Conformational transitions linked to active site ligation in human thrombin: effect on the interaction with fibrinogen and the cleavable platelet receptor.
An experimental strategy based on solution viscosity perturbation allowed us to study the energetics of amide-substrates, p-aminobenzamidine (p-ABZ) and proflavin binding to the catalytic site of two proteolyzed forms of alpha-thrombin, i.e. zeta- and gamma T-thrombin. These thrombin derivatives are cleaved at the Leu144-Gly150 loop and at the fibrinogen recognition exosite (FRS), respectively. A phenomenological analysis of thermodynamic data showed that the amide substrates and p-ABZ interactions with zeta-thrombin were respectively, associated with a chemical compensation (i.e. the linear relationship between entropy and enthalpy of binding) and a hydrophobic phenomenon (i.e. a change in the standard heat capacity). The latter was slightly lower than that previously observed for a alpha-thrombin (0.78 +/- 0.25 versus 1.01 +/- 0.17 kcal/mol K). Both phenomenon were absent in gamma T-thrombin. The interaction of a alpha-, zeta- and gamma T-thrombin with macromolecular substrates that "bridge-bind" to both the catalytic site (CS) and fibrinogen recognition exosite (FRS), such as fibrinogen and the cleavable platelet receptor (CPR), was also evaluated. These interactions were studied by following fibrinopeptide A (FpA) release and by measuring intraplatelet Ca2+ changes induced by thrombin-CPR interaction. It was found that the free energy of activation (RT ln Kcat/Km) for both fibrinogen and CPR hydrolysis followed the same hierarchy, i.e. alpha > zeta > gamma. Moreover, the values of delta Cp for alpha-, zeta- and gamma T-thrombin interaction with p-ABZ were found to be linearly correlated to the free energy of activation for both fibrinogen and CPR cleavage. In conclusion, these data demonstrate that: (1) the Leu144-Gly150 loop and the FRS are both involved in the conformational transition linked to the binding of p-aminobenzamidine to the thrombin active site; (2) the extent of thrombin's capacity to undergo conformational transitions in alpha-, zeta- and gamma T forms is positively correlated to the free energy of activation for hydrolysis of macromolecular substrates interacting with both the catalytic domain and the FRS.
Department of Internal Medicine, Catholic University, Rome, Italy.
R
R
De Cristofaro
E
E
De Candia
M
M
Picozzi
R
R
Landolfi
D000595:Amino Acid Sequence; D001550:Benzamidines / Q000378:metabolism; D001665:Binding Sites; D001792:Blood Platelets / Q000378:metabolism*; D005340:Fibrinogen / Q000378:metabolism*; D006629:Hirudins / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011956:Receptors, Cell Surface / Q000378:metabolism*; D013696:Temperature; D013816:Thermodynamics; D013917:Thrombin / Q000737:chemistry / Q000378:metabolism*
D001550:Benzamidines; D006629:Hirudins; D011956:Receptors, Cell Surface; C006500:4-aminobenzamidine; D005340:Fibrinogen; D013917:Thrombin