期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 281, 期 30, 页码 21073-21081出版社
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M602956200
关键词
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资金
- NHLBI NIH HHS [R01 HL67774] Funding Source: Medline
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 Mn2+, 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 alpha 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, alpha-D217N, and alpha-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 beta-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 Km, isocitrate. The K-m,K-NAD of alpha-D230C is increased 16-fold, whereas that of beta-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 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 Mn2+; 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.
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