Surface loop 199-204 in blood coagulation factor IX is a cofactor-dependent site involved in macromolecular substrate interaction

In factor IX residues 199-204 encompass one of six surface loops bordering its substrate-binding groove, To investigate the contribution of this loop to human factor IX function, a series of chimeric factor IX variants was co nstructed, in which residues 199-204 were replaced by the corresponding seq uence of factor VII, factor X, or prothrombin. The immunopurified and activ ated chimeras were indistinguishable from normal factor Ma in hydrolyzing a small synthetic substrate, indicating that this region is not involved in the interaction with substrate residues on the N-terminal side of the sciss ile bond. In contrast, replacement of loop 199-204 resulted in a 5-25-fold reduction in reactivity toward the macromolecular substrate factor X This r eduction was due to a combination of increased K-m and reduced k(cat). In t he presence of factor VIIIa the impaired reactivity toward factor X was lar gely restored for all factor Ma variants, resulting in a more pronounced st imulation by factor VIIIa compared with normal factor Ma (3 to 5 x 10(4)-fo ld versus 5 x 10(3)-fold). Inhibition by antithrombin was only slightly aff ected for the factor Ma variant with the prothrombin loop sequence, whereas factor Ma variants containing the analogous residues of factor VII or fact or X were virtually insensitive to antithrombin inhibition. In the presence of heparin, however, all chimeric factor Ma variants formed complexes with antithrombin, Thus the cofactors heparin and factor VIIIa have in common t hat they both alleviate the deleterious effects of mutations in the factor IX loop 199-204. Collectively, our data demonstrate that loop 199-204 plays an important role in the interaction of factor Ma with macromolecular subs trates.