EXPRESSION AND CHARACTERIZATION OF HUMAN FACTOR-IX - FACTOR-IXTHR-397 AND FACTOR-IXVAL-397

Factor IX(Long Beach) has a single amino acid substitution at 397 (Ile to Thr) in the catalytic domain which results in severe hemophilia B. Recent investigations have shown that the substitution of threonine for isoleucine at 397 may affect a part of the macromolecular substrate binding site. Because threonine has a hydroxyl group in its side chain, it is possible that this hydroxyl group makes new hydrogen bonds and disturbs the substrate binding site. We used three techniques: molecular biology, which includes site-directed mutagenesis and recombinant protein expression in tissue culture; computer-aided kinetic data analysis; and molecular modeling to study this mutation site. We have produced two mutant factor IX molecules that have isoleucine 397 replaced by valine or threonine. Factor IX(wild type) and the two mutants (factor IX(Val) and factor IX(Thr) were expressed in human kidney cells and purified using a conformation-specific monoclonal antibody column. After the activation by factor XIa, these three molecules were able to bind p-aminobenzamidine and increase its fluorescence intensity in a similar manner. Factor IX(Val) and factor IX(wild type) had indistinguishable activities in an activated partial thromboplastin time (aPTT) assay and similar kinetic parameters with factor X as a substrate. Factor IX(Thr) had only 5% clotting activity compared with normal factor IX, a slightly lower K(m) and significantly reduced k(cat), using factor X as a substrate. We developed energy-refined (AMBER v.3.1) computer models of the three factor IX molecules based on previous work. Three factor IXa models (Ile, Val, or Thr at 397) with a fragment of the factor X activation site were used to predict the effect of the mutation at 397 and evaluate the significance of the new hydrogen bond thought to form between the side chain hydroxyl group of threonine 397 and the carbonyl oxygen of tryptophan 385. This new hydrogen bond would affect the position of an amide proton of adjacent glycine 386 which has been proposed to make a hydrogen bond with a backbone carbonyl oxygen of the P3 residue of factor X. In addition to the new hydrogen bond, there is significant movement in the side chain of tryptophan 385 between the factor IXa(wild type)-factor X model and the factor IXa(Thr)-factor X model that could interfere with substrate binding. This movement could be caused by the change in the molecular volume, the orientation of the side chain at 397, and the new hydrogen bond.