PDBsum entry 4h6t

Hydrolase

PDB id: 4h6t

Contents

Protein chain

293 a.a.

Ligands

PO4 ×7

Waters ×63

PDB id:

4h6t

Name:

Hydrolase

Title:

Crystal structure of prethrombin-2 mutant e14ea/d14la/e18a/s195a

Structure:

Prothrombin. Chain: a. Synonym: coagulation factor ii, activation peptide fragment 1, activation peptide fragment 2, thrombin light chain, thrombin heavy chain. Engineered: yes. Mutation: yes

Source:

Homo sapiens. Human. Organism_taxid: 9606. Gene: f2. Expressed in: escherichia coli. Expression_system_taxid: 562

Resolution:

2.40Å R-factor: 0.214 R-free: 0.253

Authors:

N.Pozzi,Z.Chen,F.Zapata,L.A.Pelc,E.Di Cera

Key ref:

N.Pozzi et al. (2013). Autoactivation of thrombin precursors. J Biol Chem, 288, 11601-11610. PubMed id: 23467412 DOI: 10.1074/jbc.M113.451542

Date:

19-Sep-12 Release date: 13-Mar-13

Protein chain

P00734  (THRB_HUMAN) -  Prothrombin from Homo sapiens

Seq: 622 a.a.

Struc: 293 a.a.*

* PDB and UniProt seqs differ at 4 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.3.4.21.5 - thrombin.
• Reaction: Preferential cleavage: Arg-|-Gly; activates fibrinogen to fibrin and releases fibrinopeptide A and B.

DOI no: 10.1074/jbc.M113.451542

J Biol Chem 288:11601-11610 (2013)

PubMed id: 23467412

Autoactivation of thrombin precursors.

N.Pozzi, Z.Chen, F.Zapata, W.Niu, S.Barranco-Medina, L.A.Pelc, E.Di Cera.

ABSTRACT

Trypsin-like proteases are synthesized as inactive zymogens and convert to the mature form upon activation by specific enzymes, often assisted by cofactors. Central to this paradigm is that the zymogen does not convert spontaneously to the mature enzyme, which in turn does not feed back to activate its zymogen form. In the blood, the zymogens prothrombin and prethrombin-2 require the prothrombinase complex to be converted to the mature protease thrombin, which is unable to activate prothrombin or prethrombin-2. Here, we show that replacement of key residues within the activation domain causes these zymogens to spontaneously convert to thrombin. The conversion is started by the zymogen itself, which is capable of binding ligands at the active site, and is abrogated by inactivation of the catalytic residue Ser-195. The product of autoactivation is functionally and structurally equivalent to wild-type thrombin. Zymogen autoactivation is explained by conformational selection, a basic property of the trypsin fold uncovered by structural and rapid kinetics studies. Both the zymogen and protease undergo a pre-existing equilibrium between active and inactive forms. The equilibrium regulates catalytic activity in the protease and has the potential to unleash activity in the zymogen to produce autoactivation. A new strategy emerges for the facile production of enzymes through zymogen autoactivation that is broadly applicable to trypsin-like proteases of biotechnological and clinical interest.