Allosterism in thrombin

Thrombin is a protease that selectively cuts certain peptide bonds. (Details below)

The substrate is positioned in a pocket or cleft that includes the catalytic site:

  • of the catalytic site (Asp, His, Ser triad)
  • occupied by a substrate

Thrombin suffers a transition between two forms, slightly different in their structure but highly different in their enzymatic activity:

The part which is particularly altered in this conformational change includes the Trp215 residue and its neighbours Gly216 and Glu217, in a darker colour.

Using the above controls, you will notice that in the E* conformation the 215-217 segment shifts, covering up the entrance to the pocket and, particularly, the position of the tryptophan residue is incompatible with entry of the substrate. To the contrary, in the E conformation there is clear access.
This may be better appreciated with a model displaying

between both conformations.

This conformational change is an example of allosterism, since it involves a change in catalytic activity as a consequence of ligand binding far away from the active site. In the case of thrombin, several effectors are involved; among them, Na+ ions, hirudin (a peptide present in leech saliva) and heparin (a polysaccharide). The latter two are anticoagulants since they inhibit protease activity in thrombin (responsible for the formation of fibrin, the essential component of blood clots).

Enzymatic activity of thrombin

It hydrolises peptide bonds in which the CO group belongs to arginine. This is an example of a substrate (⋯FPRV⋯, or ⋯Phe-Pro-Arg-Val⋯):

chemical structure of the FPRV peptide
Green circles mark the Cα; yellow highlights the peptide bonds, and blue marks the peptide bond that will be hydrolised by thrombin.

A frequently used inhibitor in the laboratory is PPACK (phenylalanyl-prolyl-arginyl-chloromethylketone, also known as FPRCK):
chemical structure of the PPACK analog
You may notice its structural similarity with the amino-terminal moiety of the peptide; thanks to this, it binds the active site in the E form of the enzyme, but due to its lacking the peptide bond betwwen arginine and the next group it cannot be hydrolysed by the enzyme; therefore, it acts as a competitive inhibitor.

PPACK.

Image of the catalytic triad: © Loren Williams, Georgia Institute of Technology [1]. Reproduced with author's permission.

Protein structures are based on X-ray crystallography experiments of human thrombin with N143P mutation: 3qgn.pdb (E) and 3jz2.pdb (E*); the substrate is part of 5gim.pdb; the PPACK is part of 1sfq.pdb