The covalent nature of the human antithrombin III–thrombin bond

Abstract

1. Cleavage of the human antithrombin III--thrombin complex with [14C]methoxyamine hydrochloride results in inactive thrombin and 14C-labelled antithrombin III. 2. Discontinuous polyacrylamide-gel electrophoresis of the reduced dissociation fragments of the complex in the presence of sodium dodecyl sulphate reveals two antithrombin III bands that do not resolve during electrophoresis without reduction. The heavy band has the electrophoretic mobility of the native protein. The light band has an apparent mol.wt. that is approx. 4000 less than the molecular weight of native antithrombin III. 3. Treatment of the cleavage products of the complex with carboxypeptidase B yields 1 mumol of arginine, a new C-terminal amino acid, per mumol of thrombin dissociated. The results indicate that during formation of the antithrombin III--thrombin complex, the inhibitor is cleaved at an arginine--X bond; this arginine residue forms a carboxylic ester with the enzyme, while the excised polypeptide remains bound through a disulphide bridge(s).

A potent, proteolysis-resistant inhibitor of kallikrein-related peptidase 6 (KLK6) for cancer therapy, developed by combinatorial engineering

“…This finding was surprising, because, while such covalent complexes are a hallmark of the unrelated serpin family of serine protease inhibitors, which form stable acyl-enzymes with their target proteases (49,62,63), the canonical inhibitors, including APPI, follow a different mechanism, in which the stable complex is a tightly but reversibly bound Michaelis complex (55,64). Previous studies of the chymotrypsin inhibitor 2, which belongs to a different family of canonical inhibitors, identified the rapid formation of a covalent acyl-enzyme by SDS-PAGE, but this species accumulated to represent only a small fraction of the inhibited enzyme due to the reversibility of acylation and the comparatively greater thermodynamic stability of the Michaelis complex (65,66).…”

Identification of a new P1 residue mutation (444Arg→Ser) in a dysfunctional C1 inhibitor protein contained in a type II hereditary angioedema plasma

“…This may be as an acyl-enzyme intermediate, requiring the hydroxyl group of the proteinase active site serine residue and the newly-released carboxyl (of the P1 residue) of the serpin [14,15]. The specificity of inhibition is heavily influenced by the amino acid at the P1 position.…”

The Axonally Secreted Serine Proteinase Inhibitor, Neuroserpin, Inhibits Plasminogen Activators and Plasmin but Not Thrombin

“…Complex Formation of cNS with Neural Serine ProteinasesUpon binding of their target proteinases, serpins form highly stable complexes that resist dissociation by SDS in the presence of reducing agents (57). Since neuroserpin is predominantly expressed in the nervous system, we tested the serine proteinases tPA, uPA, plasmin, and thrombin, which exhibit trypsin-like substrate specificity and are expressed in the nervous system (1, 2, 16, 17), for their ability to form SDS-stable complexes with neuroserpin.…”