Binding interactions for Ac-DNLD-CHO and Ac-DEVD-CHO on the active site of caspase-3. Nitrogen, oxygen, and carbon atoms of the inhibitors are illustrated in blue, red, and green, respectively. Hydrogen bonds are shown as dashed lines. Hydrophobic interactions are shown as thick broken lines schematically. A, The binding mode of Ac-DNLD-CHO was obtained from docking simulations. B, The binding mode of Ac-DEVD-CHO was obtained from the X-ray crystal structure (1PAU). C, The time courses of liberation of fluorescence (MCA) from Ac-DNLD-MCA catalyzed by wild-type caspase-3 (●) and substituted (S209A) caspase-3 (▲). D, The time courses of liberation of fluorescence (MCA) from Ac-DEVD-MCA catalyzed by wild-type caspase-3 (○) and substituted (S209A) caspase-3 (△). The cleavage assays were performed as described in ''Methods''. Data indicate the mean of three independent experiments. E, Amounts of wild-type (lane 2) and substituted (S209A) (lane 3) active caspase-3 proteins generated by coexpression of HA-p17 and HA-p12 subunits in in vitro translation system were analyzed by Western blotting as described under ''Methods''. In this experiment, empty vector was used as control (lane 1).