CHYMOTRYPSIN INHIBITORY ACTIVITY OF NORMAL C1-INHIBITOR AND A P1 ARG TO HIS MUTANT - EVIDENCE FOR THE PRESENCE OF OVERLAPPING REACTIVE CENTERS

C1-inhibitor is a serine proteinase inhibitor that is active against C1s, C1r, kallikrein, and factor XII. Recently, it has been shown that it also has inhibitory activity against chymotrypsin. We have investigated this activity of normal human C1-inhibitor, normal rabbit C1-inhibitor, and P1 Arg to His mutant human C1-inhibitors and find that all are able to inhibit chymotrypsin and form stable sodium dodecyl sulfate-resistant complexes. The K(ass) values show that the PI His mutant is a slightly better inhibitor of chymotrypsin than normal human C1-inhibitor (3.4 x 10(4) compared with 7.3 x 10(3)). The carboxy-terminal peptide of normal human C1-inhibitor, derived from the dissociated protease-inhibitor complex, shows cleavage between the P2 and P1 residues. Therefore, as with alpha2-antiplasmin, C1-inhibitor possesses two overlapping P1 residues, one for chymotrypsin and the other for Arg-specific proteinases. In contrast, with the PI His mutant, the peptide generated from the dissociation of its complex with chymotrypsin demonstrated cleavage between the P1 and P'1 residues. Therefore, unlike alpha2-antiplasmin, chymotrypsin utilizes the P2 residue as its reactive site in normal C1-inhibitor but utilizes the P1 residue as its reactive site in the P1 His mutant protein. This suggests that the reactive center loop allows a degree of induced fit and therefore must be relatively flexible.