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- Title
Human mesotrypsin exhibits restricted S1′ subsite specificity with a strong preference for small polar side chains.
- Authors
Szepessy, Edit; Sahin-Tóth, Miklós
- Abstract
Mesotrypsin, an inhibitor-resistant human trypsin isoform, does not activate or degrade pancreatic protease zymogens at a significant rate. These observations led to the proposal that mesotrypsin is a defective digestive protease on protein substrates. Surprisingly, the studies reported here with α1-antitrypsin (α1AT) revealed that, even though mesotrypsin was completely resistant to this serpin-type inhibitor, it selectively cleaved the Lys10–Thr11 peptide bond at the N-terminus. Analyzing a library of α1AT mutants in which Thr11 was mutated to various amino acids, we found that mesotrypsin hydrolyzed lysyl peptide bonds containing Thr or Ser at the P1′ position with relatively high specificity ( kcat/ KM∼105 m−1·s−1). Compared with Thr or Ser, P1′ Gly or Met inhibited cleavage 13- and 25-fold, respectively, whereas P1′ Asn, Asp, Ile, Phe or Tyr resulted in 100–200-fold diminished rates of proteolysis, and Pro abolished cleavage completely. Consistent with the Ser/Thr P1′ preference, mesotrypsin cleaved the Arg358–Ser359 reactive-site peptide bond of α1AT Pittsburgh and was rapidly inactivated by the serpin mechanism ( ka∼106 m−1 s−1). Taken together, the results indicate that mesotrypsin is not a defective protease on polypeptide substrates in general, but exhibits a relatively high specificity for Lys/Arg–Ser/Thr peptide bonds. This restricted, thrombin-like subsite specificity explains why mesotrypsin cannot activate pancreatic zymogens, but might activate certain proteinase-activated receptors. The observations also identify α1AT Pittsburgh as an effective mesotrypsin inhibitor and the serpin mechanism as a viable stratagem to overcome the inhibitor-resistance of mesotrypsin.
- Subjects
TRYPSIN inhibitors; SERPINS; PROTEINASES; PROTEOLYTIC enzymes; BIOCHEMISTRY
- Publication
FEBS Journal, 2006, Vol 273, Issue 13, p2942
- ISSN
1742-464X
- Publication type
Article
- DOI
10.1111/j.1742-4658.2006.05305.x