Glutathione S-transferase A

 

Glutathione transferase (GST) is a detoxification enzyme found in the liver. GSTs are the most important enzymes involved in the metabolism of electrophilic xenobiotic/endobiotic compounds. These enzymes are able to catalyze the nucleophilic addition of glutathione (GSH) sulfur thiolate to a wide range of electrophilic substrates, building up a less toxic and more soluble compound. Classes alpha, pi, and mu are the most extensively studied GSTs. GSTA and GSTP are similar in active site residues and have the same mechanism for glutathione transfer.

 

Reference Protein and Structure

Sequence
P09211 UniProt (2.5.1.18) IPR003080 (Sequence Homologues) (PDB Homologues)
Biological species
Homo sapiens (Human) Uniprot
PDB
1pgt - CRYSTAL STRUCTURE OF HUMAN GLUTATHIONE S-TRANSFERASE P1-1[V104] COMPLEXED WITH S-HEXYLGLUTATHIONE (1.8 Å) PDBe PDBsum 1pgt
Catalytic CATH Domains
3.40.30.10 CATHdb (see all for 1pgt)
Click To Show Structure

Enzyme Reaction (EC:2.5.1.18)

glutathionate(1-)
CHEBI:57925ChEBI
+
organic halide
CHEBI:17792ChEBI
→
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

GSH transfers its GSH-SH proton onto its GSH-COO- through a proton transfer mediated by a water molecule. A water molecule deprotonates the thiolate and brigdes the proton to the carboxylate part of the GSH molecule. This reaction activates GSH to attack the electrophilic substrate being metabolised. The Tyr9 and Arg15 stabilize the GSH and the transition state.

Catalytic Residues Roles

UniProt PDB* (1pgt)
Arg15 Arg15A Arg15 is crucial for stabilising the GSH thiolate when it turns into a nucleophile. Without this arginine the activity drops 200 to 400 times. electrostatic stabiliser
Tyr9 Tyr9A Tyr9 modifies the pKa of the thiolate of the GSH. This helps deprotonate the GSH thiolate with a water molecule, activating GSH. modifies pKa, hydrogen bond 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

intermediate formation, proton transfer, bimolecular nucleophilic substitution, intermediate collapse, overall product formed, overall reactant used

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. Dourado DF et al. (2013), Biochemistry, 52, 8069-8078. Mechanism of glutathione transferase P1-1-catalyzed activation of the prodrug canfosfamide (TLK286, TELCYTA). DOI:10.1021/bi4005705. PMID:24066958.
  3. Dourado DFAR et al. (2010), J Phys Chem B, 114, 1690-1697. Glutathione Transferase A1-1: Catalytic Importance of Arginine 15. DOI:https://doi.org/10.1021/jp908251z.
  4. Grahn E et al. (2006), Acta Crystallogr D Biol Crystallogr, 62, 197-207. New crystal structures of human glutathione transferase A1-1 shed light on glutathione binding and the conformation of the C-terminal helix. DOI:10.1107/S0907444905039296. PMID:16421451.

Step 1. Tyr9 lowers the pKa of the thiolate and a water molecule deprotonates the thiolate bridging the proton to the GSH-COO- part of the molecule.

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Catalytic Residues Roles

Residue Roles
Arg15A electrostatic stabiliser
Tyr9A modifies pKa, hydrogen bond donor

Chemical Components

intermediate formation, proton transfer

Step 2. The hydronium intermediate collapses and the GSH sulphur atom nucleophilically attacks the electrophile which is being metabolised.

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Catalytic Residues Roles

Residue Roles
Arg15A electrostatic stabiliser
Tyr9A modifies pKa, hydrogen bond donor

Chemical Components

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

Step 1. Tyr9 lowers the pKa of the thiolate and a water molecule deprotonates the thiolate bridging the proton to the GSH-COO- part of the molecule.

Step 2. The hydronium intermediate collapses and the GSH sulphur atom nucleophilically attacks the electrophile which is being metabolised.

Products.

Introduction

This is the mechanism of the activation of the prodrug canfosfamide. A proton is transferred from the Tyr7 hydroxyl group to the canfosfamide COO- oxygen atom via a water molecule bridge. Then the negatively charged Tyr7 works as a base and receives a proton from the substrates α-C atom. Lastly, we observe the breaking of the bond between canfosfamides β-C and the adjacent oxygen and the consequent release of the phosphorodiamidate.

Catalytic Residues Roles

UniProt PDB* (1pgt)
Tyr8 Tyr7(8)A Tyr7 is deprotonated by the COO- of canfosfamide via a water molecule bridge. The deprotonated Tyr7 then deprotonates the substrates α-C atom causing it to break apart into two products. 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

proton transfer, native state of enzyme regenerated, unimolecular elimination by the conjugate base, overall product formed, intramolecular rearrangement

References

  1. Dourado DF et al. (2013), Biochemistry, 52, 8069-8078. Mechanism of glutathione transferase P1-1-catalyzed activation of the prodrug canfosfamide (TLK286, TELCYTA). DOI:10.1021/bi4005705. PMID:24066958.
  2. 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.
  3. Ji X (1997),CRYSTAL STRUCTURE OF HUMAN GLUTATHIONE S-TRANSFERASE P1-1[V104] COMPLEXED WITH S-HEXYLGLUTATHIONE. DOI:10.2210/pdb1PGT/pdb.

Step 1. Tyr7 gets deprotonated by the canfosfamide COO- end through a water bridge molecule.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Tyr7(8)A proton donor

Chemical Components

proton transfer

Step 2. Tyr7 works as a base and receives a proton from canfosfamides α-C atom.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Tyr7(8)A proton acceptor

Chemical Components

proton transfer, native state of enzyme regenerated

Step 3. The bond between canfosfamide β-C and the adjacent oxygen breaks and consequentially releases the phosphorodiamidate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles

Chemical Components

ingold: unimolecular elimination by the conjugate base, overall product formed, intramolecular rearrangement
Download: Image, Marvin File

Step 1. Tyr7 gets deprotonated by the canfosfamide COO- end through a water bridge molecule.

Step 2. Tyr7 works as a base and receives a proton from canfosfamides α-C atom.

Step 3. The bond between canfosfamide β-C and the adjacent oxygen breaks and consequentially releases the phosphorodiamidate.

Products.

Contributors

Marko Babić, Antonio Ribeiro