Figure 2.

Effect of residue 225 on thrombin interactions. Plotted are the values (in log units) of k_cat/K_m for the hydrolysis of H-D-Phe-Pro-Arg-p-nitroanilide (FPR), fibrinogen, and protein C (in the presence of 5 mM CaCl₂ and 100 nM rabbit thrombomodulin), and the k_on values for inhibition of thrombin by antithrombin III (in the presence of 0.5 United States Pharmacopeia units/ml of heparin). Experimental conditions are: 5 mM Tris, 145 mM NaCl, 0.1% polyethylene glycol, pH 7.4 at 37°C (see legend to Fig. 1 for FPR). Measurements of K_m and k_cat for FPR hydrolysis by several mutants show that the reduced specificity is the result almost entirely of increased K_m and compromised substrate binding. The four profiles are quite similar and suggest that perturbation of residue 225 affects the environment of the primary specificity site to which FPR, fibrinogen, protein C, and antithrombin III make contact.