Document Detail


Determinants of chymotrypsin C cleavage specificity in the calcium-binding loop of human cationic trypsinogen.
MedLine Citation:
PMID:  23035638     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
The pancreatic serine protease chymotrypsin C (CTRC) cleaves the Leu81-Glu82 peptide bond in the calcium-binding loop of human cationic trypsinogen and thereby promotes its degradation. This serves as a protective mechanism against ectopic trypsinogen activation in the pancreas. In the present study, we demonstrate that cleavage of the Leu81-Glu82 peptide bond by CTRC is highly specific, and other human pancreatic chymotrypsins (CTRB1, CTRB2 and CTRL1) and elastases (ELA2A, ELA3A and ELA3B) do not catalyze this reaction. To elucidate the mechanistic basis for CTRC specificity, we surveyed the primary (P1) cleavage preference of these pancreatic proteases on peptide substrates. We found that CTRC cleaved after a P1 Leu with at least tenfold higher catalytic efficiency than other enzymes tested. To assess extended sub-site interactions, we introduced Ala mutations into human cationic trypsinogen at the P3, P1' P3' and P4' amino acid positions, where P1-P1' corresponds to Leu81-Glu82. Interestingly, CTRC-mediated cleavage was stimulated threefold by mutation E82A and unaffected by mutations E79A and N84A, but all three mutations compromised specificity and resulted in increased cleavage by ELA2A. Mutation E85A decreased CTRC cleavage by twofold. Remarkably, other chymotrypsins and elastases did not cleave human cationic trypsinogen even with the L81F or L81A mutations, which introduced favorable P1 residues for these enzymes. We conclude that specific cleavage of the Leu81-Glu82 peptide bond in human cationic trypsinogen by CTRC is primarily determined by its distinctively high activity on leucyl peptide bonds, with the P1' Glu82, P3' Asn84 and P4' Glu85 residues serving as additional specificity determinants.
Authors:
András Szabó; Miklós Sahin-Tóth
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural     Date:  2012-10-30
Journal Detail:
Title:  The FEBS journal     Volume:  279     ISSN:  1742-4658     ISO Abbreviation:  FEBS J.     Publication Date:  2012 Dec 
Date Detail:
Created Date:  2012-11-22     Completed Date:  2013-01-25     Revised Date:  2013-12-04    
Medline Journal Info:
Nlm Unique ID:  101229646     Medline TA:  FEBS J     Country:  England    
Other Details:
Languages:  eng     Pagination:  4283-92     Citation Subset:  IM    
Copyright Information:
© 2012 The Authors Journal compilation © 2012 FEBS.
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MeSH Terms
Descriptor/Qualifier:
Calcium / metabolism*
Chymotrypsin / chemistry*,  metabolism*
Humans
Pancreas / enzymology
Pancreatic Elastase / chemistry,  metabolism
Protein Binding
Protein Structure, Secondary
Substrate Specificity
Trypsinogen / chemistry*,  metabolism*
Grant Support
ID/Acronym/Agency:
R01 DK058088/DK/NIDDK NIH HHS; R01 DK082412/DK/NIDDK NIH HHS; R01DK058088/DK/NIDDK NIH HHS; R01DK082412/DK/NIDDK NIH HHS; R01DK082412-S2/DK/NIDDK NIH HHS
Chemical
Reg. No./Substance:
9002-08-8/Trypsinogen; EC 3.4.21.1/Chymotrypsin; EC 3.4.21.2/chymotrypsin C; EC 3.4.21.36/Pancreatic Elastase; SY7Q814VUP/Calcium
Comments/Corrections

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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