfer, native state of enzyme regenerated
Download: Image, Marvin File

Step 1. Tyr10-out acts a base to deprotonate glutathione's thiol group, activiating it for nucleophilic attack on GST's electrophilic substrate.

Step 2. Tyr-10 is deprotonated by a water molecule or the leaving group (denoted X-).

Products.

Introduction

In the GSTs belonging to the classes alpha, mu, pi, and sigma, glutathione activation has two proposed mechanisms. The first is thought via the interaction with a Tyr10-in conformer at H-bonding distance from the sulfur of GSH which lowers the pKa of GSH to activate it for nucleophilic attack on electrophilic atoms joined to sulfate, nitrile and halide groups.

Catalytic Residues Roles

UniProt PDB* (1oe8)
Tyr10 Tyr10A Activates glutathione (GSH) to GS(-) via a hydrogen bond between the tyrosine and sulfur of GSH.
*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

overall product formed, overall reactant used, proton transfer, bimolecular nucleophilic substitution

References

  1. Angelucci F et al. (2005), Structure, 13, 1241-1246. Insights into the catalytic mechanism of glutathione S-transferase: the lesson from Schistosoma haematobium. DOI:10.1016/j.str.2005.06.007. PMID:16154081.
  2. Flanagan JU et al. (2011), Drug Metab Rev, 43, 194-214. Sigma-class glutathione transferases. DOI:10.3109/03602532.2011.560157. PMID:21425928.
  3. Baiocco P et al. (2006), J Mol Biol, 360, 678-689. Probing the mechanism of GSH activation in Schistosoma haematobium glutathione-S-transferase by site-directed mutagenesis and X-ray crystallography. DOI:10.1016/j.jmb.2006.05.040. PMID:16777141.
  4. Armstrong RN (1997), Chem Res Toxicol, 10, 2-18. Structure, Catalytic Mechanism, and Evolution of the Glutathione Transferases. DOI:10.1021/tx960072x. PMID:9074797.

Step 1. Tyr10-in acts as a hydrogen bond donor to lower the pKa of glutathione's thiol group, activiating it for nucleophilic attack on GST's electrophilic substrate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles

Chemical Components

overall product formed, overall reactant used, proton transfer, ingold: bimolecular nucleophilic substitution
Download: Image, Marvin File

Step 1. Tyr10-in acts as a hydrogen bond donor to lower the pKa of glutathione's thiol group, activiating it for nucleophilic attack on GST's electrophilic substrate.

Products.

Introduction

One of two water coordination spheres in the active site contain 2 water molecules which His107 uses as bridges to remove a proton from the sulphur group on GSH. This activates GSH to attack the electrophile that is being metabolised. The proton gained by His107 is then returned to the aqueous solution or gained by the substituted metabolite, regenerating the native state of the enzyme.

Catalytic Residues Roles

UniProt PDB* (1oe8)
Arg43 Arg42(43)A Arg42 is crucial for stabilising the transition state of the GSH substrate via its positively charged nitrogens. electrostatic stabiliser, transition state stabiliser
Tyr7 Tyr6(7)A Tyr6 stabilises the GSH molecule by hydrogen bonding to it. The mutation of this residue shows a reduction in catalytic activity. hydrogen bond donor, electrostatic stabiliser
His108 His107(108)A His107 deprotonates the SH of the glutathione through two water molecule bridges, activating it. proton acceptor, proton donor
*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, proton transfer, overall reactant used, overall product formed, native state of enzyme regenerated

References

  1. Dourado DF et al. (2010), J Phys Chem B, 114, 12972-12980. Glutathione transferase classes alpha, pi, and mu: GSH activation mechanism. DOI:10.1021/jp1053875. PMID:20853826.
  2. Patskovsky Y et al. (2006), Biochemistry, 45, 3852-3862. Transition state model and mechanism of nucleophilic aromatic substitution reactions catalyzed by human glutathione S-transferase M1a-1a. DOI:10.1021/bi051823+. PMID:16548513.

Step 1. His107 deprotonates the GSH-SH atom via 2 water bridges. This activates GSH to attack the electrophilic substance needed to be metabolised.

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Catalytic Residues Roles

Residue Roles
His107(108)A proton acceptor
Arg42(43)A electrostatic stabiliser, transition state stabiliser
Tyr6(7)A electrostatic stabiliser, hydrogen bond donor

Chemical Components

ingold: bimolecular nucleophilic substitution, proton transfer, overall reactant used, overall product formed

Step 2. it can be inferred that His107 is then deprotonated either by the metabolite X- or the aqueous solution.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His107(108)A proton donor
Tyr6(7)A electrostatic stabiliser, hydrogen bond donor
Arg42(43)A electrostatic stabiliser

Chemical Components

proton transfer, native state of enzyme regenerated
Download: Image, Marvin File

Step 1. His107 deprotonates the GSH-SH atom via 2 water bridges. This activates GSH to attack the electrophilic substance needed to be metabolised.

Step 2. it can be inferred that His107 is then deprotonated either by the metabolite X- or the aqueous solution.

Products.

Contributors

Alex Gutteridge, Craig Porter, Gemma L. Holliday, Morwenna Hall, Marko Babić