Kinetic pathway for the slow to fast transition of thrombin - Evidence of linked ligand binding at structurally distinct domains

The kinetic pathway for the Na+ -induced slow --> fast transition of thrombin was characterized, The slow form was shown to consist of two conformers in a 3:1 ratio (E-s2:E-s1) at 5 degrees C, pH 7.4, Gamma/2 0.3.E-s2 binds Na+ 3 orders of magnitude faster than does E-s1. The small molecule active site-directed inhibitor L-371,912, and the exosite I binding ligand hirugen, like Na+, bind selectively to E-s2 and induce the slow --> fast conversion of thrombin. The slow --> fast transition is limited by the rate of conversion of E-s1 to E-s2 (k similar to 28 s(-1) at 5 degrees C), Replacement of Arg-221a or Lys-224 at the Na+ binding site with Ala appears to selectively alter the slow form and reduce the apparent affinity of the mutants for Na+ and L-371,912, This replacement, however, has little effect on the affinity for the inhibitor in the presence of saturating concentrations of Na+, The kinetically linked ligand binding at the Na+ binding site, exosite I, and the active site of thrombin characterized in the present study indicates the basis for the plasticity of this important enzyme, and suggests the possibility that the substrate specificity and, therefore, the procoagulant and anticoagulant activities of thrombin may be subject to allosteric regulation by as yet unidentified physiologically important effecters.