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.
The reactive center loop of native ␣ 1 -proteinase inhibitor has been reported to be in a helical conformation and in a -strand conformation by two different studies. In the -strand loop structure the P 5 glutamic acid plays a unique role by stabilizing the loop in the predicted optimal conformation for the interaction with target proteinases and insertion into -sheet A. We hypothesize here that disrupting the interactions that stabilize the -strand conformation of the loop would result in changes in the inhibitory properties of the serpin. In addition, our earlier studies on reactive center loop mutants of ␣ 1 -proteinase inhibitor suggested that the P 5 residue was important in stabilizing the ␣ 1 -proteinase inhibitor-proteinase complexes. To address these issues we made mutants of ␣ 1 -proteinase inhibitor with glycine, glutamine, or lysine at the P 5 position and measured the rates and stoichiometries of inhibition with trypsin and human neutrophil elastase and the stabilities of the resulting complexes. In most cases the rate of inhibition was reduced by about half and the stoichiometry increased between 2-and 4-fold. The only exception was for trypsin with the lysine variant where the P 5 was now the favored site of cleavage. These data show that the P 5 Glu is important in maintaining the reactive center loop in a conformation optimal for interaction with the proteinase and for a fast rate of loop insertion. The complexes formed with trypsin and the variant serpins were less stable than that formed with wild-type serpin and resulted in up to 33% regeneration of trypsin activity over a period of 6 days, compared with 17% with wild type. Thus, the P 5 residue of ␣ 1 -proteinase inhibitor is important in all steps of the inhibitory mechanism in a manner consistent with the structural role played by this residue in the -strand loop structure of native ␣ 1 -proteinase inhibitor.One of the best studied members of the serpin family of serine proteinase inhibitors is ␣ 1 -proteinase inhibitor (␣ 1 -PI), 1 which is an important inhibitor of neutrophil elastase and is deficient in emphysema (1-3). ␣ 1 -PI inhibits its target serine proteinases by the branched suicide-substrate pathway (1, 4) characteristic of inhibitory serpins (Fig. 1). The structure of native ␣ 1 -PI has been solved by two groups (5, 6). From one study (6), the reactive center loop, which contains the scissile P 1 -P 1 Ј bond, 2 was found to be in a helical conformation similar to other native serpins, namely ovalbumin and an ␣ 1 -antichymotrypsin variant (8, 9). In the other, more detailed, structure of native ␣ 1 -PI (5) it was found that the reactive center loop is in an extended -strand conformation similar to the canonical conformation found for other classes of protein inhibitors of serine proteinases (10). In this structure the P 5 glutamic acid residue plays a crucial role in the stabilization of the -strand conformation of the reactive center loop by forming hydrogen bonds with Arg-196 and the backbone amide of Met-226. These res...
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