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Ligand Binding Shuttles Thrombin along a Continuum of Zymogen- and Proteinase-like States
Authors:Parvathi Kamath  James A. Huntington  Sriram Krishnaswamy
Affiliation:From the Research Institute, Children''s Hospital of Philadelphia, and ;the Department of Pediatrics, University of Pennsylvania, Philadelphia, Pennsylvania 19104 and ;the §Department of Haematology, Cambridge Institute for Medical Research, University of Cambridge, Hills Road, Cambridge CB2 0XY, United Kingdom
Abstract:The critical and multiple roles of thrombin in blood coagulation are regulated by ligands and cofactors. Zymogen activation imparts proteolytic activity to thrombin and also affects the binding of ligands to its two principal exosites. We have used the activation peptide fragment 1.2 (F12), a ligand for anion-binding exosite 2, to probe the zymogenicity of thrombin by isothermal titration calorimetry. We show that F12 binding is sensitive to subtle aspects of proteinase formation beyond simply reporting on zymogen cleavage. Large thermodynamic differences in F12 binding distinguish between a series of thrombin species poised along the transition of zymogen to proteinase. Active-site ligands transitioned a zymogen-like state to a proteinase-like state. Conversely, removal of Na+ converted proteinase-like thrombin to a more zymogen-like form. Thrombin mutants, with deformed x-ray structures, previously considered to be emblematic of specific regulated states of the enzyme, are instead shown to be variously zymogen-like and can be made proteinase-like by active-site ligation. Thermodynamic linkage between anion-binding exosite 2, the Na+-binding site, and the active site arises from interconversions of thrombin between a continuum of zymogen- and proteinase-like states. These interconversions, reciprocally regulated by different ligands, cast new light on the problem of thrombin allostery and provide a thermodynamic framework to explain the regulation of thrombin by different ligands.
Keywords:Allosteric Regulation   Blood Coagulation Factors   Serine Protease   Thermodynamics   Thrombin
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