The pH controls the activation and inactivation of legumain. (a) pH-dependent activation of legumain. Wild-type (black) and Δ-(Val18–Asp25) prolegumain (grey) were incubated at different pH values ranging from 7.0 to 4.0 (310 K) for 20 h. Bz-Asn-pNA turnover by the resulting activation intermediates in legumain activity-assay buffer at pH 5.5 was measured after autoprocessing was complete as judged by SDS–PAGE (4 h at pH 4.0; 20 h for all other pH values). White bars: prolegumain activity after incubation with Bz-Asn-pNA at the indicated pH. Prolegumain is inactive from pH 7 to pH 4. Activities are normalized to that of super-activated legumain. (b) Active legumain is irreversibly inactivated when incubated at neutral pH. Super-activated legumain was incubated at pH 4 and pH 7 (30 min, 310 K) and enzymatic activity was measured in legumain reaction buffer at pH 5.5. Neutral pH leads to irreversible inactivation of legumain. Activity values are averaged over three independent measurements and are shown together with the corresponding standard deviations.