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 ov albumin. alpha(1)-Proteinase inhibitor is a rapid inhibitor of a numbe r of proteinases and undergoes the characteristic serpin conformationa l change on cleavage within the reactive center loop, whereas ovalbumi n is noninhibitory and does not undergo the conformational change. To investigate if residues from P-12 to P-2 in the reactive center loop o f ovalbumin are intrinsically incapable of being in an inhibitory serp in, we have made chimeric alpha(1)-proteinase inhibitor variants conta ining residues P-12-P-7, P-6-P-2, or P-12-P-2 Of ovalbumin and determi ned their inhibitory properties with trypsin and human neutrophil elas tase. With the P-12-P-7 and P-6-P-2 variants, the steps before and aft er 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 r esidual inhibitory activity against trypsin. The properties of the var iants were consistent with them obeying the suicide-substrate mechanis m characteristic of serpins. Enzyme activity was regenerated from comp lexes formed with the P-6-P-2 variant faster than with wild-type indic ating 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 cl eaved P-12-P-2 variant, it was concluded that the variants had undergo ne complete loop insertion on cleavage, These results show that the re active center loop residues P-12-P-2 Of ovalbumin can be present in in hibitory serpins although decreasing the inhibitory properties. These data also demonstrate that the residues in the P-6-P-2 region of serpi ns are critical for rapid inhibition of proteinases and formation of s table serpin-proteinase complexes.