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Updated: May 19, 2026
High-throughput, Microscale Protocol for the Analysis of Processing Parameters and Nutritional Qualities in Maize (Zea mays L.)
Published on: June 16, 2018
Limited proteolysis of maize NADP-malic enzyme
S Pinto1, S R Rao, A S Bhagwat
1Molecular Biology and Agricultural Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400 085, India.
Abstract:
The incubation of maize malic enzyme at 37 degrees C with trypsin at a ratio of 150:1 of malic enzyme to trypsin caused rapid and complete inactivation of enzyme activity. The inactivation was caused by fairly specific cleavage of 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 microM) 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 Km value for malate in case of native and modified enzyme was found to be identical. Km 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.
Insights
Maize malic enzyme activity was rapidly inactivated by trypsin, which cleaved the enzyme into smaller fragments. Enzyme substrates like NADP protected against this inactivation, suggesting a specific interaction site.
Area of Science:
- Biochemistry
- Enzymology
Background:
- Maize malic enzyme is a crucial enzyme in plant metabolism.
- Understanding its regulation by proteolysis is important for metabolic studies.
Purpose of the Study:
- To investigate the effect of limited proteolysis on maize malic enzyme activity and structure.
- To identify proteases that can inactivate the enzyme and characterize the modified enzyme's properties.
Main Methods:
- Incubation of maize malic enzyme with trypsin and other endoproteases.
- Analysis of enzyme activity and fragmentation using SDS-PAGE.
- Kinetic analysis (Km values) and fluorescence spectroscopy of the modified enzyme.
Main Results:
- Trypsin rapidly inactivated maize malic enzyme by cleaving it into 40 kDa and 20 kDa fragments.
- Substrates, particularly NADP, protected the enzyme from inactivation.
- Endoprotease Lys-C was also effective in inactivating the enzyme.
- Limited proteolysis did not significantly alter malate Km but slightly increased NADP Km, indicating reduced affinity for NADP.
- Fluorescence properties and NADPH binding remained unaffected.
Conclusions:
- Limited proteolysis, especially by trypsin and Lys-C, significantly impacts maize malic enzyme activity.
- The NADP binding site appears to be involved in the protective effect against proteolysis.
- The enzyme's catalytic and coenzyme binding properties are altered, but not completely abolished, after proteolysis.
