title: Structural characteristics of the nonallosteric human cytosolic malic enzyme.
abstract: Human cytosolic NADP(+)-dependent malic enzyme (c-NADP-ME) is neither a cooperative nor an allosteric enzyme, whereas mitochondrial NAD(P)(+)-dependent malic enzyme (m-NAD(P)-ME) is allosterically activated by fumarate. This study examines the molecular basis for the different allosteric properties and quaternary structural stability of m-NAD(P)-ME and c-NADP-ME. Multiple residues corresponding to the fumarate-binding site were mutated in human c-NADP-ME to correspond to those found in human m-NAD(P)-ME. Additionally, the crystal structure of the apo (ligand-free) human c-NADP-ME conformation was determined. Kinetic studies indicated no significant difference between the wild-type and mutant enzymes in Km,NADP, Km,malate, and kcat. A chimeric enzyme, [51-105]_c-NADP-ME, was designed to include the putative fumarate-binding site of m-NAD(P)-ME at the dimer interface of c-NADP-ME; however, this chimera remained nonallosteric. In addition to fumarate activation, the quaternary structural stability of c-NADP-ME and m-NAD(P)-ME is quite different; c-NADP-ME is a stable tetramer, whereas m-NAD(P)-ME exists in equilibrium between a dimer and a tetramer. The quaternary structures for the S57K/N59E/E73K/S102D and S57K/N59E/E73K/S102D/H74K/D78P/D80E/D87G mutants of c-NADP-ME are tetrameric, whereas the K57S/E59N/K73E/D102S m-NAD(P)-ME quadruple mutant is primarily monomeric with some dimer formation. These results strongly suggest that the structural features near the fumarate-binding site and the dimer interface are highly related to the quaternary structural stability of c-NADP-ME and m-NAD(P)-ME. In this study, we attempt to delineate the structural features governing the fumarate-induced allosteric activation of malic enzyme.
authors.affiliation: Department of Life Sciences, National Chung Hsing University (NCHU), Taichung, Taiwan.
authors.forename: Ju-Yi
authors.initials: JY
authors.lastname: Hsieh
authors.affiliation: Department of Life Sciences, National Chung Hsing University (NCHU), Taichung, Taiwan.
authors.forename: Shao-Yu
authors.initials: SY
authors.lastname: Li
authors.affiliation: Department of Life Sciences, National Chung Hsing University (NCHU), Taichung, Taiwan.
authors.forename: Meng-Chun
authors.initials: MC
authors.lastname: Chen
authors.affiliation: Department of Life Sciences, National Chung Hsing University (NCHU), Taichung, Taiwan.
authors.forename: Pai-Chun
authors.initials: PC
authors.lastname: Yang
authors.affiliation: Biotechnology Center, NCHU, Taichung, Taiwan.
authors.forename: Hui-Yi
authors.initials: HY
authors.lastname: Chen
authors.affiliation: Institute of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University (NTU), Taipei, Taiwan. Electronic address: nlchan@ntu.edu.tw.
authors.forename: Nei-Li
authors.initials: NL
authors.lastname: Chan
authors.affiliation: Department of Life Sciences, National Chung Hsing University (NCHU), Taichung, Taiwan; Institute of Genomics and Bioinformatics, NCHU, Taichung, Taiwan; Agricultural Biotechnology Center (ABC), NCHU, Taichung, Taiwan; Rong Hsing Research Center for Translational Medicine, NCHU, Taichung, Taiwan. Electronic address: hchung@dragon.nchu.edu.tw.
authors.forename: Jyung-Hurng
authors.initials: JH
authors.lastname: Liu
authors.affiliation: Department of Life Sciences, National Chung Hsing University (NCHU), Taichung, Taiwan; Institute of Genomics and Bioinformatics, NCHU, Taichung, Taiwan; Agricultural Biotechnology Center (ABC), NCHU, Taichung, Taiwan. Electronic address: jhliu@dragon.nchu.edu.tw.
authors.forename: Hui-Chih
authors.initials: HC
authors.lastname: Hung
keywords: Allosteric activation; Fumarate-binding site; Kinetics; Malic enzyme; Mutagenesis; Quaternary structural stability