Enzyme Catalysis and Regulation
Identification of Mn2+-binding Aspartates from α, β, and γ Subunits of Human NAD-dependent Isocitrate Dehydrogenase*

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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 Km,Mn2+ for α-D230C and γ-D215N are increased 32- and 100-fold, respectively, along with elevations in Km,isocitrate. The Km,NAD of α-D230C is increased 16-fold, whereas that of β-D217N is elevated 10-fold. For all the mutants Km,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 Mn2+; 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.

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This work was supported by National Institutes of Health Grant R01 HL67774. A preliminary version of some of this work has been presented (12). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.