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 l ocated 7 residues away from the scissile bond at P-8 to P-15, on the e dge 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 acid s, most commonly alanine. Each of these residues in alpha(1)-antitryps in was mutated to a glutamate, and the effect of the mutation on the i nhibitory 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 inhibitor y characteristics of alpha(1)-antitrypsin, substitutions at positions P-7, P-8, P-9, and P-11 had no effect on inhibition, Thus, the conserv ation of residues with small side chains at the latter positions does not appear to be related to an essential function in the inhibitory me chanism. Following the glutamate substitution at P-10, alpha(1)-antitr ypsin 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 o f enzyme, and led to a 12-fold decrease in the association rate consta nt. The data could be interpreted in terms of the suicide substrate in hibition model for serpin-protease interactions and allowed a further refinement of the role of the hinge region in this process.