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Physiological fIXa activation involves a cooperative conformational rearrangement of the 99-loop
Katrin Sichler1, Erhard Kopetzki, Robert Huber
1Max-Planck-Institut für Biochemie, D-82152 Martinsried, Germany. Katrin.Sichler@roche.com
The Journal of Biological Chemistry
|November 22, 2002
Summary
Coagulation factor IXa (fIXa) has low activity due to its 99-loop. Mutating specific residues, particularly Lys-98 and Tyr-99, significantly enhances fIXa activity and alters specificity, revealing a new activation model.
Area of Science:
- Biochemistry
- Molecular Biology
- Hematology
Background:
- Coagulation factor IXa (fIXa) is crucial for blood clotting but exhibits very low enzymatic activity.
- This low activity is partly attributed to the 99-loop near the active site, which hinders substrate binding.
- Unlike other coagulation factors, fIXa's activity is not significantly enhanced by its cofactor, factor VIIIa (fVIIIa).
Purpose of the Study:
- To investigate the role of specific residues, Lys-98 and Tyr-99, in the amidolytic activity of fIXa.
- To understand how mutations affect fIXa's interaction with its substrate, factor X (fX).
- To elucidate the conformational changes involved in fIXa activation by its cofactor and substrate.
Main Methods:
- Site-directed mutagenesis of fIXa, focusing on residues Lys-98, Tyr-99, Tyr-177, and Tyr-94.
- Assessing the amidolytic activity and substrate binding properties of wild-type and mutant fIXa enzymes.
- Developing a cooperative two-step activation model based on mutant enzyme characteristics.
Main Results:
- Mutating Tyr-99 to smaller residues decreased overall activity and impaired S1 binding.
- Replacing Lys-98 with smaller, uncharged residues increased fIXa activity.
- A triple mutant (Lys-98, Tyr-177, Tyr-94) exhibited a 7000-fold increase in activity and altered specificity, mimicking physiological activation.
Conclusions:
- Residues Lys-98 and Tyr-99 are critical determinants of fIXa's amidolytic properties.
- The triple mutant provides insights into the conformational dynamics of fIXa activation.
- A cooperative two-step activation model is proposed, involving cofactor-mediated release of the 99-loop inhibition followed by substrate-induced rearrangement.