Abstract
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 alpha(1)-antichymotrypsin either one residue closer (P2) or further (P1') from the base of the RSL (Glu-342). alpha(1)-Antichymotrypsin 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 human chymase and chymotrypsin and the non-target protease human neutrophil elastase (HNE) were analyzed. The P2 variant inhibited chymase and chymotrypsin but not HNE. 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 HNE (stoichiometry of inhibition = 4) but not chymase or chymotrypsin. However, inhibition of HNE 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 SDS-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 Glu-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.
Highlights
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 (Leu358) of the serpin ␣1-antichymotrypsin either one residue closer (P2) or further (P1) from the base of the RSL (Glu-342). ␣1-Antichymotrypsin 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)
These results suggests that lengthening the distance between the beginning of the RSL and the reactive site by a single residue (17 to 18 residues) effectively abolishes the ability of rACT to form a stable complex with a protease
Summary
Vol 277, No 33, Issue of August 16, pp. 29927–29935, 2002 Printed in U.S.A. The Effects of Reactive Site Location on the Inhibitory Properties of the Serpin ␣1-Antichymotrypsin*. A number of spectroscopic studies using site-specific fluorescent labels support the location of the inhibited protease at the pole opposite from that of encounter [7, 8], other studies employing fluorescent labels [9], monoclonal antibodies [10, 11], and synthetic peptides resembling the RSL [12] place the enzyme closer to, if not at, the site of encounter The latter studies suggest that only partial insertion of the RSL is required for inhibition. The effect on inhibition of relocating the reactive site of the serpin ␣1-antichymotrypsin (ACT) one residue closer or further from Glu-342 was evaluated using three different proteases. The patterns observed were similar to those described by Zhou et al [18], suggesting that full insertion is a general requirement of protease inhibition by serpins
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