CHEMICAL COMPENSATION IN MACROMOLECULAR BRIDGE-BINDING TO THROMBIN

The binding energetics of eight synthetic peptides capable of interfering with thrombin function have been studied by steady-state measurements and clotting assays. The synthetic peptides are bifunctional inhibitors consisting of three domains: (i) a fragment of the C-terminus of recombinant hirudin, hir55-65, which binds to the fibrinogen-recognition site of thrombin; (ii) a small active site inhibitor, Ac-(DF)PRP, binding to the catalytic pocket of the enzyme, and (iii) a linker spanning these two portions with variable length and chemical composition. All these synthetic peptides are competitive inhibitors of fibrinogen. On the other hand, a linker of at least 13 carbon atoms is required for full competitive inhibition of the hydrolysis by thrombin of small synthetic substrates, which only bind to the catalytic pocket of the enzyme. The best inhibitory effect is observed with a linker of 13 carbon atoms, with a value of K(I) in the nanomolar range. Studies conducted as a function of temperature, in the range 15-40-degrees-C, have revealed the enthalpic and entropic components of inhibitor binding to thrombin. Chemical compensation is observed for all synthetic peptides that bridge-bind to the fibrinogen-recognition site and the catalytic pocket of the enzyme thereby inhibiting in a competitive fashion either fibrinogen binding or the hydrolysis of small synthetic substrates. The extrathermodynamic relationship between DELTAH and DELTAG also includes the enthalpy and free energy of binding for the natural substrate fibrinogen and the potent natural inhibitor hirudin, measured under identical solution conditions. Preferential binding of hirudin over fibrinogen is an entropy-driven process. The existence of chemical compensation suggests that a unique conformational transition of thrombin takes place upon macromolecular bridge-binding to the fibrinogen-recognition site and the catalytic pocket. This transition may have a bearing on the recently discovered Na+-induced slow --> fast transition of human alpha-thrombin.