Selective oxidative modification and affinity cleavage of pigeon liver malic enzyme by the Cu(2+)-ascorbate system.
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.
Chou
W Y
WY
Department of Biochemistry, National Defense Medical Center, Taipei, Taiwan, Republic of China.
Tsai
W P
WP
Lin
C C
CC
Chang
G G
GG
D000595:Amino Acid Sequence; D000818:Animals; D001205:Ascorbic Acid / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D002413:Cations, Divalent / Q000378:metabolism; D010856:Columbidae; D003300:Copper / Q000737:chemistry / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism*; D008099:Liver / Q000201:enzymology*; D008291:Malate Dehydrogenase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D008293:Malates / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D017386:Sequence Homology, Amino Acid
D002413:Cations, Divalent; D004791:Enzyme Inhibitors; D008293:Malates; D008345:Manganese; D009249:NADP; D003300:Copper; C030298:malic acid; D008291:Malate Dehydrogenase; C030574:malate dehydrogenase (decarboxylating); D001205:Ascorbic Acid