Site-directed mutagenesis of alanine-382 of human antithrombin III.
Antithrombin III Hamilton is a structural variant of antithrombin III (AT-III) with normal heparin affinity but impaired serine protease inhibitory activity. The molecular defect of AT-III-Hamilton is a substitution of threonine for alanine at amino acid residue 382. Recently it has been shown that both plasma-derived and cell-free-derived AT-III-Hamilton polypeptides act as substrates rather than inhibitors of thrombin and factor Xa. In the present study, the cell-free expression phagemid vector pGEM-3Zf(+)-AT-III1-432 was mutated at amino acid residue 382 of AT-III to generate 7 cell-free-derived variants. All these cell-free-derived AT-III variants were able to bind heparin as effectively as cell-free-derived normal AT-III. In terms of alpha-thrombin inhibitory activity each variant reacted differently. Variants could be grouped into 3 categories with respect to thrombin-AT-III complex formation: (1) near normal activity (glycine, isoleucine, leucine, valine); (2) low activity (threonine, glutamine); (3) no detectable activity (lysine). These data suggest that mutations at position 382 of AT-III may have a variable effect on protease inhibitory activity, depending on either the stability of the P12-P9 region of the exposed loop of AT-III, or the inability of the amino acid residue at position 382 to interact with a conserved hydrophobic pocket consisting of phenylalanine (at positions 77, 221 and 422) and isoleucine (position 412) residues.
Austin
R C
RC
Canadian Red Cross Blood Transfusion Service, McMaster University, Hamilton, Ontario.
Rachubinski
R A
RA
Blajchman
M A
MA
D000409:Alanine / Q000235:genetics*; D000595:Amino Acid Sequence; D000990:Antithrombin III / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D002474:Cell-Free System; D002846:Chromatography, Affinity; D006493:Heparin / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010447:Peptide Hydrolases / Q000235:genetics* / Q000378:metabolism; D012995:Solubility
C046193:antithrombin III-protease complex; D000990:Antithrombin III; D006493:Heparin; D010447:Peptide Hydrolases; D000409:Alanine