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 Mn2+, 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 Mn2+, a specific cutting site in the Lys-352-Gly-Arg-354 region was able to generate a unique polypeptide with Mr 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.