| Abstract
| - Insertions in surface loops bordering the substrate-binding groove have been shown to play amajor role in the interaction of serine proteases with their cognate inhibitors and substrates. In the presentstudy, we investigated the functional role of factor IX insertion loop 256−268, and in particular of residuesAsn264 and Lys265 therein. To this end, the purified and activated mutants des-(N264,K265)-FIX and FIX-K265A were compared to normal factor IXa with regard to a number of functional properties. The catalyticefficiency of des-(N264,K265)-FIXa and FIXa-K265A toward the amide substrate CH3SO2-Leu-Gly-Arg-pNA was 2−3-fold increased relative to that of normal factor IXa. Comparison of the activities ofnormal and mutant factor IXa toward a series of closely related amide substrates indicates that mutationof residues Asn264−Lys265 influences the interactions in the S2-binding site. The mutations in loop 256−268 also increased the susceptibility of factor IXa to antithrombin inhibition by approximately 3-fold.Factor X activation experiments in the absence of factor VIIIa revealed that the catalytic efficiency ofdes-(N264,K265)-FIXa and FIXa-K265A was about 20 times higher than that of normal factor IXa. Inthe presence of factor VIIIa, however, the activity toward factor X was similar to that of normal factorIXa. The reduced sensitivity of the factor IXa mutants to factor VIIIa was neither due to an increase infactor IXa-dependent inactivation of factor VIIIa, nor to a lower affinity for this cofactor. Overall, thesedata demonstrate that loop 256−268 restricts the activity of factor IXa toward both synthetic and naturalsubstrates. Complex formation with factor VIIIa alleviates the inhibitory effect of this insertion loop onthe activation of FX.
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