ROLE OF N-TERMINAL AND C-TERMINAL AMINO-ACIDS IN ANTITHROMBIN BINDINGTO PENTASACCHARIDE

We have used a monoclonal antibody-based binding procedure to determin e the dissociation constants of the interactions between the essential antithrombin-binding pentasaccharide and a series of 13 distinct N- a nd C-terminal antithrombin substitution mutation variants with defecti ve binding interaction with heparin. The reduction in binding affinity of the pentasaccharide with the N-terminal variants (with substitutio n mutations Pro-41 --> Leu, Arg-47 --> Cys and His, Leu-99 --> Val and Phe, Gln-118 --> Pro, Arg-129 --> Gln) compared to normal antithrombi n, K-d 200 nM, ranged from 15-984-fold and was generally less than 150 -fold. Reduced binding affinity is assumed to arise mostly by perturba tion, direct or indirect, of the initial contact of pentasaccharide wi th basic residues of antithrombin. Surprisingly, the binding interacti on of the pentasaccharide with the C-terminal variants (with substitut ion mutations in or near strand 1C/4B, Phe-402 --> Leu, Cys, and Ser, Ala-404 --> Thr, Pro-407 --> Thr, Pro-429 --> Leu) was more uniformly and yet substantially (135-482-fold) decreased, despite the spatial se paration between the site of mutation and the proposed primary contact site of the pentasaccharide. These results demonstrate that strand 1C /4B region integrity is required for optimum interaction with the pent asaccharide, suggesting its involvement in transmission of the induced conformation change required for high affinity binding.