Glutathione-disulfide reductase

 

The pyrimidine nucleotide-disulphide oxidoreductases are a family of proteins which transfer electrons from NAD(P)H via FAD to a redox-active disulphide bond in the enzyme active site, which then reduces the substrate.

The structure of the ubiquitous enzyme glutathione reductase (EC 1.6.4.2), which helps protect cells from oxidative stress, is similar to trypanothione reductase (EC 1.6.4.8), lipoamide dehydrogenase (EC 1.8.1.4), higher eukaryotic thioredoxin reductase (EC 1.6.4.5) and mercuric reductase (1.16.1.1).

 

Reference Protein and Structure

Sequence
P00390 UniProt (1.8.1.7) IPR006322 (Sequence Homologues) (PDB Homologues)
Biological species
Homo sapiens (Human) Uniprot
PDB
2gh5 - Crystal Structure of human Glutathione Reductase complexed with a Fluoro-Analogue of the Menadione Derivative M5 (1.7 Å) PDBe PDBsum 2gh5
Catalytic CATH Domains
3.30.390.30 CATHdb 3.50.50.60 CATHdb (see all for 2gh5)
Cofactors
Fadh2(2-) (1), Glutathione (1)
Click To Show Structure

Enzyme Reaction (EC:1.8.1.7)

glutathione disulfide(2-)
CHEBI:58297ChEBI
+
NADPH
CHEBI:16474ChEBI
+
hydron
CHEBI:15378ChEBI
→
glutathionate(1-)
CHEBI:57925ChEBI
+
NADP(+)
CHEBI:18009ChEBI
Alternative enzyme names: GSH reductase, GSSG reductase, NADPH-GSSG reductase, NADPH-glutathione reductase, NADPH:oxidized-glutathione oxidoreductase, Glutathione S-reductase, Glutathione reductase, Glutathione reductase (NADPH),

Enzyme Mechanism

Introduction

Initially, the enzyme is in the oxidised state, with its redox active cysteines 58 and 63 forming a disulphide bond to each other. A hydride is transferred from NAD(P)H to FAD, a process facilitated by Glu201, Lys66 and Tyr197, as demonstrated by mutagenesis and structural studies. From there the electron makes an SN2 attack on the sulfur atom of Cys63, causing Cys58 to be displaced as thiolate. This residue is now ready to attack the substrate, which in all cases except mercuric reductase is another disulphide bond. His467 from the other subunit of the dimer has an essential role here, as shown by mutagenesis; first it seems to withdraw a proton from Cys58, activating the latter residue for a nucleophilic attack on the substrate. The proton is then poised to polarise the resulting Cys58-substrate mixed disulphide bond, making it vulnerable to attack from Cys63 to return the enzyme to its rest state. In addition to His467, another residue from the second subunit is found in the active site: Glu472 is shown to be essential by mutagenesis, and in structures is seen to orientate the catalytic Histidine.

Catalytic Residues Roles

UniProt PDB* (2gh5)
His511 His467B Acts as a general acid/base during the reaction. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor
Glu516 Glu472B Activates His467 activator, hydrogen bond acceptor, electrostatic stabiliser
Cys102, Cys107 Cys58A, Cys63A These residues are in a disulfide bond at the start of the reaction and this bond provides the redox potential for the reduction reaction. electrophile, electrofuge, nucleofuge, nucleophile
Tyr241, Lys110, Glu245 Tyr197A, Lys66A, Glu201A Helps facilitate the hydride transfer from NAD(P) to the FAD molecule. activator
*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

aromatic unimolecular elimination by the conjugate base, hydride transfer, aromatic bimolecular nucleophilic addition, cofactor used, intermediate formation, overall product formed, overall reactant used, rate-determining step, bimolecular nucleophilic substitution, enzyme-substrate complex formation, enzyme-substrate complex cleavage, intermediate collapse, intermediate terminated, native state of cofactor regenerated, proton transfer, native state of enzyme regenerated

References

  1. Deponte M (2013), Biochim Biophys Acta, 1830, 3217-3266. Glutathione catalysis and the reaction mechanisms of glutathione-dependent enzymes. DOI:10.1016/j.bbagen.2012.09.018. PMID:23036594.
  2. Filomeni G et al. (2002), Biochem Pharmacol, 64, 1057-1064. Cell signalling and the glutathione redox system. DOI:10.1016/s0006-2952(02)01176-0.
  3. Sweet WL et al. (1991), Biochemistry, 30, 8702-8709. Human erythrocyte glutathione reductase: chemical mechanism and structure of the transition state for hydride transfer. DOI:10.1021/bi00099a031. PMID:1888731.
  4. Karplus PA et al. (1989), J Mol Biol, 210, 163-180. Substrate binding and catalysis by glutathione reductase as derived from refined enzyme: Substrate crystal structures at 2Å resolution. DOI:10.1016/0022-2836(89)90298-2. PMID:2585516.
  5. Deonarain MP et al. (1989), Biochemistry, 28, 9602-9607. Alternative proton donors/acceptors in the catalytic mechanism of the glutathione reductase of Escherichia coli: the role of histidine-439 and tyrosine-99. DOI:10.1021/bi00451a008. PMID:2558727.
  6. Karplus PA et al. (1987), J Mol Biol, 195, 701-729. Refined structure of glutathione reductase at 1.54 Å resolution. DOI:10.1016/0022-2836(87)90191-4. PMID:3656429.
  7. Pai EF et al. (1983), J Biol Chem, 258, 1752-1757. The catalytic mechanism of glutathione reductase as derived from x-ray diffraction analyses of reaction intermediates. PMID:6822532.

Step 1. NADP eliminates a hydride ion, which attacks the FAD cofactor with concomitant double bond rearrangement.

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Catalytic Residues Roles

Residue Roles
His467B hydrogen bond donor
Lys66A hydrogen bond donor
Glu472B hydrogen bond acceptor
Glu201A hydrogen bond acceptor, electrostatic stabiliser
Tyr197A activator
Lys66A activator
Glu201A activator

Chemical Components

ingold: aromatic unimolecular elimination by the conjugate base, hydride transfer, ingold: aromatic bimolecular nucleophilic addition, cofactor used, intermediate formation, overall product formed, overall reactant used, rate-determining step

Step 2. The FAD oxyanion collapses, initiating a nucleophilic attack on Cys63 in a substitution reaction that eliminates Cys58 from the disulfide bond.

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Catalytic Residues Roles

Residue Roles
His467B hydrogen bond donor
Lys66A hydrogen bond donor, electrostatic stabiliser
Glu472B hydrogen bond acceptor
Glu201A hydrogen bond acceptor, electrostatic stabiliser
Cys58A nucleofuge
Cys63A electrofuge, electrophile

Chemical Components

ingold: bimolecular nucleophilic substitution, enzyme-substrate complex formation, intermediate formation

Step 3. The FAD cofactor eliminates Cys63 with concomitant deprotonation of the nitrogen to which the hydride was added.

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Catalytic Residues Roles

Residue Roles
Cys63A hydrogen bond acceptor
His467B hydrogen bond donor
Lys66A hydrogen bond donor
Glu472B hydrogen bond acceptor
Glu201A hydrogen bond acceptor
Cys63A nucleofuge, proton acceptor

Chemical Components

ingold: aromatic unimolecular elimination by the conjugate base, enzyme-substrate complex cleavage, intermediate collapse, intermediate terminated, native state of cofactor regenerated

Step 4. His467B deprotonates Cys63A.

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Catalytic Residues Roles

Residue Roles
Cys63A hydrogen bond donor
His467B hydrogen bond acceptor, hydrogen bond donor
Lys66A hydrogen bond donor
Glu472B hydrogen bond acceptor, activator
Glu201A hydrogen bond acceptor
His467B proton acceptor
Cys63A proton donor

Chemical Components

proton transfer

Step 5. Cys58 initiates a nucleophilic attack on the disulfide bond of oxidised glutathione in a substitution reaction, eliminating glutathione with concomitant deprotonation of His467B.

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Catalytic Residues Roles

Residue Roles
Lys66A hydrogen bond donor
Glu201A hydrogen bond acceptor
His467B hydrogen bond donor
Glu472B hydrogen bond acceptor, electrostatic stabiliser
Cys58A nucleophile
His467B proton donor

Chemical Components

proton transfer, ingold: bimolecular nucleophilic substitution, enzyme-substrate complex formation, intermediate formation, overall product formed, cofactor used

Step 6. Cys63 initiates a nucleophilic attack on Cys58 in a substitution reaction, eliminating glutathione with concomitant deprotonation of water.

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Catalytic Residues Roles

Residue Roles
Lys66A hydrogen bond donor
Glu201A hydrogen bond acceptor
His467B hydrogen bond donor, hydrogen bond acceptor
Glu472B hydrogen bond acceptor
Cys58A electrofuge, electrophile
Cys63A nucleophile

Chemical Components

proton transfer, ingold: bimolecular nucleophilic substitution, enzyme-substrate complex cleavage, intermediate collapse, intermediate terminated, overall product formed, native state of enzyme regenerated, native state of cofactor regenerated
Download: Image, Marvin File

Step 1. NADP eliminates a hydride ion, which attacks the FAD cofactor with concomitant double bond rearrangement.

Step 2. The FAD oxyanion collapses, initiating a nucleophilic attack on Cys63 in a substitution reaction that eliminates Cys58 from the disulfide bond.

Step 3. The FAD cofactor eliminates Cys63 with concomitant deprotonation of the nitrogen to which the hydride was added.

Step 4. His467B deprotonates Cys63A.

Step 5. Cys58 initiates a nucleophilic attack on the disulfide bond of oxidised glutathione in a substitution reaction, eliminating glutathione with concomitant deprotonation of His467B.

Step 6. Cys63 initiates a nucleophilic attack on Cys58 in a substitution reaction, eliminating glutathione with concomitant deprotonation of water.

Products.

Contributors

Gemma L. Holliday, Daniel E. Almonacid, Gail J. Bartlett, Anna Waters, Craig Porter, Katherine Ferris