Identification of Mn super(2+)-binding Aspartates from alpha , beta , and gamma Subunits of Human NAD-dependent Isocitrate Dehydrogenase

The human NAD-dependent isocitrate dehydrogenase (IDH), with three types of subunits present in the ratio of 2 alpha :1 beta :1 gamma , requires a divalent metal ion to catalyze the oxidative decarboxylation of isocitrate. With the aim of identifying ligands of the enzyme-bound Mn super(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 mu mol of NADH/min/mg, respectively, whereas alpha -D230N and alpha -D234N enzymes showed no activity. The K sub(m) sub(,Mn2+) for alpha -D230C and gamma -D215N are increased 32- and 100-fold, respectively, along with elevations in K sub(m) sub(,isocitrate). The K sub(m) sub(,NAD) of alpha -D230C is increased 16-fold, whereas that of beta -D217N is elevated 10-fold. For all the mutants K sub(m) sub(,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 super(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.