Molecular recognition of substrates by fIXa seems to be determined by the action of the 99-loop on Tyr99. This is in contrast to other coagulation enzymes where, in general, the chemical nature of residue 99 determines molecular recognition in S2 and S3-S4. This dominant role on substrate interaction suggests that the 99-loop may be rearranged in the physiological fX activation complex of fIXa, fVIIIa, and fX.

No abstract

Coagulation factor IXa (fIXa) plays a central role in the coagulation cascade. Enzymatically, fIXa is characterized by its very low amidolytic activity that is not improved in the presence of cofactor, factor VIIIa (fVIIIa), distinguishing fIXa from all other coagulation factors. Activation of the fIXa-fVIIIa complex requires its macromolecular substrate, factor X (fX). The 99-loop positioned near the active site partly accounts for the poor activity of fIXa because it adopts a conformation that interferes with canonical substrate binding in S2-S4. Here we show that residues Lys-98 and Tyr-99 are critically linked to the amidolytic properties of fIXa. Exchange of Tyr-99 with smaller residues resulted not only in an overall decreased activity but also in impaired binding in S1. Replacement of Lys-98 with smaller and uncharged residues increased activity. Simultaneous mutagenesis of Lys-98, Tyr-177, and Tyr-94 produced an enzyme with 7000-fold increased activity and altered specificity. This triple mutant probably mimics the conformational changes that are physiologically induced by cofactor and substrate binding. It therefore provides a cooperative two-step activation model for fIXa. Tyr-177 locks the 99-loop in an inactive conformation which, in the physiologic complex, is released by cofactor fVIIIa. FX is then able to rearrange the unlocked 99-loop and subsequently binds to the active site cleft.

We examined the in£uence of Ser/Ala190 in the S1 site on P1 substrate selectivity in several serine proteases. The impact of residue 190 on the selectivity was constant, regardless of di¡erences in original selectivity or reactivity. Substrate binding in S1 was optimised in all wild-type enzymes, while the e¡ects on k cat depended on the combination of residue 190 and substrate. Mutagenesis of residue 190 did not a¡ect the S2^S4 sites. Pronounced selectivity for arginine residues was coupled with low enzymatic activity, in particular in recombinant factor IXa. This is due to the dominance of the S1^P1 interaction over substrate binding in the S2^S4 sites. ß 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.