The hydrolysis and resynthesis of a single reactive site peptide bond in recombinant antistasin by coagulation factor Xa
Research article published in Archives of biochemistry and biophysics (1992)
Abstract
Antistasin (ATS) is a 119-amino acid, leech-derived protein which exhibits selective, tight-binding inhibition of blood coagulation factor Xa. Prolonged incubation of ATS with factor Xa leads to the highly specific hydrolysis of the peptide bond between residues Arg34 and Val35, implicating this peptide bond as the putative reactive site. We report here the preparation of pure, cleaved (modified) recombinant ATS (rATS) and utilize this material to provide additional proof that the cleaved peptide bond is in fact the reactive site. Modified rATS retains strong inhibitory potency against factor Xa as evidenced by a dissociation constant of 166.3 +/- 9.6 pM; four-fold greater than that of native inhibitor, 43.4 +/- 1.4 pM. Incubation of pure, modified rATS with catalytic amounts of factor Xa results in resynthesis of the hydrolyzed peptide bond, achieving an equilibrium near unity between native and modified inhibitors. Specific removal of the newly formed carboxy-terminal Arg residue from modified rATS by carboxypeptidase B treatment obviates its conversion to native inhibitor coincident with the complete loss of inhibitory activity. These results establish that rATS inhibits factor Xa according to a standard mechanism of serine protease inhibitors and support the contention that the Arg34-Val35 peptide bond constitutes the reactive site.
Abstract sourced from PubMed (NCBI) for the cited record. See the original publication for the authoritative version.
Zusammenfassung
Antistasin (ATS) is a 119-amino acid, leech-derived protein which exhibits selective, tight-binding inhibition of blood coagulation factor Xa.
Warum dies für die Hirudotherapie relevant ist
Antistasin is a 119-amino-acid, leech-derived protein that selectively and tightly inhibits blood coagulation factor Xa. This study used purified recombinant antistasin to demonstrate that the Arg34–Val35 peptide bond is the reactive site, showing that modified (cleaved) inhibitor retains potent anti-Xa activity (Kd ~166 pM) and can resynthesize its hydrolyzed bond in the presence of catalytic factor Xa. This work is directly relevant to the leech secretome and ASH's domain, as it characterizes the mechanism of a specific leech-derived anticoagulant protein. The honest caveat is that this is a purely biochemical in vitro study using recombinant protein; no live leeches, crude saliva, or clinical or in vivo data are involved.
Zitation
The hydrolysis and resynthesis of a single reactive site peptide bond in recombinant antistasin by coagulation factor Xa
Dunwiddie CT et al. · Archives of biochemistry and biophysics, 1992
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