Protein-glutamine gamma-glutamyltransferase (eukaryotic)

 

Eukaryotic transglutaminase is able to catalyse the Ca(II) dependent acyl transfer between glutamine and the side chain amino group of lysine. It is unknown what the biological function of this is in fish, but the human homologue is blood clotting factor XIII which forms cross-links between these two residues in fibrin, thus plays a key role in the clotting cascade. The activation of the clotting factor is a subject of study in the hope of designing inhibitors which could be of pharmacological importance.

 

Reference Protein and Structure

Sequence
P00488 UniProt (2.3.2.13) IPR023608 (Sequence Homologues) (PDB Homologues)
Biological species
Homo sapiens (Human) Uniprot
PDB
1ggt - THREE-DIMENSIONAL STRUCTURE OF A TRANSGLUTAMINASE: HUMAN BLOOD COAGULATION FACTOR XIII (2.65 Å) PDBe PDBsum 1ggt
Catalytic CATH Domains
3.90.260.10 CATHdb 2.60.40.10 CATHdb (see all for 1ggt)
Click To Show Structure

Enzyme Reaction (EC:2.3.2.13)

L-glutamine residue
CHEBI:30011ChEBI
+
alkylaminium
CHEBI:17664ChEBI
→
N(5)-alkyl-L-glutamine residue
CHEBI:83697ChEBI
+
ammonium
CHEBI:28938ChEBI
Alternative enzyme names: R-glutaminyl-peptide:amine gamma-glutamyl transferase, Factor XIIIa, Fibrin stabilizing factor, Fibrinoligase, Glutaminylpeptide gamma-glutamyltransferase, Polyamine transglutaminase, Tissue transglutaminase, Transglutaminase, TGase, Protein-glutamine:amine gamma-glutamyltransferase,

Enzyme Mechanism

Introduction

The reaction proceeds in a manner analogous to a cysteine protease: the catalytic Cys314 acts as the initial nucleophile to attack the carbonyl of the glutamine side chain, resulting in a tetrahedral intermediate. Protonation of the leaving group by His337, assisted by Asp396 allows the collapse of this tetrahedral intermediate to form a thiolacyl enzyme intermediate. This intermediate is protected by a Tyr560 residue in the active form so that a nearby cysteine residue cannot form a disulphide to Cys314 as would otherwise happen. Subsequent nucleophilic attack on the intermediate from a Lysine, activated by deprotonation by His373, leads to the formation of the product and the release of the Cysteine residue.

Catalytic Residues Roles

UniProt PDB* (1ggt)
Cys315 Cys314A Acts as the nucleophile to attack the substrate forming the oxyanion intermediate which collapses to form the thioacyl enzyme intermediate. This in turn breaks down to release the Cysteine, through attack of a Lysine residue from the substrate, forming a crosslinkage. covalently attached, hydrogen bond acceptor, hydrogen bond donor, nucleofuge, nucleophile, electrostatic stabiliser
His374 His373A Protonates the leaving group to allow collapse of the tetrahedral intermediate. Then deprotonates the lysine residue from the protein to allow it to act as a nucleophile to break down the covalent intermediate. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor, electrostatic stabiliser
Asp397 Asp396A Forms the third part of the catalytic triad, thus alters the pKa of His373 so that it can act as a general acid/base. hydrogen bond acceptor, electrostatic stabiliser
Tyr561 Tyr560A In the active form, Ca(II) causes Tyr560 to move so that it prevents a second cysteine residue from forming a disulphide to the nucleophilic Cys314; thus can be described as stabilising the thioacyl enzyme intermediate or as activating Cys314. hydrogen bond donor, electrostatic stabiliser
Cys315 (main-N), Trp280 Cys314A (main-N), Trp279A The NE1 atom of Trp279 and the backbone NH of Cys314 act as he hydrogen bond donors that form the oxyanion hole, stabilising the negatively charged intermediates. electrostatic stabiliser
*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 addition, enzyme-substrate complex formation, intermediate formation, overall reactant used, unimolecular elimination by the conjugate base, proton transfer, enzyme-substrate complex cleavage, intermediate collapse, overall product formed, deamination, intermediate terminated, native state of enzyme regenerated

References

  1. Pedersen LC et al. (1994), Protein Sci, 3, 1131-1135. Transglutaminase factor XIII uses proteinase-like catalytic triad to crosslink macromolecules. DOI:10.1002/pro.5560030720. PMID:7920263.
  2. Thomas A et al. (2016), Hum Mutat, 37, 1030-1041. Coagulation Factor XIIIA Subunit Missense Mutations Affect Structure and Function at the Various Steps of Factor XIII Action. DOI:10.1002/humu.23041. PMID:27363989.
  3. Gupta S et al. (2016), Sci Rep, 6, 30105-. Revisiting the mechanism of coagulation factor XIII activation and regulation from a structure/functional perspective. DOI:10.1038/srep30105. PMID:27453290.
  4. Stieler M et al. (2013), Angew Chem Int Ed Engl, 52, 11930-11934. Structure of Active Coagulation Factor XIII Triggered by Calcium Binding: Basis for the Design of Next-Generation Anticoagulants. DOI:10.1002/anie.201305133. PMID:24115223.
  5. Iismaa SE et al. (2003), Proc Natl Acad Sci U S A, 100, 12636-12641. Evolutionary specialization of a tryptophan indole group for transition-state stabilization by eukaryotic transglutaminases. DOI:10.1073/pnas.1635052100. PMID:14566064.
  6. Murthy SN et al. (2002), Proc Natl Acad Sci U S A, 99, 2738-2742. Conserved tryptophan in the core domain of transglutaminase is essential for catalytic activity. DOI:10.1073/pnas.052715799. PMID:11867764.
  7. Noguchi K et al. (2001), J Biol Chem, 276, 12055-12059. Crystal Structure of Red Sea Bream Transglutaminase. DOI:10.1074/jbc.m009862200. PMID:11080504.
  8. Fox BA et al. (1999), J Biol Chem, 274, 4917-4923. Identification of the calcium binding site and a novel ytterbium site in blood coagulation factor XIII by x-ray crystallography. PMID:9988734.
  9. Yee VC et al. (1994), Proc Natl Acad Sci U S A, 91, 7296-7300. Three-dimensional structure of a transglutaminase: human blood coagulation factor XIII. PMID:7913750.

Step 1. The thiolate of Cys314 attacks the carbonyl carbon of the protein glutamine substrate. The formed oxyanion is stabilised through a H-bond with Trp279.

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Catalytic Residues Roles

Residue Roles
His373A hydrogen bond donor, electrostatic stabiliser
Asp396A hydrogen bond acceptor, electrostatic stabiliser
Tyr560A hydrogen bond donor, electrostatic stabiliser
Cys314A hydrogen bond acceptor, hydrogen bond donor
Trp279A hydrogen bond donor
Cys314A (main-N) electrostatic stabiliser
Cys314A nucleophile

Chemical Components

ingold: bimolecular nucleophilic addition, enzyme-substrate complex formation, intermediate formation, overall reactant used

Step 2. The oxyanion initiates the elimination of ammonia, which achieves the correct protonation state through the deprotonation of His373.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His373A hydrogen bond donor
Asp396A hydrogen bond acceptor, electrostatic stabiliser
Tyr560A hydrogen bond donor
Cys314A covalently attached, hydrogen bond acceptor, hydrogen bond donor, electrostatic stabiliser
Trp279A hydrogen bond donor, electrostatic stabiliser
Cys314A (main-N) electrostatic stabiliser
His373A proton donor

Chemical Components

ingold: unimolecular elimination by the conjugate base, proton transfer, enzyme-substrate complex cleavage, intermediate collapse, intermediate formation, overall product formed, deamination

Step 3. His373 deprotonates the substrate protein lysine, which attacks the carbonyl carbon of the cysteine-bound intermediate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His373A hydrogen bond donor, hydrogen bond acceptor
Asp396A hydrogen bond acceptor
Tyr560A hydrogen bond donor
Cys314A covalently attached, hydrogen bond acceptor, hydrogen bond donor
Trp279A hydrogen bond donor
Cys314A (main-N) electrostatic stabiliser
His373A proton acceptor

Chemical Components

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

Step 4. The oxyanion initiates the elimination of Cys314, producing the protein N5-alkylglutamine product and regenerating the enzyme's native state.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His373A hydrogen bond donor, electrostatic stabiliser
Asp396A hydrogen bond acceptor, electrostatic stabiliser
Tyr560A hydrogen bond donor
Cys314A hydrogen bond acceptor, hydrogen bond donor, electrostatic stabiliser
Trp279A hydrogen bond donor, electrostatic stabiliser
Cys314A (main-N) electrostatic stabiliser
Cys314A nucleofuge

Chemical Components

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

Step 1. The thiolate of Cys314 attacks the carbonyl carbon of the protein glutamine substrate. The formed oxyanion is stabilised through a H-bond with Trp279.

Step 2. The oxyanion initiates the elimination of ammonia, which achieves the correct protonation state through the deprotonation of His373.

Step 3. His373 deprotonates the substrate protein lysine, which attacks the carbonyl carbon of the cysteine-bound intermediate.

Step 4. The oxyanion initiates the elimination of Cys314, producing the protein N5-alkylglutamine product and regenerating the enzyme's native state.

Products.

Contributors

Gemma L. Holliday, Daniel E. Almonacid, Peter Sarkies