The P6-P2 region of serpins is critical for proteinase inhibition and complex stability

Two of the prototypic serpins are alpha1-proteinase inhibitor and ovalbumin. alpha1-Proteinase inhibitor is a rapid inhibitor of a number of proteinases and undergoes the characteristic serpin conformational change on cleavage within the reactive center loop, whereas ovalbumin is noninhibitory and does not undergo the conformational change. To investigate if residues from P12 to P2 in the reactive center loop of ovalbumin are intrinsically incapable of being in an inhibitory serpin, we have made chimeric alpha1-proteinase inhibitor variants containing residues P12-P7, P6-P2, or P12-P2 of ovalbumin and determined their inhibitory properties with trypsin and human neutrophil elastase. With the P12-P7 and P6-P2 variants, the steps before and after the fork of the branched suicide-substrate pathway were affected as reflected by changes in rates and stoichiometries of inhibition with both proteinases. The P12-P2 variant showed that those effects were nonadditive, with exclusive substrate behavior for elastase and only residual inhibitory activity against trypsin. The properties of the variants were consistent with them obeying the suicide-substrate mechanism characteristic of serpins. Enzyme activity was regenerated from complexes formed with the P6-P2 variant faster than with wild-type indicating that the rate of turnover of the complex was increased. Based on proteinase susceptibility in the reactive center loops of the P6-P2 and P12-P2 variants, and on an increase in heat stability of the cleaved P12-P2 variant, it was concluded that the variants had undergone complete loop insertion on cleavage. These results show that the reactive center loop residues P12-P2 of ovalbumin can be present in inhibitory serpins although decreasing the inhibitory properties. These data also demonstrate that the residues in the P6-P2 region of serpins are critical for rapid inhibition of proteinases and formation of stable serpin-proteinase complexes.