The contribution of the conserved hinge region residues of alpha1-antitrypsin to its reaction with elastase.

The hinge region of serpins is a conserved sequence of 8 amino acids located 7 residues away from the scissile bond at P8 to P15, on the edge of the protease-binding domain. In the inhibitory serpins the P8 to P12 residues of this motif are usually small side-chain amino acids, most commonly alanine. Each of these residues in alpha1-antitrypsin was mutated to a glutamate, and the effect of a hinge-region glutamic acid substitution was found. While substitutions at positions P10 and P12 affected the inhibitory characteristics of alpha1-antitrypsin, substitutions at positions P7, P8, P9, and P11 had no effect on inhibition. Thus, the conservation of residues with small side chains at the latter positions does not appear to be related to an essential function in the inhibitory mechanism. Following the glutamate substitution at P10, alpha1-antitrypsin remained a rapid inhibitor of elastase, but the elastase--serpin complex slowly broke down to yield active elastase and cleaved alpha1-antitrypsin. The glutamate substitution at P12 caused the resultant molecule (P12 Ala-->Glu) to become a partial substrate of elastase such that four moles of inhibitor were required to inhibit one mole of enzyme, and led to a 12-fold decrease in the association rate constant. The data could be interpreted in terms of the suicide substrate inhibition model for serpin-protease interactions and allowed a further refinement of the role of the hinge region in this process.