The Cleavage of a Serine Protease Inhibitor at the Reactive Center by Its Target Protease: Analysis of the Substrate-Like Form of a Serpin

Abstract

Many enzymes involved in coagulation and fibrinolysis have a serine residue at their active site and they are therefore defined as serine proteases. In plasma the activity of such proteases are controlled by specific inhibitors belonging to the serine protease inhibitor (SERPIN) family that forms stoichiometric tight complexes with the target proteases. The inhibitors of the SERPIN family are thought to have a common tertiary structure[1] with a reactive center located on an exposed loop denoted “the strained loop”, situated near the carboxyl terminus of the inhibitor molecule[2–4]. These inhibitors interact with their target proteases by providing a so called “bait” residue (P1 residue), that mimics the normal substrate of the target proteases[2,3,5]. The complex is supposed to exist either as Michaelis type, tetrahedral type[6,7] or acylintermediate[8,9], although which stage is the stable state is not clear. Recently, we discovered that the SERPIN plasminogen activator inhibitor 1 (PAI-1) can be cleaved by plasminogen activators at its reactive site and that treatment with low concentrations of sodium dodecyl sulphate (SDS) converts active PAI-1 to a form that is cleaved like a substrate[10]. Since the conformation of the cleavable PAI-1 is completely different from its native form we defined it as the substrate-like form of PAI-1. The conversion of the inhibitor to a substrate like form is caused by a conformational change without alteration of the amino acid sequence and is of interest in order to study the mechanism of the interaction between serine protease and SERPIN. In this manuscript we demonstrate the cleavage of PAI-1 and discuss the possible mechanism by which a SERPIN is cleaved by its target protease.