Thrombin-thrombomodulin interaction: energetics and potential role of water as an allosteric effector.
The interaction of rabbit lung thrombomodulin (TM) and C-terminal hirudin 54-65 fragment (Hir54-65) with human alpha-thrombin were investigated by exploiting their competitive inhibition of thrombin-fibrinogen interaction. Measurements of Ki values for TM and Hir54-65 interactions with human alpha-thrombin performed over a temperature range spanning from 10 to 40 degrees C showed a constant enthalpy for both ligands. The enthalpic and entropic contributions to the free energy of binding, however, are different for TM and the hirudin peptide. The calculated values of delta H and delta S, in fact, were -47.3 +/- 2.51 kJ (-11.3 +/- 0.6 kcal)/mol and -42.7 +/- 7.9 J (-10.2 +/- 1.9 cal)/mol.K for the hirudin peptide, while being -22.9 +/- 2.09 kJ (-5.47 +/- 0.5 kcal)/mol and 102.50 +/- 6.69 J (24.5 +/- 1.6 cal)/mol.K respectively for TM binding. These findings indicate that the interaction between thrombin and Hir54-65 is largely driven by the enthalpic contribution, whereas the positive entropy change is the driving force for the formation of the thrombin-TM complex. In other experiments performed in the presence of various concentrations of either sorbitol or sucrose it could be demonstrated that the value of the equilibrium association constant for thrombin-TM interaction increases as a function of the osmotic pressure, while the thrombin-Hir54-65 interaction was not affected by the same conditions. Moreover, control experiments showed that no major conformational changes are produced on TM by osmotic pressures used in the present study. From these experiments it was calculated that roughly 35 water molecules are released into the bulk water upon TM binding. Such a phenomenon, which is likely to be responsible for the entropic change described above, indicates the relevance of hydration processes for the formation of the thrombin-TM adduct.
De Cristofaro
R
R
Department of Internal Medicine, Catholic University, Rome, Italy.
Picozzi
M
M
De Candia
E
E
Rocca
B
B
Landolfi
R
R
D000494:Allosteric Regulation; D000818:Animals; D004734:Energy Metabolism; D006629:Hirudins / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D009997:Osmotic Pressure; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011486:Protein C / Q000378:metabolism; D011817:Rabbits; D013816:Thermodynamics; D013917:Thrombin / Q000737:chemistry / Q000378:metabolism*; D018180:Thrombomodulin / Q000737:chemistry / Q000378:metabolism*
D006629:Hirudins; D010446:Peptide Fragments; D011486:Protein C; D018180:Thrombomodulin; C091596:hirudin (54-65); D013917:Thrombin