Identification of metal binding site of maize NADP malic enzyme by affinity cleavage by Fe2+-ascorbate

Abstract

Maize malic enzyme (EC 1.1.1.40) was irreversibly inactivated by micromolar concentrations of ferrous sulphate in the presence of ascorbate at 25 °C. Inclusion of substrate, malate and NADP alone or together, in the reaction medium did not influence the inactivation rate, however, Mg2+ offered considerable protection against Fe2+ inactivation. The presence of 4 mM EDTA completely prevented the inactivation of the enzyme. The loss of enzyme activity is also associated with the cleavage of the subunit polypeptide into 2 fragments with Mr of 31 000 and 34 000. The cleavage site was identified as the peptide bond between Asp352 and Ile353 by N-terminal sequences of both the fragments using automated gas phase sequencer. The mechanism of inactivation and affinity cleavage of maize malic enzyme by Fe2+-ascorbate is explained on the basis of our data including protection by Mg2+ and catalase. Our studies suggest that reactive species like H2O2, superoxide or hydroxyl radicals may interact with the essential amino acid residues at the metal binding sites causing enzyme inactivation and cleavage, in that order. This corroborates the model proposed earlier by others for the mechanism of inactivation of other metal requiring enzymes by the MCO system The results suggest that Asp352 may function as the coordination site for metal binding in maize NADP malic enzyme.