title: Identification of Mn2+-binding aspartates from alpha, beta, and gamma subunits of human NAD-dependent isocitrate dehydrogenase.
abstract: The human NAD-dependent isocitrate dehydrogenase (IDH), with three types of subunits present in the ratio of 2alpha:1beta:1gamma, requires a divalent metal ion to catalyze the oxidative decarboxylation of isocitrate. With the aim of identifying ligands of the enzyme-bound Mn(2+), we mutated aspartates on the alpha, beta, or gamma subunits. Mutagenesis target sites were based on crystal structures of metal-isocitrate complexes of Escherichia coli and pig mitochondrial NADP-IDH and sequence alignments. Aspartates replaced by asparagine or cysteine were 206, 230, and 234 of the alpha subunit and those corresponding to alpha-Asp-206: 217 of the beta subunit and 215 of the gamma subunit. Each expressed, purified mutant enzyme has two wild-type subunits and one subunit with a single mutation. Specific activities of WT, alpha-D206N, alpha-D230C, alpha-D234C, beta-D217N, and gamma-D215N enzymes are 22, 29, 1.4, 0.2, 7.3 and 3.7 micromol of NADH/min/mg, respectively, whereas alpha-D230N and alpha-D234N enzymes showed no activity. The K(m,Mn(2+)) for alpha-D230C and gamma-D215N are increased 32- and 100-fold, respectively, along with elevations in K(m,isocitrate). The K(m,NAD) of alpha-D230C is increased 16-fold, whereas that of beta-D217N is elevated 10-fold. For all the mutants K(m,isocitrate) is decreased by ADP, indicating that these aspartates are not needed for normal ADP activation. This study demonstrates that alpha-Asp-230 and alpha-Asp-234 are critical for catalytic activity, but alpha-Asp-206 is not needed; alpha-Asp-230 and gamma-Asp-215 may interact directly with the Mn(2+); and alpha-Asp-230 and beta-Asp-217 contribute to the affinity of the enzyme for NAD. These results suggest that the active sites of the human NAD-IDH are shared between alpha and gamma subunits and between alpha and beta subunits.
authors.affiliation: Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716.
authors.forename: Sambanthamurthy
authors.initials: S
authors.lastname: Soundar
authors.affiliation: Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716.
authors.forename: Molly
authors.initials: M
authors.lastname: O'Hagan
authors.affiliation: Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716.
authors.forename: Kenneth S
authors.initials: KS
authors.lastname: Fomulu
authors.affiliation: Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716. Electronic address: rfcolman@udel.edu.
authors.forename: Roberta F
authors.initials: RF
authors.lastname: Colman
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid / Q000737:chemistry*; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D007521:Isocitrate Dehydrogenase / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008345:Manganese / Q000737:chemistry*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010100:Oxygen / Q000737:chemistry; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013552:Swine
chemical_list: D001224:Aspartic Acid; D008345:Manganese; D007521:Isocitrate Dehydrogenase; D010100:Oxygen