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• W2000694401 endingPage "8270" @default.
• W2000694401 startingPage "8261" @default.
• W2000694401 abstract "The binding of thrombomodulin (TM) to exosite-1 and the binding of Na+ to 225-loop allosterically modulate the catalytic activity and substrate specificity of thrombin. To determine whether the conformation of these two cofactor-binding loops are energetically linked to each other and to the active site, we rationally designed two thrombin mutants in which either the 70−80 loop of exosite-1 or the 225-loop of the Na+-binding site was stabilized by an engineered disulfide bond. This was possible by replacing two residues, Arg-67 and Ile-82, in the first mutant and two residues, Glu-217 and Lys-224, in the second mutant with Cys residues. These mutants were expressed in mammalian cells as monomeric molecules, purified to homogeneity and characterized with respect to their ability to bind TM and Na+ by kinetic and direct binding approaches. The Cys-67/Cys-82 mutant did not bind TM and exhibited a normal amidolytic activity, however, the activity of Cys-217/Cys-224 was dramatically impaired, though TM interacted with this mutant with >20-fold elevated KD to partially restore its activity. Both mutants exhibited ∼2−3-fold higher KD for interaction with Na+, and neither mutant clotted fibrinogen or activated protein C in the presence of TM. Both mutants interacted with heparin with a normal affinity. These results suggest that, while exosite-2 of thrombin is an independent cofactor binding-site, both Na+-binding and exosite-1 are energetically linked. Further studies with the fluorescein labeled Cys-195 mutant of thrombin revealed that the catalytic residue of thrombin is modulated by Na+, but TM has no effect on the conformation of this residue." @default.
• W2000694401 created "2016-06-24" @default.
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• W2000694401 date "2009-08-06" @default.
• W2000694401 modified "2023-09-27" @default.
• W2000694401 title "Mutagenesis Studies toward Understanding Allostery in Thrombin" @default.
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• W2000694401 doi "https://doi.org/10.1021/bi900921t" @default.
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