Figure 2

Rapid kinetics of PPACK binding to the thrombin mutants W215A (closed circles), E192A (mixed circles), E217A (open circles) and W215A/E217A (closed triangles). Binding to W215A and E192A obeys a saturable relaxation that increase hyperbolically with PPACK, as seen for wild-type (Fig. 1A). Binding to E217A and W217A/E217A obeys a simple straight line consistent with a lock-and-key mechanism of interaction. Values for W215A/E217A were increased 100-fold to enable comparison in the plot. Continuous lines were drawn according to Eq. 2 in the text (W215A and E192A) or with the lock-and-key expression^16,49 α = k_off + k_on[L] (E217A and W215A/E217A) with best-fit parameter values listed in Table 1. Experimental conditions are: 400 mM ChCl, 50 mM Tris, 0.1% PEG8000, pH 8.0, at 15 °C. Experimental errors are 5% or less.