Identification of Mn²⁺-binding aspartates from α, β, and γ subunits of human NAD-dependent isocitrate dehydrogenase

The human NAD-dependent isocitrate dehydrogenase (IDH), with three types of subunits present in the ratio of 2α:1β:1γ, requires a divalent metal ion to catalyze the oxidative decarboxylation of isocitrate. With the aim of identifying ligands of the enzyme-bound Mn²⁺, we mutated aspartates on the α, β, or γ 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 α subunit and those corresponding to α-Asp-206: 217 of the β subunit and 215 of the γ subunit. Each expressed, purified mutant enzyme has two wild-type subunits and one subunit with a single mutation. Specific activities of WT, α-D206N, α-D230C, α-D234C, β-D217N, and γ-D215N enzymes are 22, 29, 1.4, 0.2, 7.3 and 3.7 μmol of NADH/min/mg, respectively, whereas α-D230N and α-D234N enzymes showed no activity. The K _m,Mn2+ for α-D230C and γ-D215N are increased 32- and 100-fold, respectively, along with elevations in K_m,isocitrate. The K_m,NAD of α-D230C is increased 16-fold, whereas that of β-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 α-Asp-230 and α-Asp-234 are critical for catalytic activity, but α-Asp-206 is not needed; α-Asp-230 and γ-Asp-215 may interact directly with the Mn ²⁺; and α-Asp-230 and β-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 α and γ subunits and between α and β subunits.