The active site region plays a critical role in Na+ binding to thrombin.

Abstract The catalytic activity of thrombin and other enzymes of the blood coagulation and complement cascades is enhanced significantly by binding of Na+ to a site >15 A away from the catalytic residue S195, buried within the 180- and 220- loops that also contribute to the primary specificity of the enzyme. Rapid kinetics support a binding mechanism of conformational selection where the Na+ binding site is in equilibrium between open (N) and closed (N*) forms and the cation binds selectively to the N form. Allosteric transduction of this binding step produces enhanced catalytic activity. Molecular details on how Na+ gains access to this site and communicates allosterically with the active site remain poorly defined. In this study, we show that the rate of the N ∗ → N transition is strongly correlated with the analogous E ∗ → E transition that governs the interaction of synthetic and physiologic substrates with the active site. This correlation supports the active site as the likely point of entry for Na+ to its binding site. Mutagenesis and structural data rule out an alternative path through the pore defined by the 180- and 220- loops. We suggest that the active site communicates allosterically with the Na+ site through a network of H-bonded water molecules that embeds the primary specificity pocket. Perturbation of the mobility of S195 and its H-bonding capabilities alters interaction with this network and influences the kinetics of Na+ binding and allosteric transduction. These findings may have general mechanistic relevance for Na+-activated proteases and allosteric enzymes.