BINDING OF HIRUDIN TO HUMAN ALPHA-THROMBIN, BETA-THROMBIN AND GAMMA-THROMBIN - A COMPARATIVE KINETIC AND THERMODYNAMIC STUDY

Thermodynamic parameters for the binding of hirudin to human α, β and γ-thrombin have been determined between pH 5.0 and 9.0, and from 10 °C to 40 °C; kinetic data for the association and dissociation of the proteinase-inhibitor complex were obtained at pH 7.5 and 21 °C. These results have been analysed in parallel with the inhibitor-binding properties of human α, β and γ-thrombin for the bovine basic pancreatic trypsin inhibitor (Kunitz-type inhibitor; BPTI). For the purpose of an homogeneous comparison, values of the apparent association equilibrium constant for BPTI binding to human γ-thrombin have been determined between pH 5.0 and 9.0, at 21 °C. The different binding behaviour of hirudin and BPTI with respect to human α, β and γ-thrombin has been related to the inferred stereochemistry of the proteinase-inhibitor contact regions. In particular, whereas the β and γ-loops play an appreciable role in the stabilization of the enzyme-hirudin complexes, they contribute to impairment of the adduct formation for the proteinase/BPTI system. © 1992.