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Short-lived protease serpin complexes: partial disruption of the rat trypsin active site.

Protein science : a publication of the Protein Society (2007-10-27)
Lu Liu, Nicole Mushero, Lizbeth Hedstrom, Anne Gershenson
ABSTRACT

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 beta-sheet in the body of the serpin, translocating the attached protease approximately 70 A and deforming the protease active site, thereby trapping the acylenzyme. Loop insertion (approximately 1 s(-1)) is an order of magnitude slower than hydrolysis of a typical substrate acylenzyme (approximately 50 s(-1)), indicating that the protease is inhibited during translocation. We have previously trapped a partially translocated covalent complex of rat trypsin and alpha1-proteinase inhibitor (EpartI*) resulting from attractive interactions between cationic dyes and anionic rat trypsin. Here, using single pair Förster resonance energy transfer, we demonstrate that EpartI* is a metastable complex that can dissociate to free protease and cleaved serpin (I*) as well as convert to the canonical fully translocated complex EfullI*. 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 EpartI* (approximately 3 h at pH 7.4) and the pH dependence of EpartI* dissociation suggest that, unlike in EfullI*, the catalytic triad is intact in EpartI*. 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.

MATERIALS
Product Number
Brand
Product Description

Supelco
Atto 610-NHS ester, BioReagent, suitable for fluorescence, ≥90.0% (coupling to amines)
Sigma-Aldrich
Atto 610, BioReagent, suitable for fluorescence, ≥85.0% (HPCE/HPLC)
Sigma-Aldrich
Atto 610-Biotin, BioReagent, suitable for fluorescence, ≥85% (HPCE)
Sigma-Aldrich
Atto 610 maleimide, BioReagent, suitable for fluorescence, ≥90% (coupling to thiols)

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