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Physiological fIXa activation involves a cooperative conformational rearrangement of the 99-loop

Sichler, Katrin, Kopetzki, Erhard, Huber, Robert, Bode, Wolfram, Hopfner, Karl-Peter and Brandstetter, Hans 2002. Physiological fIXa activation involves a cooperative conformational rearrangement of the 99-loop. Journal of Biological Chemistry 278 (6) , pp. 4121-4125. 10.1074/jbc.M210722200

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Coagulation factor IXa (fIXa) plays a central role in the coagulation cascade. Enzymatically, fIXa is characterized by its very low amidolytic activity that is not improved in the presence of cofactor, factor VIIIa (fVIIIa), distinguishing fIXa from all other coagulation factors. Activation of the fIXa-fVIIIa complex requires its macromolecular substrate, factor X (fX). The 99-loop positioned near the active site partly accounts for the poor activity of fIXa because it adopts a conformation that interferes with canonical substrate binding in S2-S4. Here we show that residues Lys-98 and Tyr-99 are critically linked to the amidolytic properties of fIXa. Exchange of Tyr-99 with smaller residues resulted not only in an overall decreased activity but also in impaired binding in S1. Replacement of Lys-98 with smaller and uncharged residues increased activity. Simultaneous mutagenesis of Lys-98, Tyr-177, and Tyr-94 produced an enzyme with 7000-fold increased activity and altered specificity. This triple mutant probably mimics the conformational changes that are physiologically induced by cofactor and substrate binding. It therefore provides a cooperative two-step activation model for fIXa. Tyr-177 locks the 99-loop in an inactive conformation which, in the physiologic complex, is released by cofactor fVIIIa. FX is then able to rearrange the unlocked 99-loop and subsequently binds to the active site cleft.

Item Type: Article
Date Type: Publication
Status: Published
Schools: Biosciences
Subjects: Q Science > QD Chemistry
Q Science > QH Natural history > QH301 Biology
Publisher: American Society for Biochemistry and Molecular Biology
ISSN: 0021-9258
Last Modified: 24 Jun 2017 11:00

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