Molecular recognition by thrombin. Role of the slow-->fast transition, site-specific ion binding energetics and thermodynamic mapping of structural components.

The interaction of thrombin with the potent natural inhibitor hirudin is controlled in a complex fashion by the binding of Na+ and Cl- to the enzyme and allosteric transitions. Binding of hirudin is positively linked to Na+ binding, but is opposed in a competitive fashion by the binding of Cl-. Since Na+ binding induces the slow-->fast transition of thrombin, it follows from linkage principles that hirudin binds to the fast form with higher affinity. Hence, the slow-->fast transition is a key component of molecular recognition of hirudin by thrombin. We propose a three-step mechanism for molecular recognition of hirudin by thrombin, which is also relevant for recognition of fibrinogen and possibly the platelet receptor and thrombomodulin. First, the C-terminal acidic tail of hirudin binds to the fibrinogen recognition site of thrombin displacing one Cl ion from the thrombin surface. Then, the enzyme undergoes a conformational transition that gives rise to increased accessibility of the catalytic pocket to small synthetic substrates through movement of the Trp148 loop. The changes in the catalytic moiety triggered allosterically by binding to the fibrinogen recognition site are linked to the uptake of Na+ and are similar to, if not identical with, those observed in the Na(+)-induced slow-->fast transition. Finally, the compact N-terminal domain is accommodated in the region surrounding the catalytic pocket. Hirudin binding is also used as a probe of site-specific ion-binding interactions of Na+ and Cl- with the enzyme, characterized by cooperativity between the Na+ and Cl- binding domains. The structural components directly involved or linked to Na+ and Cl- binding have been explored in terms of free energy perturbations of the binding of hirudin and a number of ligands. The fibrinogen recognition site stores most of the free energy of coupling with Cl- binding, while regions surrounding the access to the catalytic pocket provide most of the free energy of coupling with Na+ binding and the slow-->fast transition of thrombin. It is concluded that thrombin is an allosteric enzyme that exists in two forms, slow and fast, and that the allosteric transition slow-->fast represents the key element of molecular recognition of important physiological substrates. The allosteric nature of the enzyme also enables different structural domains to communicate and give rise to biological function.