Linkage between proton binding and amidase activity in human alpha-thrombin: effect of ions and temperature.

The amidase activity of human alpha-thrombin has been studied at steady state in the pH range 6-10, as a function of NaCl concentration from 1 mM to 1 M and temperature from 10 to 40 degrees C. The Michaelis-Menten constant, Km, shows a bell-shaped dependence over this pH range with a minimum around pH 7.5 in the presence of 0.1 M NaCl at 25 degrees C. The catalytic constant, kcat, also has a bell-shaped pH dependence with multiple inflection points that are more evident at low NaCl concentrations and a maximum around pH 8.2 in the presence of 0.1 M NaCl at 25 degrees C. A detailed analysis of the results in terms of a general linkage scheme has allowed a thorough characterization of the linkage between proton and substrate binding and its dependence on NaCl concentration, as well as the relevant entropic and enthalpic contributions to binding and catalytic events. Formulation of detailed partition functions for each enzyme intermediate involved in the catalytic cycle suggests that (at least) three groups are responsible for the control of thrombin amidase activity as a function of pH. One group is to be identified with the active site His, due to its pK values in the free enzyme and the adduct and its enthalpy of ionization. The effect of NaCl concentration on amidase activity seems to be extremely specific. Comparative steady-state measurements carried out in the presence of NaCl, NaBr, NaI, KCl, and MgCl2 show that human alpha-thrombin is capable of discriminating among different cations and anions. This suggests that small ions participate as allosteric effectors in the regulation of thrombin activity. The linkage with NaCl is strongly pH dependent and increases with decreasing pH. The present results provide information on the basic aspects of human alpha-thrombin activity and regulation and enable a rigorous thermodynamic approach to other important regulatory interactions in human alpha-thrombin and its structurally perturbed derivatives.