Affinity cleavage at the putative metal-binding site of pigeon liver malic enzyme by the Fe(2+)-ascorbate system.
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.
Graduate Institutes of Life Sciences and Biochemistry, National Defense Medical Center, Taipei, Taiwan, Republic of China.
C H
CH
Wei
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Chou
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SM
Huang
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Lin
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Chang
D000595:Amino Acid Sequence; D000818:Animals; D001205:Ascorbic Acid / Q000737:chemistry*; D001224:Aspartic Acid / Q000737:chemistry; D010856:Columbidae; D005296:Ferrous Compounds / Q000737:chemistry*; D008099:Liver / Q000201:enzymology; D008291:Malate Dehydrogenase / Q000037:antagonists & inhibitors / Q000737:chemistry*; D008345:Manganese / Q000737:chemistry*; D008667:Metalloproteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D005296:Ferrous Compounds; D008667:Metalloproteins; D001224:Aspartic Acid; D008345:Manganese; D008291:Malate Dehydrogenase; C030574:malate dehydrogenase (decarboxylating); D001205:Ascorbic Acid