title: Effect of sodium on the energetics of thrombin-thrombomodulin interaction and its relevance for protein C hydrolysis.
abstract: Measurements of the apparent affinity constant for thrombomodulin (TM) binding to human alpha-thrombin as a function of both NA+ and temperature at constant ionic strength (0.15 M) showed that TM affinity increases in the presence of Na+ and vice versa. Moreover, this experimental strategy allowed us to accurately split the free energy of sodium binding into its entropic and enthalpic components for both the TM-free and TM-bound enzyme. Namely, at 25 degrees C, the value of delta G of sodium binding was found equal to -2.4 kcal/mol in the absence of TM and -3.6 kcal/mol for the thrombin-TM complex. The enthalpic contribution to the free energy of sodium binding is equal to -27 kcal/mol and -21 kcal/mol in the TM-free and TM-bound thrombin forms, respectively. Finally, the entropy change for sodium binding was also affected by TM, being equal to -83 cal/(mol deg) and -58 cal/(mol deg) in TM-free and TM-bound thrombin species, respectively. Moreover, the thermodynamic parameters for TM binding to Na+-free thrombin species were solved. TM binding is characterized by an enthalpy and entropy change equal to -10 kcal/mol and 2 cal/(mol deg), respectively, for Na+-free thrombin. It is well known that Na+ binding to thrombin causes conformational transitions and functional activation of the enzyme molecule. The finding that binding of thrombomodulin enhances thrombin affinity for sodium and vice versa raises the question as to whether the change of Na+ ligation induced by TM binding could contribute to the change in thrombin specificity for the hydrolysis of Protein C. Therefore, the effect of sodium binding to thrombin on the hydrolysis of human Protein C was extensively investigated. At both 25 and 37 degrees C the value of kcat/Km for Protein C hydrolysis by thrombin in the absence of TM was found to be enhanced by Na+ over a concentration ranging from 0 to 150 mM. Application of thermodynamic principles demonstrated that the Na+-thrombomodulin linkage contributes, under physiological conditions of sodium activity and temperature, to reduce significantly the transition-state stabilization free energy for Protein C hydrolysis.
authors.affiliation: Department of Internal Medicine, Catholic University, Rome, Italy.
authors.forename: R
authors.initials: R
authors.lastname: De Cristofaro
authors.forename: M
authors.initials: M
authors.lastname: Picozzi
authors.forename: R
authors.initials: R
authors.lastname: Morosetti
authors.forename: R
authors.initials: R
authors.lastname: Landolfi
mesh_terms: D000595:Amino Acid Sequence; D004789:Enzyme Activation; D006801:Humans; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009994:Osmolar Concentration; D011485:Protein Binding; D011486:Protein C / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism / Q000494:pharmacology*; D013816:Thermodynamics; D013917:Thrombin / Q000378:metabolism*; D018180:Thrombomodulin / Q000378:metabolism*
chemical_list: D011486:Protein C; D018180:Thrombomodulin; D012964:Sodium; D013917:Thrombin