Insertion loop 256-268 in coagulation factor IX restricts enzymatic activity in the absence but not in the presence of factor VIII

Insertions in surface loops bordering the substrate-binding groove have bee n shown to play a major role in the interaction of serine proteases with th eir cognate inhibitors and substrates. In the present study, we investigate d the functional role of factor IX insertion loop 256-268, and in particula r of residues Asn(264) and Lys(265) therein. To this end, the purified and activated mutants des-(N264,K265)-FIX and FIX-K265A were compared to normal factor Ma with regard to a number of functional properties. The catalytic efficiency of des-(N264,K265)-FIXa and FIXa-K265A toward the amide substrat e CH3SO2-Leu-GlyArg-pNA was 2-3-fold increased relative to that of normal f actor IXa. Comparison of the activities of normal and mutant factor IXa tow ard a series of closely related amide substrates indicates that mutation of residues Asn(264)-Lys(265) influences the interactions in the S2-binding s ite. The mutations in loop 256-268 also increased the susceptibility of fac tor XIa to antithrombin inhibition by approximately S-fold, Factor X activa tion experiments in the absence of factor Vma revealed that the catalytic e fficiency of des-(N264,K265)-FIXa and FIXa-K265A was about 20 times higher than that of normal factor IXa. In the presence of factor VIIIa, however, t he activity toward factor X was similar to that of normal factor IXa. The r educed sensitivity of the factor IXa mutants to factor VIIIa was neither du e to an increase in factor IXa-dependent inactivation of factor VIIIa, nor to a lower affinity for this cofactor. Overall, these data demonstrate that loop 256-268 restricts the activity of factor IXa toward both synthetic an d natural substrates. Complex formation with factor villa alleviates the in hibitory effect of this insertion loop on the activation of FX.