Pigeon liver malic enzyme was rapidly inactivated by micromolar concentrations of ferrous sulfate in the presence of ascorbate at neutral pH and 0 or 25 degrees C. Omitting the ascorbate or replacing the ferrous ion with manganese ion did not lead to any inactivation. Manganese, magnesium, zinc, cobalt, or calcium ion at 200 molar excess over ferrous ion offered complete protection of the enzyme from Fe(2+)-induced inactivation. Ni2+ provided partial protection, while Ba2+ or imidazole was ineffective in protection. Addition of 4 mM Mn2+ or 5 mM EDTA into a partially modified enzyme stopped further inactivation of the enzyme. Inclusion of substrates (L-malate or NADP+, singly or in combination) in the incubation mixture did not affect the inactivation rate. The enzyme inactivation was demonstrated to be followed by protein cleavage. Native pigeon liver malic enzyme had a subunit M(r) of 65,000. The inactivated enzyme with residual activity of only 0.3% was cleaved into two fragments with M(r) of 31,000 and 34,000, respectively. The cleavage site was identified as the peptide bond between Asp258 and Ile259. Native pigeon liver malic enzyme was blocked at the N-terminus. Cleavage at the putative metal-binding site exposed a new N-terminus, which was identified to be at the 34-kDa fragment containing the C-terminal half of original sequence 259-557. Our results indicated that Fe2+ catalyzed a specific oxidation of pigeon liver malic enzyme at Asp258 and/or some other essential amino acid residues that caused enzyme inactivation. The modified enzyme was then affinity cleaved at the Mn(2+)-binding site.

Pigeon liver malic enzyme was inactivated by ferrous sulfate in the presence of ascorbate. Manganese and some other divalent metal ions provided complete protection of the enzyme against the Fe(2+)-induced inactivation. The inactivated enzyme was subsequently cleaved by the Fe(2+)-ascorbate system at Asp258-Ile259, which was presumably the Mn(2+)-binding site of the enzyme [Wei, C. H., Chou, W. Y., Huang, S. M., Lin, C. C., & Chang, G. G. (1994) Biochemistry 33, 7793-7936]. For identification of Asp258 as the putative metal-binding site of the enzyme, we prepared four mutant enzymes substituted at Asp258 with glutamate (D258E), asparagine (D258N), lysine (D258K), or alanine (D258A), respectively. These mutant proteins were recombinantly expressed in a bacterial expression system (pET-15b) with a stretch of histidine residues attached at the N-terminus and were successfully purified to apparent homogeneity by a single Ni-chelated affinity column. Among the four mutants, only D258E possessed 0.8% residual activity after purification; all other purified mutants had < 0.0001% residual activity in catalyzing the oxidative decarboxylation of L-malate. The D258E mutant was susceptible to inactivation by the Fe(2+)-ascorbate system, albeit with much slower inactivation rate, and was protected by the Mn2+ to a lesser extent as compared to the wild-type enzyme. None of the mutants were cleaved by the Fe(2+)-ascorbate system under conditions that cleaved the natural or wild-type enzyme at Asp258.(ABSTRACT TRUNCATED AT 250 WORDS)