The distinct roles that Gln-192 and Glu-217 of factor IX play in selectivity for macromolecular substrates and inhibitors

In this paper, we report functional characterization of positions 192 and 2 17 (chymotrypsinogen numbering system) in human factor IX and discuss the d istinction and similarity of these two sites among the blood coagulation fa ctors. Recombinant factor IXQ192E (residue glutamine at position 192 replac ed by glutamic acid), IXQ192K, IXE217D, and IXE217R proteins exhibited 11%, 46%, 39%, and 2% of the wild-type factor IX's clotting activity, respectiv ely. Binding of these variants to factor VIIIa (FVIIIa) was inefficient com pared to that of wild-type factor IX, and the dissociation constants double d for IXQ192E, 3-fold higher for IXQ192K and 4-fold higher for both IXE217D and IXE217R. In the presence of FVIIIa, all variant factor IX hydrolyzed f actor X at the catalytic efficiencies correlating with respective clotting activities. However, FVIIIa greatly enhanced the catalytic efficiency of bo th IXE217 variants to a greater extent (similar to7 x 10(4)-fold) as compar ed to its effect on the wild-type factor IXa and the other two IXQ192 varia nts [by a factor of (1-2) x 10(4)]. Moreover, while both IXQ192 variants de monstrated small substrate selectivity similar to that of wild-type factor IXa, the selectivity of both IXE217 variants was greatly altered. Mutations at position 192 disturbed the interaction of factor IXa with physiological inhibitors. Although all variants formed an SDS-stable complex with antith rombin III (ATIII) equally well in the presence of heparin and were readily inhibited by ATIII in the absence of heparin, activated IXQ192K exhibited a slower stable complex formation with ATIII without heparin. On the other hand, only IXQ192E showed decreased interaction with TFPI. Our results demo nstrate that positions 192 and 217 play different roles unique to factor IX in specifying the interaction of factor IX with substrates and inhibitors.