T-plasminogen activator

 

Tissue-type plasminogen activator is a serine proteinase of the trypsin-family. It catalyses the activation of the zymogen plasminogen to the fibrin-degrading proteinase plasmin in fibrinolysis. Human tissue-type plasminogen activator is being studied for therapeutic use in cases such as myocardial infarctions and other thromboembolic disorders.

 

Reference Protein and Structure

Sequence
P00750 UniProt (3.4.21.68) IPR026280 (Sequence Homologues) (PDB Homologues)
Biological species
Homo sapiens (Human) Uniprot
PDB
1rtf - COMPLEX OF BENZAMIDINE WITH THE CATALYTIC DOMAIN OF HUMAN TWO CHAIN TISSUE PLASMINOGEN ACTIVATOR [(TC)-T-PA] (2.3 Å) PDBe PDBsum 1rtf
Catalytic CATH Domains
2.40.10.10 CATHdb (see all for 1rtf)
Click To Show Structure

Enzyme Reaction (EC:3.4.21.68)

water
CHEBI:15377ChEBI
+
Arg-Val
CHEBI:73823ChEBI
→
L-arginine
CHEBI:16467ChEBI
+
L-valine
CHEBI:16414ChEBI
Alternative enzyme names: Plasminogen activator, tissue-type, T-PA, tPA, Tissue plasminogen activator, Tissue-type plasminogen activator,

Enzyme Mechanism

Introduction

Tissue-type plasminogen activator employs a classical serine protease mechanism. Ser 195 acts as a nucleophile to attack the peptide bond and form a tetrahedral intermediate that is stabilised by the backbone NH of Ser 195 and Gly 193. His 57 promotes the nucleophilic attack by deprotonating Ser 195, while Asp 102 functions to modify the pKa of His 57. Collapse of the tetrahedral intermediate with protonation of the departing amine leaving group by His 57 generates an acyl-enzyme intermediate. This is then hydrolysed by a water molecule that is deprotonated by His 57.

Catalytic Residues Roles

UniProt PDB* (1rtf)
Gly511 (main-N), Ser513 (main-N) Gly193(201)B (main-N), Ser195(203)B (main-N) Forms part of the oxyanion hole. electrostatic stabiliser
His357 His57(47)B His 57 acts as a general acid/base catalyst to activate Ser 195 and water to nucleophilic attack and facilitates collapse of the intermediates by proton donation. proton acceptor, proton donor
Asp406 Asp102(96)B Modifies the pKa of His 57 to allow it to act as a general acid/base catalyst. electrostatic stabiliser
Ser513 Ser195(203)B Acts as a nucleophile to attack the substrate. 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, intermediate formation, overall reactant used, rate-determining step, unimolecular elimination by the conjugate base, intermediate collapse, overall product formed, native state of enzyme regenerated

References

  1. Lamba D et al. (1996), J Mol Biol, 258, 117-135. The 2.3 Å Crystal Structure of the Catalytic Domain of Recombinant Two-chain Human Tissue-type Plasminogen Activator. DOI:10.1006/jmbi.1996.0238. PMID:8613982.
  2. Gong L et al. (2015), J Biol Chem, 290, 25795-25804. Crystal Structure of the Michaelis Complex between Tissue-type Plasminogen Activator and Plasminogen Activators Inhibitor-1. DOI:10.1074/jbc.M115.677567. PMID:26324706.

Step 1. His57 deprotonates Ser195 which activates it to attack the carbon of the peptide bond in a nucleophilic addition.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp102(96)B electrostatic stabiliser
Gly193(201)B (main-N) electrostatic stabiliser
Ser195(203)B (main-N) electrostatic stabiliser
His57(47)B proton acceptor
Ser195(203)B proton donor, nucleophile

Chemical Components

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

Step 2. Collapse of the tetrahedral intermediate with protonation of the departing amine leaving group by His 57 generates an acyl-enzyme intermediate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp102(96)B electrostatic stabiliser
Gly193(201)B (main-N) electrostatic stabiliser
Ser195(203)B (main-N) electrostatic stabiliser
His57(47)B proton donor

Chemical Components

ingold: unimolecular elimination by the conjugate base, proton transfer, intermediate collapse, overall product formed

Step 3. His57 deprotonates a water which activates it to attack the carbon of the ester bond in a nucleophilic addition.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp102(96)B electrostatic stabiliser
Gly193(201)B (main-N) electrostatic stabiliser
Ser195(203)B (main-N) electrostatic stabiliser
His57(47)B proton acceptor

Chemical Components

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

Step 4. The tetrahedral intermediate collapses which results in the cleavage of the ester bond releasing Ser195 and an Arginine. The released Ser195 then accepts a proton from His57 which returns the enzyme to its native state.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp102(96)B electrostatic stabiliser
Gly193(201)B (main-N) electrostatic stabiliser
Ser195(203)B (main-N) electrostatic stabiliser
Ser195(203)B proton acceptor
His57(47)B proton donor
Ser195(203)B nucleofuge

Chemical Components

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

Step 1. His57 deprotonates Ser195 which activates it to attack the carbon of the peptide bond in a nucleophilic addition.

Step 2. Collapse of the tetrahedral intermediate with protonation of the departing amine leaving group by His 57 generates an acyl-enzyme intermediate.

Step 3. His57 deprotonates a water which activates it to attack the carbon of the ester bond in a nucleophilic addition.

Step 4. The tetrahedral intermediate collapses which results in the cleavage of the ester bond releasing Ser195 and an Arginine. The released Ser195 then accepts a proton from His57 which returns the enzyme to its native state.

Products.

Contributors

Gary McDowell, Gemma L. Holliday, Charity Hornby