Identification of Mn2+-binding aspartates from alpha, beta, and gamma subunits of human NAD-dependent isocitrate dehydrogenase.

Article Details

Citation

Copy to clipboard

Soundar S, O'hagan M, Fomulu KS, Colman RF

Identification of Mn2+-binding aspartates from alpha, beta, and gamma subunits of human NAD-dependent isocitrate dehydrogenase.

J Biol Chem. 2006 Jul 28;281(30):21073-81. Epub 2006 May 31.

PubMed ID

16737955 [ View in PubMed

]

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.

DrugBank Data that Cites this Article

Drug Targets

Drug	Target	Kind	Organism	Pharmacological Action	Actions
Manganese	Isocitrate dehydrogenase [NAD] subunit alpha, mitochondrial	Protein	Humans	Unknown	Not Available	Details
Manganese	Isocitrate dehydrogenase [NAD] subunit gamma, mitochondrial	Protein	Humans	Unknown	Not Available	Details