The role of Arg46 and Arg47 of antithrombin in heparin binding

Heparin greatly accelerates the reaction between antithrombin and its targe t proteinases, thrombin and factor Xa, by virtue of a specific pentasacchar ide sequence of heparin binding to antithrombin. The binding occurs in two steps, an initial weak interaction inducing a conformational change of anti thrombin that increases the affinity for heparin and activates the inhibito r. Arg46 and Arg47 of antithrombin have been implicated in heparin binding by studies of natural and recombinant variants and by the crystal structure of a pentasaccharide-antithrombin complex. We have mutated these two resid ues to Ala or His to determine their role in the heparin-binding mechanism. The dissociation constants for the binding of both full-length heparin and pentasaccharide to the R46A and R47H variants were increased 3-4-fold and 20-30-fold, respectively, at pH 7.4. Arg46 thus contributes only little to the binding, whereas Arg47 is of appreciable importance. The ionic strength , dependence of the dissociation constant for pentasaccharide binding to th e R47H variant showed that the decrease in affinity was due to the loss of both one charge interaction and nonionic interactions. Rapid-kinetics studi es further revealed that the affinity loss was caused by both a somewhat lo wer forward rate constant and a greater reverse rate constant of the confor mational change step, while the affinity of the initial binding step was un affected. Arg47 is thus not involved in the initial weak binding of heparin to antithrombin but is important for the heparin-induced conformational ch ange. These results are in agreement with a previously proposed model, in w hich an initial low-affinity binding of the nonreducing-end trisaccharide o f the heparin pentasaccharide induces the antithrombin conformational chang e. This change positions Arg47 and other residues for optimal interaction w ith the reducing-end disaccharide, thereby locking the inhibitor in the act ivated state.