THE CONTRIBUTION OF THE CONSERVED HINGE REGION RESIDUES OF ALPHA(1)-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 P-8 to P-15, on the edge of the protease-binding domain. In the inhibitory serpins the P-8 to P-12 residues of this motif are usually small side-chain amino acids, most commonly alanine. Each of these residues in alpha(1)-antitrypsin was mutated to a glutamate, and the effect of the mutation on the inhibitory characteristics was assessed. A strong positional dependence of the effect of a hinge-region glutamic acid substitution was found. While substitutions at positions P-10 and P-12 affected the inhibitory characteristics of alpha(1)-antitrypsin, substitutions at positions P-7, P-8, P-9, and P-11 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 P-10, alpha(1)-antitrypsin remained a rapid inhibitor of elastase, but the elastase-serpin complex slowly broke down to yield active elastase and cleaved alpha(1)-antitrypsin, The glutamate substitution at P-12 caused the resultant molecule (P-12 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.