title: Hydrophobic contact between the two epidermal growth factor-like domains of blood coagulation factor IX contributes to enzymatic activity.
abstract: The three-dimensional structure of activated factor IX comprises multiple contacts between the two epidermal growth factor (EGF)-like domains. One of these is a salt bridge between Glu(78) and Arg(94), which is essential for binding of factor IXa to its cofactor factor VIII and for factor VIII-dependent factor X activation (Christophe, O. D., Lenting, P. J., Kolkman, J. A., Brownlee, G. G., and Mertens, K. (1998) J. Biol. Chem. 273, 222-227). We now addressed the putative hydrophobic contact at the interface between the EGF-like domains. Recombinant factor IX chimeras were constructed in which hydrophobic regions Phe(75)-Phe(77) and Lys(106)-Val(108) were replaced by the corresponding sites of factor X and factor VII. Activated factor IX/factor X chimeras were indistinguishable from normal factor IXa with respect to factor IXa enzymatic activity. In contrast, factor IXa(75-77)/factor VII displayed approximately 2-fold increased factor X activation in the presence of factor VIII, suggesting that residues 75-77 contribute to cofactor-dependent factor X activation. Activation of factor X by factor IX(106-108)/factor VII was strongly decreased, both in the absence and presence of factor VIII. Activity could be restored by simultaneous substitution of the hydrophobic sites in both EGF-like domains for factor VII residues. These data suggest that factor IXa enzymatic activity requires hydrophobic contact between the two EGF-like domains.
authors.affiliation: Department of Plasma Protein Technology, CLB, 1066 CX Amsterdam, The Netherlands.
authors.forename: P H
authors.initials: PH
authors.lastname: Celie
authors.forename: P J
authors.initials: PJ
authors.lastname: Lenting
authors.forename: K
authors.initials: K
authors.lastname: Mertens
mesh_terms: D003029:Coagulants / Q000737:chemistry / Q000378:metabolism*; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000378:metabolism*; D004815:Epidermal Growth Factor / Q000737:chemistry / Q000235:genetics; D005164:Factor IX / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015944:Factor VIIIa / Q000378:metabolism; D006801:Humans; D009154:Mutation; D010743:Phospholipids; D017434:Protein Structure, Tertiary / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism*
chemical_list: D003029:Coagulants; D004792:Enzyme Precursors; D010743:Phospholipids; D011994:Recombinant Proteins; D004815:Epidermal Growth Factor; D015944:Factor VIIIa; D005164:Factor IX; D012697:Serine Endopeptidases