Thrombin has dual trypsin-like and chymotrypsin-like specificity

Area of Science:

• Biochemistry
• Enzymology
• Evolutionary Biology

Background:

• Protein C activation typically involves Arginine (Arg) cleavage site.
• Ray-finned fish possess a Tryptophan (Trp) residue at this site, posing an evolutionary question.
• The physiological activator of protein C across vertebrates remains under investigation.

Purpose of the Study:

• To determine if thrombin can cleave Tryptophan (Trp) residues.
• To investigate thrombin's role in activating protein C in ray-finned fish.

Main Methods:

• Assessed wild-type thrombin and D189S mutant activity using chromogenic substrates.
• Tested thrombin's cleavage of wild-type protein C and mutants with altered cleavage sites.

Main Results:

• Thrombin exhibits dual trypsin-like and chymotrypsin-like specificity, cleaving substrates at both Arginine (Arg) and Tryptophan (Trp) residues.
• Cleavage at Tryptophan (Trp) by thrombin is significant, comparable to chymotrypsin.
• Thrombin activates protein C across vertebrates, including ray-finned fish, with activation rates influenced by flanking sequences.
• The D189S mutant displayed broad specificity, cleaving at basic and aromatic residues.

Conclusions:

• This study supports thrombin as the physiological activator of protein C in all vertebrates.
• The findings resolve the paradox of Tryptophan (Trp) at the protein C cleavage site in ray-finned fish.
• Thrombin's dual specificity suggests a broader range of physiological substrates than previously recognized.