The important role of furin in the proteolytic activation of many pathogenic molecules has made this endoprotease a target for the development of potent and selective antiproteolytic agents. Here, we demonstrate the utility of the protein-based inhibitor ␣ 1 -antitrypsin Portland (␣ 1 -PDX) as an antipathogenic agent that can be used prophylactically to block furin-dependent cell killing by Pseudomonas exotoxin A. Biochemical analysis of the specificity of a bacterially expressed Hisand FLAG-tagged ␣ 1 -PDX (␣ 1 -PDX͞hf) revealed the selectivity of the ␣ 1 -PDX͞hf reactive site loop for furin (K i , 600 pM) but not for other proprotein convertase family members or other unrelated endoproteases. Kinetic studies show that ␣ 1 -PDX͞hf inhibits furin by a slow tight-binding mechanism characteristic of serpin molecules and functions as a suicide substrate inhibitor. Once bound to furin's active site, ␣ 1 -PDX͞hf partitions with equal probability to undergo proteolysis by furin at the Cterminal side of the reactive center -Arg 355 -Ile-Pro-Arg 358 -2 or to form a kinetically trapped SDS-stable complex with the enzyme. This partitioning between the complex-forming and proteolytic pathways contributes to the ability of ␣ 1 -PDX͞hf to differentially inhibit members of the proprotein convertase family. Finally, we propose a structural model of the ␣ 1 -PDX-reactive site loop that explains the high degree of enzyme selectivity of this serpin and which can be used to generate small molecule furin inhibitors.

No abstract