The active site of factor IXa is located far above the membrane surface and its conformation is altered upon association with factor VIIIa. A fluorescence study

• Published in: The Journal of biological chemistry 1992-08, Vol.267 (24), p.17012-17021
• Main Authors: MUTUCUMARANA, V. P, DUFFY, E. J, LOLLAR, P, JOHNSON, A. E
• Format: Article
• Language: English
• Subjects: Animals; association; Binding Sites; Biological and medical sciences; blood coagulation; Blood coagulation. Blood cells; Calcium - pharmacology; Cattle; Cell Membrane - metabolism; changes; coagulation factor IXa; coagulation factor VIIIa; Coagulation factors; conformation; Factor IXa - chemistry; Factor IXa - metabolism; Factor VIIIa - chemistry; Factor VIIIa - metabolism; Fluorescent Dyes; Fundamental and applied biological sciences. Psychology; induction; Kinetics; Liposomes; Mathematics; Molecular and cellular biology; Phosphatidylcholines - pharmacology; Phosphatidylserines - pharmacology; Protein Conformation; Spectrometry, Fluorescence; Swine
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)41886-8

The topography of membrane-bound blood coagulation factor IXa (fIXa) and the nature of its interaction with its cofactor, factor VIIIa (fVIIIa), were examined using fluorescent derivatives of fIXa. A fluorescein dye was covalently attached to the active-site histidine of fIXa via a D-Phe-Pro-Arg tripeptide tether to form Fl-A-FPR-fIXa; similarly, a 5-dimethylaminonaphthalene-1-sulfonyl (dansyl) dye was covalently attached via Glu-Gly-Arg to form DEGR-fIXa. When either Fl-A-FPR-fIXa or DEGR-fIXa was titrated with phosphatidylcholine-phosphatidylserine vesicles containing octadecylrhodamine in the presence of Ca2+, fluorescence energy transfer was observed. Assuming a random orientation of dyes, the distance of closest approach between the donor dyes in the active sites of the membrane-bound enzymes and the acceptor dyes at the membrane surface was found to be 89 +/- 3 A for Fl-A-FPR-fIXa and 73 +/- 4 A for DEGR-fIXa. Although the exact distance remains uncertain, it is clear that the active site of fIXa is positioned more than 70 A above the surface, and hence that the elongated fIXa molecule projects approximately perpendicularly from the surface when bound to the membrane. The binding of fVIIIa to membrane-bound Fl-A-FPR-fIXa or DEGR-fIXa did not alter the location of the active site relative to the membrane surface, but did alter both the emission intensity and anisotropy of the fluorescein and dansyl probes and hence their environments. Cofactor stimulation of fIXa activity therefore appears to be mediated, at least in part, by a conformational change in the active site that occurs when fVIIIa binds to the enzyme on the phospholipid surface.