title: The contribution of arginine residues within the P6-P1 region of alpha 1-antitrypsin to its reaction with furin.
abstract: 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.
authors.affiliation: Gladstone Institute of Cardiovascular Disease, San Francisco, California 94141, USA.
authors.forename: E K
authors.initials: EK
authors.lastname: Dufour
authors.forename: J B
authors.initials: JB
authors.lastname: Denault
authors.forename: L
authors.initials: L
authors.lastname: Bissonnette
authors.forename: P C
authors.initials: PC
authors.lastname: Hopkins
authors.forename: P
authors.initials: P
authors.lastname: Lavigne
authors.forename: R
authors.initials: R
authors.lastname: Leduc
mesh_terms: D000595:Amino Acid Sequence; D001120:Arginine / Q000737:chemistry*; D001665:Binding Sites; D004591:Electrophoresis, Polyacrylamide Gel; D045683:Furin; D015151:Immunoblotting; D007700:Kinetics; D007930:Leucine / Q000737:chemistry; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D013381:Subtilisins / Q000737:chemistry* / Q000378:metabolism; D013997:Time Factors; D000515:alpha 1-Antitrypsin / Q000737:chemistry* / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D000515:alpha 1-Antitrypsin; D012967:Sodium Dodecyl Sulfate; D001120:Arginine; D013381:Subtilisins; D045683:Furin; D007930:Leucine