Protein disulfide oxidoreductase

 

Deoxyribonucleotide synthesis requires the reduction of the the C2' atom of ribonucleotides. Ribonucleotide reductase (RNR) catalyses this reaction via the formation of a disulfide bond in RNR itself. To regenerate active RNR, this disulfide must be reduced; glutaredoxin (Grx) is one of the enzymes which does this.

 

Reference Protein and Structure

Sequence
P68688 UniProt IPR011902 (Sequence Homologues) (PDB Homologues)
Biological species
Escherichia coli K-12 (Bacteria) Uniprot
PDB
1qfn - GLUTAREDOXIN-1-RIBONUCLEOTIDE REDUCTASE B1 MIXED DISULFIDE BOND (solution nmr Å) PDBe PDBsum 1qfn
Catalytic CATH Domains
3.40.30.10 CATHdb (see all for 1qfn)
Click To Show Structure

Enzyme Reaction (EC:1.8.4.-)

L-cysteine residue
CHEBI:29950ChEBI
+
L-cystine residue
CHEBI:50058ChEBI
→
L-cystine residue
CHEBI:50058ChEBI
+
L-cysteine residue
CHEBI:29950ChEBI

Enzyme Mechanism

Introduction

Oxidoreduction is via the formation and breaking of disulfide bonds between cysteine side groups: Oxidised RNR has a disulfide bond between Cys 143 and Cys 148. Cys 11 of Grx is nucleophilic and attacks Cys 148 in an SN2 fashion to break the Cys 143-Cys 148 bond. Cys 14 is in a low pKa environment and is nucleophilic; it attacks Cys 11 in an SN2 fashion to break the Cys 148-Cys 11 bond. Thus, the disulfide bond of RNR is transferred to Grx.

Catalytic Residues Roles

UniProt PDB* (1qfn)
Arg8 Arg8A Arg8 decreases the pKa of Cys11, making it more nucleophilic. It also increases the negative electrostatic potential surrounding Cys14, making it more nucleophilic. modifies pKa, enhance reactivity
Tyr13 Tyr13A Tyr13 stabilises the transition state for the formation of the Cys754 thiol and helps to prevent the reaction from reversing. electrostatic stabiliser
Lys18 Lys18A Lys18 increases the negative electrostatic potential surrounding Cys14, making it more nucleophilic. enhance reactivity
Tyr72 Tyr72A Tyr72 stabilises the transition state for the formation of the Cys754 thiol and helps to prevent the reaction from reversing. electrostatic stabiliser
Cys11, Cys14 Cys11A, Ser14A Act as catalytic nucleophiles. electrofuge, electrophile, nucleophile
Gly10 (main-N) Gly10A (main-N) Gly10 is thought to be involved in proton transfer from Cys14 to the solvent. proton acceptor, electrostatic stabiliser, 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, intermediate formation, enzyme-substrate complex formation, proton transfer, overall product formed, inferred reaction step, native state of enzyme regenerated

References

  1. Berardi MJ et al. (1999), J Mol Biol, 292, 151-161. Binding specificity and mechanistic insight into glutaredoxin-catalyzed protein disulfide reduction. DOI:10.1006/jmbi.1999.3067. PMID:10493864.
  2. Foloppe N et al. (2004), Structure, 12, 289-300. The Glutaredoxin -C-P-Y-C- Motif: Influence of Peripheral Residues. DOI:10.1016/j.str.2004.01.009. PMID:14962389.
  3. Albright TD (1999), Proc Natl Acad Sci U S A, 96, 7611-7613. More than one way to see it move? DOI:10.1073/pnas.96.14.7611. PMID:10393864.

Step 1. In a bimolecular nucleophilic substitution reaction Cys11 attacks the S-S bond of RNR and displaces Cys143.

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Catalytic Residues Roles

Residue Roles
Tyr13A electrostatic stabiliser
Arg8A enhance reactivity, modifies pKa
Gly10A (main-N) electrostatic stabiliser
Lys18A enhance reactivity
Tyr72A electrostatic stabiliser
Cys11A nucleophile

Chemical Components

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

Step 2. Gly14 attacks Cys11 in an SN2 fashion to break the Cys148-Cys11 bond. Gly10 may be involved in deprotonating the Cys14 residue.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Tyr13A electrostatic stabiliser
Tyr72A electrostatic stabiliser
Lys18A enhance reactivity
Arg8A enhance reactivity
Ser14A nucleophile
Gly10A (main-N) proton acceptor
Ser14A proton donor
Cys11A electrophile, electrofuge

Chemical Components

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

Step 3. In an inferred reaction step Gly10 is deprotonated to regenerate the native state of the enzyme.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Gly10A (main-N) proton donor

Chemical Components

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

Step 1. In a bimolecular nucleophilic substitution reaction Cys11 attacks the S-S bond of RNR and displaces Cys143.

Step 2. Gly14 attacks Cys11 in an SN2 fashion to break the Cys148-Cys11 bond. Gly10 may be involved in deprotonating the Cys14 residue.

Step 3. In an inferred reaction step Gly10 is deprotonated to regenerate the native state of the enzyme.

Products.

Contributors

Jonathan T. W. Ng, Gemma L. Holliday, Amelia Brasnett