| Factor IXa is a critical enzyme to coagulation, yet little is known about how it interacts with its reactants (i.e. substrates, inhibitors). In this study, a novel approach was taken to obviate difficulties in using the fXa activation complex to study flXa activity toward its major substrate fX. Kunitz inhibitors are known to inhibit serine proteases (ex. flXa) with substrate-like binding that mimics active-site interactions with fX. Four native Kunitz-type inhibitors, PN2-KPI, BPTI, TFPI K1 and K2, were constructed, expressed, purified, and their inhibition of flXa was compared. Factor IXa: kunitz inhibitor structural models were assembled to simulate the human flXa: kunitz inhibitor interactions. Comparing the inhibitor affinities for flXa in conjunction with these structural models pointed to a clash between the Lys98-Tyr99 residues of flXa and the 39th (Arg, Ile, or Gly) residue of the kunitz inhibitors. This information will lead toward future mutagenesis experiments between the inhibitors and flXa. |
