P1-Asp is preferred at pH 4.0 over pH 5.5. The inhibition profile suggests that P1-Asp is only tolerated in its protonated form. Mature legumain (15 µM) was incubated for 1 min with YVAD-CMK and AAN-CMK (15 µM each) at pH 4 and pH 5.5 and residual activity was measured. Activity was normalized to the activity of the uninhibited control at the respective pH. Black, uninhibited; grey, inhibited with YVAD-CMK; white, inhibited with AAN-CMK (n = 3, standard deviations indicated).