The role of the insertion loop around tryptophan 148 in tthe activity of thrombin.

Thrombin has trypsin-like specificity for Arg-Xaa and Lys-Xaa peptide bonds; however, it is much more specific than trypsin, cleaving far fewer peptide bonds in macromolecular substrates. To probe the nature of the specificity of thrombin, a mutant has been constructed in which the Trp148 loop of thrombin has been replaced with the same loop of bovine trypsin. This mutant was expressed in Escherichia coli as prethrombin-2(148) using a T7 expression system previously described for wild-type prethrombin-2 [DiBella et al. (1995) J. Biol. Chem. 270, 163-169]. After refolding and purification, prethrombin-2(148) was activated to thrombin(148) with Echis carinatus snake venom. The k(cat)/K(m) for the release of fibrinopeptide A from fibrinogen was 4.5 +/- 0.5 microM(-1)s(-1) for thrombin(148), which was approximately 20% of that of recombinant thrombin (25 +/- 2.0 microM(-1)s(-1)). Thrombin(148) was inhibited less well by hirudin with a K(i) of 500 pM compared to a value of 12 pM determined for recombinant thrombin. The mutant thrombin was also compared to trypsin and wild-type recombinant thrombin for the ability to cleave small peptide substrates. The Michaelis constants (K(m)) were found to be between 5- and 10-fold higher for thrombin(148) relative to wild-type recombinant thrombin, although the catalytic constants (k(cat)) for thrombin(148) and recombinant thrombin remained relatively unchanged for all three substrates. Thrombin(148) had a specificity constant (k(cat)/K(m)) 2-fold higher for the hydrolysis of H-D-phenyalanyl-L- pipecolyl-L-arginine-p-nitroaniline (a thrombin substrate) than that of trypsin. For N-benzoyl-L-isoleucyl-L-glutamylglycyl-L-arginine- p-nitroaniline (a trypsin substrate) and N-carbobenzoxyglycylprolyl-L-arginine-p-nitroaniline (a substrate for both enzymes), the specificity constants for trypsin were 1000- and 16-fold higher, respectively. Although replacement of the Trp(148) loop does not yield an enzyme with more trypsin-like specificity, the Trp(148) loop is important in the substrate binding and specificity of thrombin (on the basis of K(m) and K(i)).

Abstract sourced from PubMed (NCBI) for the cited record. See the original publication for the authoritative version.

Thrombin has trypsin-like specificity for Arg-Xaa and Lys-Xaa peptide bonds; however, it is much more specific than trypsin, cleaving far fewer peptide bonds in macromolecular substrates. To probe the nature of the specificity of thrombin, a mutant has been constructed in which the Trp148 loop of...

Leech-derived molecules this paper discusses, each with a full profile in the ASH compound registry:

Explore cómo esta investigación se conecta con la práctica clínica

More peer-reviewed studies in Desarrollo de fármacos.

• Novel Leech Antimicrobial Peptides — Hirunipins — Kumar et al. (2025)
• Medicinal Leech Genome Assembly — Anticoagulant Gene Catalog — Kvist et al. (2020)
• Effects of hirudotherapy on liver functions, lipid profile, and insulin sensitivity in rats with metabolic syndrome — Bilden A et al. (2026)
• HIRUDOTHERAPY IN PRESBYACUZIS PRAECOX IN CLEAN-UP WORKERS AFTER THE CHORNOBYL ACCIDENT DURING THE POST-CATASTROPHE YEARS — Zabolotnyi D, Mishchanchuk N (2019)
• [Hirudo medicinalis-leech applications in plastic and reconstructive microsurgery--a literature review] — Knobloch K et al. (2007)
• Recombinant hirudin in clinical practice: focus on lepirudin — Greinacher A, Lubenow N (2001)
• In-vitro antileishmanial potential of peptide drug hirudin — Khan H et al. (2017)
• Parenteral anticoagulants: Antithrombotic Therapy and Prevention of Thrombosis, 9th ed: American College of Chest Physicians Evidence-Based Clinical Practice Guidelines — Garcia DA, Baglin TP, Weitz JI, Samama MM (2012)
• Direct thrombin inhibitors — Lee CJ, Ansell JE (2011)
• Updates on Use and Monitoring of Direct Thrombin Inhibitors — Navaei A et al. (2026)

Browse all ASH-analysed research →