Surface-loop residue Lys316 in blood coagulation Factor IX is a major determinant for Factor X but not antithrombin recognition

The active site of activated Factor IX (FIXa) and related blood-coagulation enzymes is surrounded by a number of highly variable surface loops, which contribute to the characteristic substrate specificity of each individual enzyme. FIX residue Lys316 is located in one of these loops and mutation of this residue to Glu is associated with haemophilia B. In the present study we investigated the functional role of Lys316 in human FIXa by analysing the purified and activated FIX mutants FIXa-K316E and FIXa-K316A. FIXa-K316E was indistinguishable from normal FIXa in binding the competitive active-site inhibitor p-aminobenzamidine. In addition, substitution of Glu for Lys316 had no significant effect on the reactivity towards various synthetic tripeptide substrates. Inhibition by the macromolecular inhibitor antithrombin was only slightly reduced for both FIXa mutants (less than 2-fold). In contrast, proteolytic activity of FIXa-K316E towards the natural substrate Factor X (FX) was virtually lacking, while the Lys316 to Ala mutation resulted in a more than 10-fold reduction in FX activation. Thus residue Lys316 plays a key role in FIXa activity towards FX. The requirement for Lys at position 316 for FX activation was also evident in the presence of the cofactor activated Factor VIII, although to a lesser extent than in its absence. These data demonstrate that Lys316 specifically determines the reactivity of FIXa towards its natural substrate FX, but not to synthetic peptide substrates or antithrombin.