Role of the P-2 residue in determining the specificity of serpins

The importance of the P-2 residue in determining serpin specificity was examined by making a series of substitutions in the P-2 position of recombinant alpha(1)-antichymotrypsin that contained an arginine P-1 residue. The importance of the P-2 residue in governing the association rate constant (k(on)) of the serpin varied with the protease examined. For trypsin, the P-2 residue played a relatively minor role, whereas the nature of this residue markedly influenced the rates of inhibition of thrombin, factor Xa, and APC. A 1000-fold difference in k(on) values was observed between the fastest (P-2 proline) and the slowest (P-2 threonine) inhibitors of thrombin. Similar differences were observed with factor Xa; the best inhibitor (P-2 glycine) displayed a 200-fold higher k(on) value than the poorest (P-2 threonine). The nature of the P-2 residue also affected whether the interaction of the serpin with the protease resulted in inhibition of the protease or cleavage of the serpin; a P-2 proline residue increased the rate of cleavage of alpha(1)-antichymotrypsin by trypsin. By using mutants of thrombin, it was possible to show that the B-insertion loop, which partially occludes the active site, is important in determining the P-2 specificity of this enzyme. Deletion of three amino acids from this loop yielded a protease (des-PPW) that became more like trypsin in its specificity. In addition, it was shown that Glu(192) dramatically restricts thrombin's ability to accommodate a threonine in the P-2 position. Taken together, the results demonstrated the importance of complementary interactions between the P-2 residue of the serpin and the S-2 binding site of the protease in regulating the specific interaction between serpin and protease.