Metal ions stabilize a dimeric molten globule state between the open and closed forms of malic enzyme.
Malic enzyme is a tetrameric protein with double dimer quaternary structure. In 3-5 M urea, the pigeon cytosolic NADP(+)-dependent malic enzyme unfolded and aggregated into various forms with dimers as the basic unit. Under the same denaturing conditions but in the presence of 4 mM Mn(2+), the enzyme existed exclusively as a molten globule dimer in solution. Similar to pigeon enzyme (Chang, G. G., T. M. Huang, and T. C. Chang. 1988. Biochem. J. 254:123-130), the human mitochondrial NAD(+)-dependent malic enzyme also underwent a reversible tetramer-dimer-monomer quaternary structural change in an acidic pH environment, which resulted in a molten globule state that is also prone to aggregate. The aggregation of pigeon enzyme was attributable to Trp-572 side chain. Mutation of Trp-572 to Phe, His, Ile, Ser, or Ala abolished the protective effect of the metal ions. The cytosolic malic enzyme was completely digested within 2 h by trypsin. In the presence of Mn(2+), a specific cutting site in the Lys-352-Gly-Arg-354 region was able to generate a unique polypeptide with M(r) of 37 kDa, and this polypeptide was resistant to further digestion. These results indicate that, during the catalytic process of malic enzyme, binding metal ion induces a conformational change within the enzyme from the open form to an intermediate form, which upon binding of L-malate, transforms further into a catalytically competent closed form.
Chang
Hui-Chuan
HC
Department of Life Sciences and Institute of Genome Sciences, National Yang-Ming University, Taipei, Taiwan.
Chen
Liang-Yu
LY
Lu
Yi-Hang
YH
Li
Meng-Ying
MY
Chen
Yu-Hou
YH
Lin
Chao-Hsiung
CH
Chang
Gu-Gang
GG
D004795:Enzyme Stability; D008291:Malate Dehydrogenase / Q000737:chemistry* / Q000648:ultrastructure*; D008670:Metals / Q000737:chemistry*; D044367:Phase Transition; D011487:Protein Conformation; D017510:Protein Folding
D008670:Metals; D008291:Malate Dehydrogenase; C495394:malic enzyme 2; human
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