Site-directed mutagenesis of the P2 residue of human antithrombin.
Antithrombin (AT) is the principal inhibitor of thrombin in human plasma, and a member of the serine proteinase (serpin) family of proteins. Previously, we have described a point mutation in the human AT gene that converted amino acid 392 from glycine to aspartic acid which was associated with thrombotic disease in a Swedish family [(1992) Blood 79, 1428-1434]. This observation prompted us to investigate the consequences of other substitutions at this position, termed P2 with respect to the reactive centre. Site-directed mutagenesis was employed to generate seven mutants (Pro, Met, Gln, Val, Lys, Glu, and Asp), whose properties were compared with wild-type recombinant AT, following in vitro transcription and cell-free expression in a rabbit reticulocyte lysate system. With only one exception, the variant forms were less active than the wild-type in forming complexes with either alpha-thrombin, factor Xa, or trypsin. Hydrophobic (Val) or negatively charged (Asp or Glu) substitutions were particularly disruptive, in that these variants exhibited less than 10% wild-type antithrombin or antitrypsin activity. In contrast, the formation of complexes with the various proteases of the Pro variant was essentially unimpaired. We conclude that the P2 residue of AT plays a role in optimal presentation of the reactive centre to its cognate protease, and propose that the observed requirement of Gly or Pro at this position is suggestive of a bend in the polypeptide backbone that aids in this presentation.
Sheffield
W P
WP
Canadian Red Cross Blood Services, McMaster University Medical Centre, Hamilton, Ont., Canada.
Blajchman
M A
MA
D000818:Animals; D000991:Antithrombins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D015951:Factor Xa / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D012333:RNA, Messenger / Q000378:metabolism; D011817:Rabbits; D011994:Recombinant Proteins / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism; D013917:Thrombin / Q000378:metabolism; D013927:Thrombosis / Q000235:genetics; D014158:Transcription, Genetic; D014357:Trypsin / Q000378:metabolism
D000991:Antithrombins; D012333:RNA, Messenger; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; D014357:Trypsin; D013917:Thrombin; D015951:Factor Xa