Interaction of site specific hirudin variants with α-thrombin

The kinetics of complex formation between recombinant hirudin or recombinant hirudin mutants with thrombin were analyzed. In order to elucidate the inhibitor's reactive site peptide bond predetermined amino acid substitutions were introduced at positions of basic amino acid residues by means of...

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Bibliographic Details
Published in:FEBS letters 1988-02, Vol.229 (1), p.87-90
Main Authors: Dodt, Johannes, Köhler, Stefanie, Baici, Antonio
Format: Article
Language:English
Subjects:
4-toluoslsulfonyl
Algorithms
alpha -thrombin
Animals
Binding Sites
Biological and medical sciences
Blood coagulation. Blood cells
Cattle
Fundamental and applied biological sciences. Psychology
General aspects, investigation methods, hemostasis, fibrinolysis
high-performance liquid chromatography
Hirudin
Hirudin variant
Hirudins - metabolism
HPLC
IPTG
isopropyl-β-D-thiogalactopyranoside
Kinetics
Molecular and cellular biology
Mutation
Putative reactive site
Recombinant Proteins - metabolism
reversed-phase
Site-directed mutagenesis
Thrombin - metabolism
Tos
α-Thrombin
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Summary:The kinetics of complex formation between recombinant hirudin or recombinant hirudin mutants with thrombin were analyzed. In order to elucidate the inhibitor's reactive site peptide bond predetermined amino acid substitutions were introduced at positions of basic amino acid residues by means of site-directed mutagenesis of a hirudin gene. In comparison to recombinant hirudin ( K i = 19 pM) only those mutant inhibitors which were modified at amino acid position Lys 47 showed a higher K i value for their complexes with thrombin. The observed effects are mainly due to increased k off rate constants.
ISSN:0014-5793
1873-3468
DOI:10.1016/0014-5793(88)80803-2