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Short-lived protease–serpin complexes: Partial disruption of the rat trypsin active site

¹ Department of Chemistry, Brandeis University, Waltham, Massachusetts 02454, USA
² Graduate Program in Biochemistry, Brandeis University, Waltham, Massachusetts 02454, USA
³ Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02454, USA

(RECEIVED July 7, 2007; FINAL REVISION August 10, 2007; ACCEPTED August 10, 2007)

Serpins inhibit serine proteases by mechanically disrupting the protease active site. The protease first reacts with the serpin's reactive center loop (RCL) to form an acylenzyme. Then the RCL inserts into a -sheet in the body of the serpin, translocating the attached protease 70 Å and deforming the protease active site, thereby trapping the acylenzyme. Loop insertion (1 s^–1) is an order of magnitude slower than hydrolysis of a typical substrate acylenzyme (50 s^–1), indicating that the protease is inhibited during translocation. We have previously trapped a partially translocated covalent complex of rat trypsin and ₁-proteinase inhibitor (E_partI*) resulting from attractive interactions between cationic dyes and anionic rat trypsin. Here, using single pair Förster resonance energy transfer, we demonstrate that E_partI* is a metastable complex that can dissociate to free protease and cleaved serpin (I*) as well as convert to the canonical fully translocated complex E_fullI*. The partitioning between these two pathways is pH dependent, with conversion favored at low pH and dissociation favored at high pH. The short lifetime of E_partI* (3 h at pH 7.4) and the pH dependence of E_partI* dissociation suggest that, unlike in E_fullI*, the catalytic triad is intact in E_partI*. These results also demonstrate that interactions between target proteases and the body of the serpin can hinder protease translocation leading to short-lived covalent complexes.

Keywords: conformational changes; fluorescence; FRET; serpin; protease; single molecule fluorescence

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