The remarkably high specificity of the coagulation proteases towards macromolecular substrates is provided by numerous interactions involving the catalytic groove and remote exosites. For FVIIa [activated FVII (Factor VII)], the principal initiator of coagulation via the extrinsic pathway, several exosites have been identified, whereas only little is known about the specificity dictated by the active-site architecture. In the present study, we have profiled the primary P4–P1 substrate specificity of FVIIa using positional scanning substrate combinatorial libraries and evaluated the role of the selective active site in defining specificity. Being a trypsin-like serine protease, FVIIa had P1 specificity exclusively towards arginine and lysine residues. In the S2 pocket, threonine, leucine, phenylalanine and valine residues were the most preferred amino acids. Both S3 and S4 appeared to be rather promiscuous, however, with some preference for aromatic amino acids at both positions. Interestingly, a significant degree of interdependence between the S3 and S4 was observed and, as a consequence, the optimal substrate for FVIIa could not be derived directly from a subsite-directed specificity screen. To evaluate the role of the active-site residues in defining specificity, a series of mutants of FVIIa were prepared at position 239 (position 99 in chymotrypsin), which is considered to be one of the most important residues for determining P2 specificity of the trypsin family members. This was confirmed for FVIIa by marked changes in primary substrate specificity and decreased rates of antithrombin III inhibition. Interestingly, these changes do not necessarily coincide with an altered ability to activate Factor X, demonstrating that inhibitor and macromolecular substrate selectivity may be engineered separately.
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August 2007
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Research Article|
July 13 2007
Engineering the substrate and inhibitor specificities of human coagulation Factor VIIa
Katrine S. Larsen;
Katrine S. Larsen
1
*Haemostasis Biochemistry, Novo Nordisk A/S, Novo Nordisk Park, DK-2760 Måløv, Denmark
1To whom correspondence should be addressed (email [email protected]).
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Henrik Østergaard;
Henrik Østergaard
*Haemostasis Biochemistry, Novo Nordisk A/S, Novo Nordisk Park, DK-2760 Måløv, Denmark
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Jais R. Bjelke;
Jais R. Bjelke
†Protein Structure and Biophysics, Novo Nordisk A/S, Novo Nordisk Park, DK-2760 Måløv, Denmark
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Ole H. Olsen;
Ole H. Olsen
*Haemostasis Biochemistry, Novo Nordisk A/S, Novo Nordisk Park, DK-2760 Måløv, Denmark
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Hanne B. Rasmussen;
Hanne B. Rasmussen
†Protein Structure and Biophysics, Novo Nordisk A/S, Novo Nordisk Park, DK-2760 Måløv, Denmark
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Leif Christensen;
Leif Christensen
‡Biopharmaceutical Protein and Peptide Chemistry, Novo Nordisk A/S, Novo Nordisk Park, DK-2760 Måløv, Denmark
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Birthe B. Kragelund;
Birthe B. Kragelund
§Structural Biology and NMR Laboratory, Institute of Molecular Biology and Physiology, University of Copenhagen, DK-1353 Copenhagen K, Denmark
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Henning R. Stennicke
Henning R. Stennicke
*Haemostasis Biochemistry, Novo Nordisk A/S, Novo Nordisk Park, DK-2760 Måløv, Denmark
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Publisher: Portland Press Ltd
Received:
December 20 2006
Revision Received:
April 24 2007
Accepted:
April 25 2007
Accepted Manuscript online:
April 25 2007
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© 2007 Biochemical Society
2007
Biochem J (2007) 405 (3): 429–438.
Article history
Received:
December 20 2006
Revision Received:
April 24 2007
Accepted:
April 25 2007
Accepted Manuscript online:
April 25 2007
Citation
Katrine S. Larsen, Henrik Østergaard, Jais R. Bjelke, Ole H. Olsen, Hanne B. Rasmussen, Leif Christensen, Birthe B. Kragelund, Henning R. Stennicke; Engineering the substrate and inhibitor specificities of human coagulation Factor VIIa. Biochem J 1 August 2007; 405 (3): 429–438. doi: https://doi.org/10.1042/BJ20061901
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