AFFINITY CLEAVAGE AT THE PUTATIVE METAL-BINDING SITE OF PIGEON LIVER MALIC ENZYME BY THE FE2-ASCORBATE SYSTEM()

Pigeon liver malic enzyme was rapidly inactivated by micromolar concen trations of ferrous sulfate in the presence of ascorbate at neutral pH and 0 or 25 degrees C. Omitting the ascorbate or replacing the ferrou s ion with manganese ion did not lead to any inactivation. Manganese, magnesium, zinc, cobalt, or calcium ion at 200 molar excess over ferro us ion offered complete protection of the enzyme from Fe2+-induced ina ctivation. Ni2+ provided partial protection, while Ba2+ or imidazole w as 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 combinati on) in the incubation mixture did not affect the inactivation rate. Th e enzyme inactivation was demonstrated to be followed by protein cleav age. Native pigeon liver malic enzyme had a subunit M(r) of 65 000. Th e inactivated enzyme with residual activity of only 0.3% was cleaved i nto two fragments with M(r) of 31 000 and 34 000, respectively. The cl eavage site was identified as the peptide bond between Asp(258) and Il e(259). 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-t erminal half of original sequence 259-557. Our results indicated that Fe2+ catalyzed a specific oxidation of pigeon liver malic enzyme at As p(258) and/or some other essential amino acid residues that caused enz yme inactivation. The modified enzyme was then affinity cleaved at the Mn2+-binding site.