Affinity Cleavage at the Putative Metal-Binding Site of Pigeon Liver Malic Enzyme by the Fe2+-Ascorbate System

Abstract

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.

Selective Oxidative Modification and Affinity Cleavage of Pigeon Liver Malic Enzyme by the Cu2+-Ascorbate System

“…1), i.e. in correlation with our previous observation (Wei et al, 1994 -ascorbate system between pH 4.0 -9.0 in which the enzyme was stable. There are two optima for the inactivation rate: one at pH 6.0 -7.0, and the other at approximately pH 4.0 (Fig.…”

“…Without a three-dimensional crystal structure available, affinity cleavage at the putative metal-binding site by the metal-catalyzed oxidation system (MCO) 1 may be the optimal approach of reaching the above goal. Using this technique with the Fe 2ϩ -ascorbate system, Asp 258 is successfully identified in our previous study as one of the metal-binding sites (Wei et al, 1994), as confirmed by site-directed mutagenesis (Wei et al, 1995). In that study, some divalent metal ions were found to be capable of providing protection of the enzyme against Fe 2ϩ -induced inactivation.…”

“…Next, the protein samples were subjected to SDS-PAGE for separating the peptide fragments. The resulting gel pattern was quantified by densitometric analysis and the isolated peptide fragments were then transblotted to an Immobilon-P membrane and subjected to automatic amino acid sequence analysis as described previously (Wei et al, 1994).…”

“…Enzyme-Pigeon liver malic enzyme was highly sensitive to metal-catalyzed oxidation (Wei et al, 1994). The inactivation rate of the enzyme by the MCO system was highly pH-dependent.…”

Comparison of partial malolactic enzyme gene sequences for phylogenetic analysis of some lactic acid bacteria species and relationships with the malic enzyme

“…The box I11 sequence is exactly the consensus between all malic and malolactic enzymes. Asp"' (second D in FNDDIQGT sequence) in pigeon liver malic enzyme is the metal coordinate (Wei et al, 1994). Four mutant enzymes substituted at Asp25s with glutamate, asparagine, lysine or alanine were inactive.…”