Abstract
Pigeon liver malic enzyme was rapidly inactivated by micromolar concentration of Fe2+ in the presence of ascorbate at neutral pH. The inactivated enzyme was subsequently cleaved by the Fe(2+)-ascorbate system at the chemical bond between Asp258 and Ile259 (Wei, C.H., Chou, W.Y., Huang, S.M., Lin, C.C., and Chang, G.G. (1994) Biochemistry, 33, 7931-7936), which was confirmed by site-specific mutagenesis (Wei, C.H., Chou, W.Y., and Chang, G.G. (1995) Biochemistry 34, 7949-7954). In the present study, at neutral pH, Cu2+ was found to be more reactive in the oxidative modification of malic enzyme and the enzyme was cleaved in a similar manner as Fe2+ did. At acidic pH, however, Fe2+ was found to be ineffective in oxidative modification of the enzyme. Nevertheless, Cu2+ still caused enzyme inactivation and cleaved the enzyme at Asp141-Gly142, Asp194-Pro195, or Asp464-Asp465. Mn2+ and L-malate synergistically protect the enzyme from Cu2+ inactivation at acidic pH. Cu2+ is also a competitive inhibitor versus Mn2+ in the malic enzyme-catalyzed reaction with Ki value 70.3 +/- 5.8 microM. The above results indicated that, in addition to the previously determined Asp258 at neutral pH, Asp141, Asp194, and Asp464 are also the coordination sites for the metal binding of malic enzyme. We suggest that the mechanism of affinity modification and cleavage of malic enzyme by the Cu(2+)-ascorbate system proceed in the following sequence. First, Cu2+ binds with the enzyme at the Mn2+ binding site and reduces to Cu+ by ascorbate. Next, the local oxygen molecules are reduced by Cu+, thereby generating superoxide or other reactive free radicals. These radicals interact with the susceptible essential amino acid residues at the metal-binding site, ultimately causing enzyme inactivation. Finally, the modified enzyme is cleaved into several peptide fragments, allowing the identification of metal site of the enzyme. The pH-dependent different specificities of metal-catalyzed oxidation system may be generally applicable for other enzymes or proteins.
Highlights
§ To whom correspondence should be addressed: Dept. of Biochemistry, National Defense Medical Center, P
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
Selective Inactivation of Pigeon Liver Malic Enzyme—Pigeon liver malic enzyme was highly sensitive to metal-catalyzed oxidation (Wei et al, 1994)
Summary
Enzyme Purification and Assay—Malic enzyme from pigeon liver was purified to apparent homogeneity according to published procedure (Chang and Chang, 1982). The enzyme activity was assayed by monitoring the formation of NADPH at 30 °C as described previously (Chang et al, 1992). Enzyme Modification and Cleavage—The inactivation experiments were performed at 0 °C by adding freshly prepared solutions of cupric nitrate (6 M) and ascorbate (20 mM) into the enzyme solution (0.97 M) in sodium acetate buffer (66.5 mM, pH 5.0). The progress of enzyme inactivation was monitored by assaying the enzyme activity in small aliquots withdrawn at the designated time intervals. For detecting the peptide bond cleavage, the samples withdrawn from the reaction mixture were added to EDTA solution (4 mM) to.
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