ROLE OF THE P6-P3' REGION OF THE SERPIN REACTIVE LOOP IN THE FORMATION AND BREAKDOWN OF THE INHIBITORY COMPLEX

Serpins have a large external peptide loop known as the reactive loop. Part of the reactive loop functions as the primary recognition site f or target proteases; however, the complete role of the reactive loop i n determining serpin specificity is unclear. In the current study, we investigated the reactive loop region that could potentially interact with the extended binding site of target proteases; the P6-P3' region. We utilized a reactive loop switching strategy to determine the exten t to which the inhibitory activity of alpha-1-protease inhibitor (PI) against human neutrophil elastase (HNE) could be transferred to alpha- 1-antichymotrypsin (ACT), a serpin that does not inhibit HNE. A series of ACT-PI chimeras were constructed in which segments of increasing l ength taken from the P6-P3' region of PI replaced the corresponding re sidues of ACT. The effectiveness of each chimera as an inhibitor of HN E was assessed by measuring (1) the rate of inhibitory complex formati on and (2) the rate of complex breakdown (complex stability). Although all the ACT-PI chimeras were fully functional against chymotrypsin-li ke proteases, the series of chimeras showed no consistent progress tow ard the production of an inhibitor with the inhibitory properties of P I. The most rapid complex formation and most stable complexes were obs erved for chimeras with the P3-P1 residues of PI, whereas extending th e replacement region to the P6 residue resulted in a considerable decr ease in both inhibitory parameters. In order to study two additional f eatures of the PI reactive loop that may play a role in the presentati on of the P6-P3' region to HNE, we constructed variants that contained a P4' proline and deleted the P6'-P9' residues. Changes on the prime side appeared to have little effect on rates of inhibition or complex stability. Overall, even the most effective chimeras demonstrated an i nhibition rate constant at least 60-fold less than that observed for P I inhibition of HNE and the most long lived chimera-HNE complexes brok e down more rapidly than PI-HNE complexes. These results indicate that residues in the reactive loop region predicted to contact a specific target protease cannot fully transfer inhibitory activity from one ser pin to another, suggesting that specific reactive loop-serpin body and serpin body-protease body interactions play a significant role in det ermining serpin inhibitory activity against target proteases.