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.
Schulze
A J
AJ
Max-Planck-Institut für Biochemie, Martinsried, Federal Republic of Germany.
Huber
R
R
Degryse
E
E
Speck
D
D
Bischoff
R
R
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D002872:Chromosome Deletion; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D014644:Genetic Variation; D006801:Humans; D008961:Models, Structural; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D012689:Sequence Homology, Nucleic Acid; D013050:Spectrometry, Fluorescence; D013917:Thrombin / Q000378:metabolism; D000515:alpha 1-Antitrypsin / Q000737:chemistry / Q000235:genetics* / Q000494:pharmacology
D000515:alpha 1-Antitrypsin; D013917:Thrombin