Site-directed mutagenesis of the reactive center (serine 394) of antithrombin III.
Human antithrombin III (AT) shares significant sequence homology and a common inhibitory mechanism with the serine protease inhibitor (serpin) superfamily. AT has a reactive site in which the P1 residue is primarily responsible for protease specificity. The P1' residue, almost invariably serine, is critical in the inactive natural variant AT-Denver, which has a leucine substitution in that position (Stephens, A.W., Thalley, B.S., and Hirs, C.H.W. (1987) J. Biol. Chem. 262, 1044-1048). In the present study site-directed mutagenesis was used to generate eight variants with altered P1' residues. All were secreted efficiently by COS cells transiently transfected with the AT cDNA in a eukaryotic shuttle vector. All variants also bound heparin as effectively as wild-type AT. Variants were grouped into three categories with respect to thrombin-AT complex formation: 1) no detectable inhibitory activity (proline, methionine); 2) low activity (cysteine, valine, leucine); and 3) near normal activity (glycine, alanine, threonine). The leucine variant, which is in the low activity group, exhibited the same physical and functional properties as AT-Denver. We conclude that the serine hydroxyl is not critical for functional activity and that there is a side chain size optimum which is modulated by hydrophobic effects.
D000595:Amino Acid Sequence; D000818:Animals; D000990:Antithrombin III / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D014644:Genetic Variation; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D012694:Serine; D014162:Transfection
D012694:Serine; D000990:Antithrombin III; D004247:DNA