Chou W Y, Tsai W P, Lin C C, Chang G G
Department of Biochemistry, National Defense Medical Center, Taipei, Taiwan, Republic of China.
J Biol Chem. 1995 Oct 27;270(43):25935-41. doi: 10.1074/jbc.270.43.25935.
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.
在中性pH条件下，在抗坏血酸盐存在时，微摩尔浓度的Fe2+能迅速使鸽肝苹果酸酶失活。随后，失活的酶在Fe(2+)-抗坏血酸盐体系作用下，在天冬氨酸258和异亮氨酸259之间的化学键处被裂解（Wei, C.H., Chou, W.Y., Huang, S.M., Lin, C.C., and Chang, G.G. (1994) Biochemistry, 33, 7931 - 7936），这一点通过定点诱变得到了证实（Wei, C.H., Chou, W.Y., and Chang, G.G. (1995) Biochemistry 34, 7949 - 7954）。在本研究中，发现在中性pH条件下，Cu2+在苹果酸酶的氧化修饰中更具活性，并且该酶以与Fe2+类似的方式被裂解。然而，在酸性pH条件下，Fe2+在该酶的氧化修饰中无效。尽管如此，Cu2+仍会导致酶失活，并在天冬氨酸141 - 甘氨酸142、天冬氨酸194 - 脯氨酸195或天冬氨酸464 - 天冬氨酸465处裂解该酶。在酸性pH条件下，Mn2+和L - 苹果酸协同保护该酶免受Cu2+失活的影响。在苹果酸酶催化的反应中，Cu2+也是Mn2+的竞争性抑制剂，其Ki值为70.3±5.8微摩尔。上述结果表明，除了先前在中性pH条件下确定的天冬氨酸258外，天冬氨酸141、天冬氨酸194和天冬氨酸464也是苹果酸酶金属结合的配位位点。我们认为，Cu(2+)-抗坏血酸盐体系对苹果酸酶进行亲和修饰和裂解的机制按以下顺序进行。首先，Cu2+在Mn2+结合位点与酶结合，并被抗坏血酸盐还原为Cu+。接下来，局部氧分子被Cu+还原，从而产生超氧化物或其他活性自由基。这些自由基与金属结合位点处易感的必需氨基酸残基相互作用，最终导致酶失活。最后，修饰后的酶被裂解成几个肽片段，从而可以鉴定该酶的金属位点。金属催化氧化体系的pH依赖性不同特异性可能普遍适用于其他酶或蛋白质。
