Journal
FEBS JOURNAL
Volume 273, Issue 13, Pages 2942-2954Publisher
WILEY
DOI: 10.1111/j.1742-4658.2006.05305.x
Keywords
serpin; alpha(1)-antitrypsin; antitrypsin Pittsburgh; trypsin inhibitor; proteinase-activated receptors
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Funding
- NIDDK NIH HHS [R01 DK058088, DK058088] Funding Source: Medline
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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 alpha(1)-antitrypsin (alpha 1AT) 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 alpha 1AT 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 (k(cat)/K-M similar to 10(5) 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 alpha 1AT Pittsburgh and was rapidly inactivated by the serpin mechanism (k(a)similar to 10(6) 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 alpha 1AT Pittsburgh as an effective mesotrypsin inhibitor and the serpin mechanism as a viable stratagem to overcome the inhibitor-resistance of mesotrypsin.
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