INHIBITORY ACTIVITY AND CONFORMATIONAL TRANSITION OF ALPHA-1-PROTEINASE INHIBITOR VARIANTS

Several variants of alpha(1)-proteinase inhibitor (alpha(1)-PI) were investigated by spectroscopic methods and characterized according to their inhibitory activity. Replacement of Thr345 (P14) with Arg in alpha(1)-PI containing an Arg residue in position 358 (yielding [Thr345 --> Arg, Met358 --> Arg]alpha(1)-PI) results in complete loss of its inhibitory activity against human alpha-thrombin; whereas an exchange of residue Met351 (P8) by Glu ([Met351 --> Glu, Met358 --> Arg]alpha(1)-PI) does not alter activity. [Thr345 --> Arg, Met358 --> Arg]alpha(1)-PI is rapidly cleaved by thrombin, while [Met358 --> Arg]alpha(1)-PI and [Met351 --> Glu, Met358 --> Arg]alpha(1)-PI form stable proteinase-inhibitor complexes. The stability of [Thr345 --> Arg, Met358 --> Arg]alpha(1)-PI against guanidinium chloride denaturation is significantly enhanced compared to wild-type alpha(1)-PI, and does not change after cleavage, resembling ovalbumin, a serpin with no inhibitory activity, from which the Thr345 --> Arg amino acid exchange had been derived. [Met351 --> Glu, Met358 --> Arg]alpha(1)-PI and [Met358 --> Arg]alpha(1)-PI resemble the wild-type protein in this respect. The CD spectra of intact and cleaved alpha(1)-PI variants do not compare well with the wild-type protein, probably reflecting local structural differences. Insertion of a synthetic peptide, which corresponds to residues Thr345 --> Met358 of human alpha(1)-PI, leads to the formation of binary complexes with all variants having the characteristic features of the binary complex between peptide and wild-type protein.