INFLUENCE OF THE P-5 RESIDUE ON ALPHA(1)-PROTEINASE INHIBITOR MECHANISM

The reactive center loop of native alpha(1)-proteinase inhibitor has b een reported to be in a helical conformation and in a beta-strand conf ormation by two different studies. In the beta-strand loop structure t he P-5 glutamic acid plays a unique role by stabilizing the loop in th e predicted optimal conformation for the interaction with target prote inases and insertion into beta-sheet A. We hypothesize here that disru pting the interactions that stabilize the beta-strand conformation of the loop would result in changes in the inhibitory properties of the s erpin. In addition, our earlier studies on reactive center loop mutant s 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)-protein ase inhibitor with glycine, glutamine, or lysine at the P-5 position a nd measured the rates and stoichiometries of inhibition with trypsin a nd human neutrophil elastase and the stabilities of the resulting comp lexes, In most cases the rate of inhibition was reduced by about half and the stoichiometry increased between 2- and 4-fold, The only except ion was for trypsin with the lysine variant where the P, was now the f avored 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 w ith 17% with wild type, Thus, the P-5 residue of alpha(1)-proteinase i nhibitor is important in all steps of the inhibitory mechanism in a ma nner consistent with the structural role played by this residue in the beta-strand loop structure of native alpha(1)-proteinase inhibitor.