SITE-DIRECTED MUTAGENESIS OF THE P2 RESIDUE OF HUMAN ANTITHROMBIN

Antithrombin (AT) is the principal inhibitor of thrombin in human plas ma, and a member of the serine proteinase (serpin) family of proteins. Previously, we have described a point mutation in the human AT gene t hat converted amino acid 392 from glycine to aspartic acid which was a ssociated with thrombotic disease in a Swedish family [(1992) Blood 79 , 1428-1434]. This observation prompted us to investigate the conseque nces of other substitutions at this position, termed P2 with respect t o the reactive centre. Site-directed mutagenesis was employed to gener ate seven mutants (Pro, Met, Gln, Val, Lys, Glu, and Asp), whose prope rties 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 t han the wild-type in forming complexes with either a-thrombin, factor Xa, or trypsin. Hydrophobic (Val) or negatively charged (Asp or Glu) s ubstitutions were particularly disruptive, in that these variants exhi bited less than 10% wild-type antithrombin or antitrypsin activity. In contrast, the formation of complexes with the various proteases of th e Pro variant was essentially unimpaired. We conclude that the P2 resi due 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.