tailieunhanh - Báo cáo khoa học: 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 acti-vate 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 a1 -antitrypsin (a1AT) revealed that, even though mesotrypsin was com- | iFEBS Journal Human mesotrypsin exhibits restricted S1 subsite specificity with a strong preference for small polar side chains Edit Szepessy and Miklos Sahin-Toth Department of Molecular and Cell Biology Boston University Goldman Schoolof DentalMedicine MA USA Keywords serpin a1-antitrypsin antitrypsin Pittsburgh trypsin inhibitor proteinase-activated receptors Correspondence M. Sahin-Toth 715 Albany Street Evans-433 Boston MA 02118 USA Fax 1 617 414 1041 Tel 1 617 414 1070 E-mail miklos@ Received 12 March 2006 revised 29 April 2006 accepted 3 May 2006 doi 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 a1-antitrypsin a1AT revealed that even though mesotrypsin was completely resistant to this serpin-type inhibitor it selectively cleaved the LyslO-Thrll peptide bond at the N-terminus. Analyzing a library of a1AT mutants in which Thr11 was mutated to various amino acids we found that mesotrypsin hydrolyzed lysyl peptide bonds containing Thr or Ser at the Pl 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 a1AT 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 thrombinlike subsite specificity explains why .