INHIBITORY MECHANISM OF SERPINS - MOBILITY OF THE C-TERMINAL REGION OF THE REACTIVE-SITE LOOP

The reactive-site loops of serpins are characterized by a defined mobi lity where the loop adopts a new secondary structure as an essential p art of the inhibitory process. While the importance of mobility in the N-terminal region of the reactive-site loop has been well studied, th e role of mobility in the C-terminal portion has not been investigated . The requirements for mobility of the C-terminal portion of the react ive-site loop of alpha(1)-antitrypsin were investigated by creating a disulfide bridge between the P'(3) residue and residue 283 near the to p of strand 2C; this disulfide would restrict the mobility of the C-te rminal portion of the reactive-site loop by locking together strands 1 and 2 of the C beta-sheet. The engineered disulfide bond had no effec t on the inhibitory activity of alpha(1)-antitrypsin, indicating that there is no requirement for mobility in this region of the molecule. M oreover, these results, coupled with those from molecular modeling, in dicate that insertion into the A beta-sheet of the intact reactive-loo p beyond P-12 is not rate-limiting for the formation of the stable com plex. The engineered disulfide bond should also prove useful in the cr eation of more stable serpin variants; for example, such a bond in pla sminogen activator inhibitor-1 would prevent it from becoming latent b y locking strand 1C onto the C beta-sheet.