The effects of reactive site location on the inhibitory properties of the serpin XXXX .

@entity1 @entity3920 @entity8698 @entity367 @entity13789 @entity214
The large size of the serpin reactive site loop (RSL) suggests that the role of the RSL in protease inhibition is more complex than that of presenting the reactive site (P1 residue) to the protease. This study examines the effect on inhibition of relocating the reactive site (Leu-358) of the serpin @entity8698 either one residue closer (P2) or further (P1') from the base of the RSL ( @entity367 -342). @entity8698 variants were produced by mutation within the P4-P2' region; the sequence ITLLSA was changed to ITLSSA to relocate the reactive site to P2 (Leu-357) and to ITITLS to relocate it to P1' (Leu-359). Inhibition of the chymotrypsin-like proteases @entity1 @entity3920 and chymotrypsin and the non-target protease @entity1 neutrophil elastase ( @entity13789 ) were analyzed. The P2 variant inhibited @entity3920 and chymotrypsin but not @entity13789 . Relative to P1, interaction at P2 was characterized by greater complex stability, lower inhibition rate constants, and increased stoichiometry of inhibition values. In contrast, the P1' variant inhibited @entity13789 (stoichiometry of inhibition = 4) but not @entity3920 or chymotrypsin. However, inhibition of @entity13789 was by interaction with Ile-357, the P2 residue. The P1' site was recognized by all proteases as a cleavage site. Covalent-complexes resistant to @entity214 -PAGE were observed in all inhibitory reactions, consistent with the trapping of the protease as a serpin-acyl protease complex. The complete loss in inhibitory activity associated with lengthening the @entity367 -342-reactive site distance by a single residue and the enhanced stability of complexes associated with shortening this distance by a single residue are compatible with the distorted-protease model of inhibition requiring full insertion of the RSL into the body of the serpin and translocation of the linked protease to the pole opposite from that of encounter.
