Importance of Tryptophan 49 of Antithrombin in Heparin Binding and Conformational Activation†

Bernhard H. Monien, Chandravel Krishnasamy, Steven T. Olson, Umesh R. Desai
2005 Biochemistry  
Tryptophan 49 of antithrombin, the primary inhibitor of blood clotting proteinases, has previously been implicated in binding the allosteric activator, heparin, by chemical modification and mutagenesis studies. However, the X-ray cocrystal structure of the antithrombin-pentasaccharide complex shows that Trp49 does not contact the bound saccharide. Here, we provide a detailed thermodynamic and kinetic characterization of heparin binding to a Trp49 to Lys variant of antithrombin and suggest a
more » ... l for how Trp49 participates in heparin binding and activation. Mutation of Trp49 to Lys resulted in substantial losses of 16-24% in heparin-binding energy at pH 7.4; I, 0.15; and 25°C. These losses were due to both the loss of one ionic interaction (∼30%) and the loss of nonionic interactions (∼70%). Rapid kinetics analyses showed that the mutation minimally affected the initial weak binding of heparin to antithrombin or the rate constant for the subsequent conformational activation of the serpin. Rather, the principal effect of the mutation was to increase the rate constant for reversal of the conformational activation step by 70-100-fold, thereby destabilizing the activated conformation. This destabilization could be accounted for by the disruption of a network of interactions involving Trp49, Glu50, and Lys53 of helix A and Ser112 of helix P, which stabilizes the activated conformation. 1 Abbreviations: AT, antithrombin; BHK, baby hamster kidney; DEFGH, heparin pentasaccharide DEFGH; ∆G°, standard free energy of binding; ∆G°i onic , standard free energy of binding because of ionic interactions only; ∆G°n onionic , standard free energy of binding because of nonionic interactions only; H, heparin; HAH, high-affinity heparin; Kd, equilibrium dissociation constant; Kd,nonionic, equilibrium dissociation constant associated with nonionic interactions only; PEG, poly(ethylene glycol); RCL, reactive center loop; Wt, wild-type.
doi:10.1021/bi050741i pmid:16128566 fatcat:clplgbzxfbfidh7lkmojjgg6u4