The Contribution of Arginine Residues within the P6–P1 Region of α1-Antitrypsin to Its Reaction with Furin
| Autor: | Pierre Lavigne, Lyne Bissonnette, Erick K. Dufour, Paul C.R. Hopkins, Jean-Bernard Denault, Richard Leduc |
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| Rok vydání: | 2001 |
| Předmět: |
Models
Molecular Time Factors Stereochemistry Proteolysis Immunoblotting Molecular Sequence Data Mutant Context (language use) Arginine Biochemistry Protein structure Leucine medicine Amino Acid Sequence Subtilisins Binding site Molecular Biology Furin Peptide sequence Binding Sites Sequence Homology Amino Acid medicine.diagnostic_test biology Chemistry Wild type Sodium Dodecyl Sulfate Cell Biology Recombinant Proteins Protein Structure Tertiary Kinetics Models Chemical alpha 1-Antitrypsin biology.protein Electrophoresis Polyacrylamide Gel Protein Binding |
| Zdroj: | Journal of Biological Chemistry. 276:38971-38979 |
| ISSN: | 0021-9258 |
| DOI: | 10.1074/jbc.m102959200 |
| Popis: | A series of mutants incorporating furin recognition sequences within the P6-P1 region of the reactive site loop of alpha(1)-antitrypsin were constructed. Variants containing different combinations of basic residues in the P1, P2, P4, and P6 positions replacing the wild type (P6)LEAIPM(P1) sequence were evaluated for their capacity to establish SDS-resistant complexes with furin, to affect association rate constants (k(ass) and k'(ass)), or to inhibit furin-dependent proteolysis of a model precursor in vivo. Each variant abolished processing of pro-von Willebrand factor in transfected hEK293 cells. The k(ass) of all variants were found to be similar (1.1-1.7 x 10(6) m(-1) s(-1)) except for one mutant, RERIRR, which had a k(ass) of 3.3 x 10(5) m(-1) s(-1). However, the stoichiometry of inhibition varied with values ranging from 2.9 to >24, indicating rapid formation of the acyl-enzyme intermediate (high k'(ass)). Moreover, those variants having high stoichiometry of inhibition values were accompanied by the rapid formation of cleaved forms of the inhibitors. The data suggest that the rate of conversion of the acyl-enzyme (EI') into the highly stable complex (EI*) was affected by replacement of specific residues within the reactive site loop. Taken together, the results reveal how furin recognition sequences within the context of the biochemical properties of serpins will play a role in the capacity of the protein to follow either the inhibitory or the substrate pathway. |
| Databáze: | OpenAIRE |
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