AN EXAMINATION OF THE INHIBITORY MECHANISM OF SERPINS BY ANALYZING THE INTERACTION OF TRYPSIN AND CHYMOTRYPSIN WITH ALPHA(2)-ANTIPLASMIN

Human alpha2-antiplasmin (alpha2-AP) has previously been shown to poss ess overlapping inhibitory sites for trypsin and chymotrypsin [Potempa , Shieh and Travis (1988) Science 241, 699-700]. Since this is current ly unique among active-site-directed inhibitors of proteinases, and di fficult to explain in terms of accepted inhibitory mechanisms, we re-e xamined the claim. Initial characterization of purified alpha2-AP reve aled an additional 12 residues preceding the published N-terminus, pro mpting us to revise the previous numbering. We found that trypsin caus ed cleavage of the Arg376-Met377 bond in the reactive-site loop of the inhibitor, whereas chymotrypsin caused cleavage at two sites in appro x. equal amounts at 37-degrees-C: Met374-Ser375 (site 1) and Met377-Se r378 (site 2). At 0-degrees-C alpha2-AP became a more efficient inhibi tor of chymotrypsin, and the proportion of cleavage at site 1 declined , indicating that chymotrypsin prefers to react with site 2 at 0-degre es-C. Inhibitors of the alpha2-AP type are inactivated when cleaved in their reactive-site loops by proteinases that they do not inhibit, so we conclude that site 1 is treated as a substrate by chymotrypsin. Si te 2 is the inhibitory site for chymotrypsin. We confirm that alpha2-A P does indeed have overlapping reactive sites for trypsin and chymotry psin, and since the locations of chymotrypsin-interaction sites vary w ith temperature, we suggest that alpha2-AP cannot have rigid reactive- site geometry. More likely, it has a mobile reactive-site loop of the type that has been recently demonstrated for eglin C.