The amidase activity of human alpha-thrombin has been studied at steady state as a function of the concentration of several chloride salts, at a constant ionic strength I = 0.2 M. All kinetic steps of the catalytic mechanism of the enzyme have been solved by studies conducted as a function of relative viscosity of the solution. Among all monovalent cations, Na+ is the most effective in activating thrombin catalysis. This effect is observed with different amide substrates and also with gamma-thrombin, a proteolytic derivative of the native enzyme which has little clotting activity but retains amidase activity toward small synthetic substrates. The specific effects observed as a function of Na+ concentration are indicative of a binding interaction of this monovalent cation with the enzyme. The basis of this interaction has been explored by measurements of substrate hydrolysis collected in a three-dimensional matrix of substrate concentration, relative viscosity, and Na+ concentration, keeping the ionic strength constant with an inert cation such as choline or tetraethylammonium. The data have globally been analyzed in terms of a kinetic linkage scheme where Na+ plays the role of an allosteric effector. The properties of the enzyme change drastically upon binding of Na+, with substrate binding and dissociation, as well as deacylation, occurring on a time scale which is 1 order of magnitude faster. The apparent association constants for Na+ binding to the various intermediate forms of the enzyme have all been resolved from analysis of experimental data and are in the range of 50-100 M-1 at 25-degrees-C. Studies conducted at different temperatures, in the range 15-35-degrees-C, have revealed the enthalpic and entropic components of Na+ binding to the enzyme. The results obtained from steady-state measurements are supported by independent measurements of the intrinsic fluorescence of the enzyme as a function of Na+ concentration at a constant ionic strength I = 0.2 M, over the temperature range 15-35-degrees-C. These measurements are indicative of a drastic conformational change of the enzyme upon Na+ binding to a single site. The energetics of Na+ binding derived from analysis of fluorescence measurements agree very well with those derived independently from steady-state determinations. It is proposed that thrombin exists in two conformations, slow and fast, and that the slow --> fast transition is triggered by binding of a monovalent cation. The high specificity in thrombin activation found in the case of Na+ is the result of its higher affinity compared to all other monovalent cations. The structural domain responsible for Na+ binding controls the energetics of the catalytic pocket of the enzyme in an allosteric fashion and is distinct and seemingly independent from the fibrinogen recognition site.
