Lysine 114 of antithrombin is of crucial importance for the affinity and kinetics of heparin pentasaccharide binding.

Lys(114) of the plasma coagulation proteinase inhibitor, antithrombin, has been implicated in binding of the glycosaminoglycan activator, heparin, by previous mutagenesis studies and by the crystal structure of antithrombin in complex with the active pentasaccharide unit of heparin. In the present work, substitution of Lys(114) by Ala or Met was shown to decrease the affinity of antithrombin for heparin and the pentasaccharide by approximately 10(5)-fold at I 0.15, corresponding to a reduction in binding energy of approximately 50%. The decrease in affinity was due to the loss of two to three ionic interactions, consistent with Lys(114) and at least one other basic residue of the inhibitor binding cooperatively to heparin, as well as to substantial nonionic interactions. The mutation minimally affected the initial, weak binding of the two-step mechanism of pentasaccharide binding to antithrombin but appreciably (>40-fold) decreased the forward rate constant of the conformational change in the second step and greatly (>1000-fold) increased the reverse rate constant of this step. Lys(114) is thus of greater importance for the affinity of heparin binding than any of the other antithrombin residues investigated so far, viz. Arg(47), Lys(125), and Arg(129). It contributes more than Arg(47) and Arg(129) to increasing the rate of induction of the activating conformational change, a role presumably exerted by interactions with the nonreducing end trisaccharide unit of the heparin pentasaccharide. However, its major effect, also larger than that of these two residues, is in maintaining antithrombin in the activated state by interactions that most likely involve the reducing end disaccharide unit.