Mechanism of Action of α-1-Antitrypsin

Abstract

Comparison of the biochemical characteristics of enzymes inhibited by α-1-antitrypsin shows that thrombin, elastase, trypsin, chymotrypsin, and plasmin are serine proteases with serine, histidine, and aspartic acid residues at their active center and cleave proteins at lysine and arginine or at aromatic or leucine residues. The present study was designed to test the hypothesis that α-1-antitrypsin has two inhibitor sites, one containing a positively charged residue and the other containing an aromatic or leucine residue, and that both sites have a binding site for the active site of serine proteases. Experiments designed to test this hypothesis showed the following: 1. Trypsin and chymotrypsin compete for inhibitory sites on purified α-1-antitrypsin, suggesting that the inhibitor has the same or overlapping inhibitory sites for these two enzymes. 2. Trypsin inactivated by diisopropylphosphorofluoridate fails to occupy inhibiting sites on α-1-antitrypsin. 3. α-1-Antitrypsin inhibits subtilisin, a proteolytic enzyme that contains serine, histidine, and aspartic acid residues at its active site in common with mammalian serine proteases. 4. α-1-Antitrypsin fails to inactivate acetylcholinesterase, a nonproteolytic enzyme whose active site contains a reactive serine residue, suggesting that this residue alone is not sufficient for inhibition by α-1-antitrypsin. 5. Treatment of α-1-antitrypsin with phenylglyoxal hydrate blocks the action of α-1-antitrypsin on trypsin but not on chymotrypsin. The change in activity is presumptive evidence that arginine residues were modified by the treatment. Antitryptic activity can be regenerated with removal of the blocking groups. These data indicate that trypsin and chymotrypsin are inhibited at two different sites on α-1-antitrypsin and suggest that the trypsin inhibitory site contains a positively charged amino acid. While these experiments have not proved this hypothesis, they are consistent with it.