INTERACTIONS OF HIRUDIN-BASED INHIBITOR WITH THROMBIN - CRITICAL ROLEOF THE ILE(H59) SIDE-CHAIN OF THE INHIBITOR

Hirudin is the most potent and specific thrombin inhibitor from medici nal leech with a K-i value of 2.2 x 10(-14) M. It consists of an activ e site inhibitor segment, hirudin(1-48), a fibrinogen-recognition exos ite inhibitor segment, hirudin(55-65), and linker, hirudin(49-54), con necting these inhibitor segments. The role of the side chain of the hi rudin 59th residue, Ile, is studied by using a series of synthetic biv alent thrombin inhibitors, which mimic the binding mode of hirudin. Th e synthetic inhibitors based on the hirudin sequence have a general se quence of Ac-(D-Phe)-Pro-Arg-Pro-(4-aminobutyric acid)-(7-aminoheptano ic d)-Asp-Phe-Glu-Glu-Xaa-Pro-Glu-Glu-Tyr-Leu-Gln-OH, in which the 59t h residue, Xaa, is substituted by various natural and unnatural L-amin o acids. For example, substitution of Ile(H59) by Val, which is equiva lent to removing the delta-methyl group of Ile(H59), reduces the affin ity of the inhibitor 5.7-fold (Delta Delta G(o) = 1.0 kcal/mol) to a K -i value of 4.7 nM compared to that (K-i = 0.82 nM) of the correspondi ng inhibitor with Ile(H59). Removal of the entire side chain of Ile(H5 9), i.e., a substitution of Ile(H59) by Gly, reduces the affinity of t he inhibitor 6300-fold, revealing the critical role of the Ile(H59) si de chain in the inhibitor binding, Theoretical free energy calculation successfully reproduces the binding free energy of most of the analog s. It suggests that intra- and intermolecular van der Waals interactio ns of delta-CH3, gamma-CH3, and gamma-CH2 of Ile(H59) play the major r ole in the binding affinity. Further search of possible methyl group(s ) which may be incorporated in the side chain of the 59th residue resu lts in the substitution of Ile(H59) with tert-butylalanine, improving the inhibitor affinity 2.1-fold (K-i = 0.39 nM).