Kinetic mechanism of protease inhibition by α1-antitrypsin

Abstract

The native form of serine protease inhibitor (serpin) is kinetically trapped in a metastable state. Metastability in these proteins is critical to inhibit target protease by forming a stable covalent complex. Despite recent determination of the crystal structures of a Michaelis protease–serpin complex as well as a stable covalent complex, details on the kinetic mechanism remain unsolved. In this report, we examined the reaction mechanism of α1-antitrypsin toward elastase by a combination of stopped-flow experiments via fluorescence resonance energy transfer and rapid-quench studies. The results suggest a non-covalent complex intermediate other than Michaelis complex as an intermediate before the cleavage of P1–P1′ scissile bond, whose formation is the rate-determining step of the overall reaction. This rate-limiting step represents rearrangement of the reactive site loop, and is regulated by a salt bridge between E354 and R196. The ionic interaction is unique to α1-antitrypsin, which suggests that protease inhibition mechanisms are varied among serpins.