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PDBsum entry 2puq
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Hydrolase/hydrolase inhibitor
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PDB id
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2puq
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Contents |
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94 a.a.
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254 a.a.
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190 a.a.
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References listed in PDB file
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Key reference
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Title
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Engineering the substrate and inhibitor specificities of human coagulation factor viia.
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Authors
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K.S.Larsen,
H.Østergaard,
J.R.Bjelke,
O.H.Olsen,
H.B.Rasmussen,
L.Christensen,
B.B.Kragelund,
H.R.Stennicke.
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Ref.
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Biochem J, 2007,
405,
429-438.
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PubMed id
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Abstract
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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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