The P-6-P-2 region of serpins is critical for proteinase inhibition and complex stability

Two of the prototypic serpins are alpha(1)-proteinase inhibitor and ovalbumin. alpha(1)-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 P-12 to P-2 in the reactive center loop of ovalbumin are intrinsically incapable of being in an inhibitory serpin, we have made chimeric alpha(1)-proteinase inhibitor variants containing residues P-12-P-7, P-6-P-2, or P-12-P-2 Of ovalbumin and determined their inhibitory properties with trypsin and human neutrophil elastase. With the P-12-P-7 and P-6-P-2 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 P-12-P-2 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 P-6-P-2 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 P-6-P-2 and P-12-P-2 variants, and on an increase in heat stability of the cleaved P-12-P-2 variant, it was concluded that the variants had undergone complete loop insertion on cleavage, These results show that the reactive center loop residues P-12-P-2 Of ovalbumin can be present in inhibitory serpins although decreasing the inhibitory properties. These data also demonstrate that the residues in the P-6-P-2 region of serpins are critical for rapid inhibition of proteinases and formation of stable serpin-proteinase complexes.