title: Biophysical characterization of the dimer and tetramer interface interactions of the human cytosolic malic enzyme.
abstract: The cytosolic NADP(+)-dependent malic enzyme (c-NADP-ME) has a dimer-dimer quaternary structure in which the dimer interface associates more tightly than the tetramer interface. In this study, the urea-induced unfolding process of the c-NADP-ME interface mutants was monitored using fluorescence and circular dichroism spectroscopy, analytical ultracentrifugation and enzyme activities. Here, we demonstrate the differential protein stability between dimer and tetramer interface interactions of human c-NADP-ME. Our data clearly demonstrate that the protein stability of c-NADP-ME is affected predominantly by disruptions at the dimer interface rather than at the tetramer interface. First, during thermal stability experiments, the melting temperatures of the wild-type and tetramer interface mutants are 8-10°C higher than those of the dimer interface mutants. Second, during urea denaturation experiments, the thermodynamic parameters of the wild-type and tetramer interface mutants are almost identical. However, for the dimer interface mutants, the first transition of the urea unfolding curves shift towards a lower urea concentration, and the unfolding intermediate exist at a lower urea concentration. Third, for tetrameric WT c-NADP-ME, the enzyme is first dissociated from a tetramer to dimers before the 2 M urea treatment, and the dimers then dissociated into monomers before the 2.5 M urea treatment. With a dimeric tetramer interface mutant (H142A/D568A), the dimer completely dissociated into monomers after a 2.5 M urea treatment, while for a dimeric dimer interface mutant (H51A/D90A), the dimer completely dissociated into monomers after a 1.5 M urea treatment, indicating that the interactions of c-NADP-ME at the dimer interface are truly stronger than at the tetramer interface. Thus, this study provides a reasonable explanation for why malic enzymes need to assemble as a dimer of dimers.
authors.affiliation: Department of Life Sciences, National Chung Hsing University, Taichung, Taiwan.
authors.forename: Sujithkumar
authors.initials: S
authors.lastname: Murugan
authors.forename: Hui-Chih
authors.initials: HC
authors.lastname: Hung
mesh_terms: D000817:Anilino Naphthalenesulfonates / Q000378:metabolism; D055592:Biophysical Phenomena; D003600:Cytosol / Q000201:enzymology*; D004795:Enzyme Stability; D006801:Humans; D050538:Malate Dehydrogenase (NADP+) / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D016296:Mutagenesis; D009154:Mutation; D011489:Protein Denaturation / Q000187:drug effects; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D013696:Temperature; D014508:Urea / Q000494:pharmacology
chemical_list: C515594:8-anilino-1-naphthalenesulfonic acid; D000817:Anilino Naphthalenesulfonates; D014508:Urea; D050538:Malate Dehydrogenase (NADP+)