Influence of the P5 residue on α1-proteinase inhibitor mechanism

The reactive center loop of native alpha(1)-proteinase inhibitor has been reported to be in a helical conformation and in a beta-strand conformation by two different studies. In the beta-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 beta-sheet A. We hypothesize here that disrupting the interactions that stabilize the beta-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 alpha(1)-proteinase inhibitor suggested that the P-5 residue was important in stabilizing the alpha(1)-proteinase inhibitor-proteinase complexes. To address these issues we made mutants of alpha(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, 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 alpha(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 beta-strand loop structure of native alpha(1)-proteinase inhibitor.