Limited proteolysis of maize NADP-malic enzyme

Abstract

The incubation of maize malic enzyme at 37°C with trypsin at a ratio of 150:1 of malic enzyme to trypsin caused rapid and complete in activation of enzyme activity. The inactivation was caused by fairly specific cleavage or the enzyme monomer (62 kDa) into 40 kDa and 20 kDa fragments. The intensity of 40 kDa band increased with the time of treatment of enzyme with trypsin from 2 to 30 min. Substrates, especially NADP (25<i>μM) </i>provided almost total protection against trypsin inactivation of the enzyme activity. The studies carried out with various other endoproteases indicated that endoprotease Lys-C was most effective in inactivating malic enzyme activity. The kinetic properties of the truncated

enzyme have been studied. The <i>K</i>_m value for malate in case of native and modified enzyme was found to be identical. <i>K</i>_m NADP for the modified enzyme was slightly higher indicating that after proteolysis the enzyme affinity for NADP had decreased. Limited proteolysis with trypsin did not show any appreciable change in fluorescence properties of the modified

enzyme. Binding of NADPH to the enzyme was not affected after modification.