Protein-arginine deiminase

 

In the presence of calcium ions, Protein-arginine deiminase (PAD) enzymes catalyse the post-translational modification reaction responsible for the formation of citrulline residues from protein-bound arginine residues. The gene for this enzyme is a susceptibility locus for rheumatoid arthritis.

 

Reference Protein and Structure

Sequence
Q9UM07 UniProt (3.5.3.15) IPR004303 (Sequence Homologues) (PDB Homologues)
Biological species
Homo sapiens (Human) Uniprot
PDB
1wd8 - Calcium free form of human peptidylarginine deiminase type4 (PAD4) (2.8 Å) PDBe PDBsum 1wd8
Catalytic CATH Domains
3.75.10.10 CATHdb (see all for 1wd8)
Cofactors
Calcium(2+) (5)
Click To Show Structure

Enzyme Reaction (EC:3.5.3.15)

water
CHEBI:15377ChEBI
+
L-argininium residue
CHEBI:29965ChEBI
→
L-citrulline residue
CHEBI:83397ChEBI
+
ammonium
CHEBI:28938ChEBI
Alternative enzyme names: Peptidylarginine deiminase, PAD,

Enzyme Mechanism

Introduction

Calcium binding causes conformational changes that order the reaction centre. A nucleophilic attack is made by the thiol group of Cys 645 onto the zeta-carbon of peptidyl-L-arginine. The carboxyl groups of Asp 350 and Asp 473 activate the substrate by formation of hydrogen bonds and a salt bridge to increase the nucleophilicity of the zeta carbon. Cleavage of the bond between the zeta-carbon and the nu-nitrogen-2 to generate ammonia. The general base His 471 then activates water so that the water may undertake a nucleophilic attack. Hydrolysis of the tetrahedral adduct yields peptidyl-L-citrulline.

Catalytic Residues Roles

UniProt PDB* (1wd8)
Asp350 Asp350(357)A The carboxyl group activates the substrate by hydrogen bond and salt bridge formation. electrostatic stabiliser
His471 His471(478)A Acts as a general base and activates Cys 645 and water to allow them to attack as a nucleophile. proton acceptor, proton donor
Asp473 Asp473(480)A Activates substrate by hydrogen bond and salt bridge formation. electrostatic stabiliser
Cys645 Cys645(652)A Thiol group attacks zeta-carbon of peptidyl-L-arginine as a nucleophile. nucleofuge, nucleophile, 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, bimolecular nucleophilic addition, enzyme-substrate complex formation, intermediate formation, overall reactant used, rate-determining step, unimolecular elimination by the conjugate base, intermediate collapse, enzyme-substrate complex cleavage, native state of enzyme regenerated, overall product formed

References

  1. Arita K et al. (2004), Nat Struct Mol Biol, 11, 777-783. Structural basis for Ca2+-induced activation of human PAD4. DOI:10.1038/nsmb799. PMID:15247907.
  2. Slade DJ et al. (2015), ACS Chem Biol, 10, 1043-1053. Protein arginine deiminase 2 binds calcium in an ordered fashion: implications for inhibitor design. DOI:10.1021/cb500933j. PMID:25621824.
  3. Dreyton CJ et al. (2014), Biochemistry, 53, 4426-4433. Mechanistic studies of protein arginine deiminase 2: evidence for a substrate-assisted mechanism. DOI:10.1021/bi500554b. PMID:24989433.
  4. Kubota K et al. (2005), Rapid Commun Mass Spectrom, 19, 683-688. Determination of sites citrullinated by peptidylarginine deiminase using18O stable isotope labeling and mass spectrometry. DOI:10.1002/rcm.1842. PMID:15700232.

Step 1. His471 deprotonates Cys645 to activate it for nucleophilic attack.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp473(480)A electrostatic stabiliser
Asp350(357)A electrostatic stabiliser
His471(478)A proton acceptor
Cys645(652)A proton donor

Chemical Components

proton transfer

Step 2. Cys647 nucleophilically attacks the guanidinum carbon of arginine. Within this step, His471 acts as a general acid, protonating the guanidinium group

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp350(357)A electrostatic stabiliser
Asp473(480)A electrostatic stabiliser
Cys645(652)A nucleophile
His471(478)A proton donor

Chemical Components

ingold: bimolecular nucleophilic addition, proton transfer, enzyme-substrate complex formation, intermediate formation, overall reactant used, rate-determining step

Step 3. The tetrahedral collapses as ammonia leaves.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp350(357)A electrostatic stabiliser
Asp473(480)A electrostatic stabiliser

Chemical Components

ingold: unimolecular elimination by the conjugate base, intermediate collapse

Step 4. His471 abstracts a proton from a water which activates it to nucleophilically attack the carbon of the C=N bond.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp350(357)A electrostatic stabiliser
Asp473(480)A electrostatic stabiliser
His471(478)A proton acceptor

Chemical Components

proton transfer, ingold: bimolecular nucleophilic addition, intermediate formation, overall reactant used

Step 5. The second tetrahedral intermediate formed protonates Cys645 which initiates the elimination resulting in the cleavage of the acyl-enzyme bond and the release of the citrullinated product.The ammonia released prior can accept a proton from His471 to return the active site to its native state.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp350(357)A electrostatic stabiliser
Asp473(480)A electrostatic stabiliser
His471(478)A proton donor
Cys645(652)A nucleofuge, proton acceptor

Chemical Components

proton transfer, ingold: unimolecular elimination by the conjugate base, proton transfer, enzyme-substrate complex cleavage, intermediate collapse, native state of enzyme regenerated, overall product formed
Download: Image, Marvin File

Step 1. His471 deprotonates Cys645 to activate it for nucleophilic attack.

Step 2. Cys647 nucleophilically attacks the guanidinum carbon of arginine. Within this step, His471 acts as a general acid, protonating the guanidinium group

Step 3. The tetrahedral collapses as ammonia leaves.

Step 4. His471 abstracts a proton from a water which activates it to nucleophilically attack the carbon of the C=N bond.

Step 5. The second tetrahedral intermediate formed protonates Cys645 which initiates the elimination resulting in the cleavage of the acyl-enzyme bond and the release of the citrullinated product.The ammonia released prior can accept a proton from His471 to return the active site to its native state.

Products.

Contributors

Gary McDowell, Gemma L. Holliday, Charity Hornby