Human mesotrypsin exhibits restricted S1' subsite specificity with a strong preference for small polar side chains.
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 alpha1-antitrypsin (alpha1AT) 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 alpha1AT 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 approximately 10(5) m(-1) x 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 alpha1AT Pittsburgh and was rapidly inactivated by the serpin mechanism (ka approximately 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 alpha1AT Pittsburgh as an effective mesotrypsin inhibitor and the serpin mechanism as a viable stratagem to overcome the inhibitor-resistance of mesotrypsin.
Szepessy
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Department of Molecular and Cell Biology, Boston University, Goldman School of Dental Medicine, MA, USA.
Sahin-Tóth
Miklós
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D001665:Binding Sites; D001671:Biochemistry / Q000379:methods; D002412:Cations; D004305:Dose-Response Relationship, Drug; D006801:Humans; D007700:Kinetics; D008956:Models, Chemical; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D020033:Protein Isoforms; D013379:Substrate Specificity; D013997:Time Factors; D014357:Trypsin / Q000737:chemistry*; D014362:Trypsinogen / Q000737:chemistry; D000515:alpha 1-Antitrypsin / Q000737:chemistry
D002412:Cations; D010455:Peptides; D020033:Protein Isoforms; D000515:alpha 1-Antitrypsin; D014362:Trypsinogen; C040749:PRSS3 protein, human; D014357:Trypsin