Ce. Chaillanhuntington et Pa. Patston, INFLUENCE OF THE P-5 RESIDUE ON ALPHA(1)-PROTEINASE INHIBITOR MECHANISM, The Journal of biological chemistry, 273(8), 1998, pp. 4569-4573
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.