Pig heart NADP-dependent isocitrate dehydrogenase requires a divalent metal cation for catalysis. On the basis of affinity cleavage studies [Soundar and Colman (1993) J. Biol. Chem. 268, 5267] and analysis of the crystal structure of E. coli NADP-isocitrate dehydrogenase [Hurley et al. (1991) Biochemistry 30, 8671], the residues Asp(253), Asp(273), Asp(275), and Asp(279) were selected as potential ligands of the divalent metal cation in the pig heart enzyme. Using a megaprimer PCR method, the Asp at each of these positions was mutated to Asn. The wild-type and mutant enzymes were expressed in Escherichia coli and purified. D253N has a specific activity, K(m) values for Mn(2+), isocitrate, and NADP, and also a pH-V(max) profile similar to those of the wild-type enzyme. Thus, Asp(253) is not involved in enzyme function. D273N has an increased K(m) for Mn(2+) and isocitrate with a specific activity 5% that of wild type. The D273N mutation also prevents the oxidative metal cleavage seen with Fe(2+) alone in the wild-type enzyme. As compared to wild type, D275N has greatly increased K(m) values for Mn(2+) and isocitrate, with a specific activity <0.1% that of wild type, and a large increase in pK(a) for the enzyme-substrate complex. D279N has only small increases in K(m) for Mn(2+) and isocitrate, but a specific activity <0.1% that of wild type and a major change in the shape of its pH-V(max) profile. These results suggest that Asp(273) and Asp(275) contribute to metal binding, whereas Asp(279), as well as Asp(275), is critical for catalysis. Asp(279) may function as the catalytic base. Using the Modeler program of Insight II, a structure for porcine NADP-isocitrate dehydrogenase was built based on the X-ray coordinates of the E. coli enzyme, allowing visualization of the metal-isocitrate site.

Mammalian NAD-dependent isocitrate dehydrogenase is an allosteric enzyme, activated by ADP and composed of 3 distinct subunits in the ratio 2␣:1␤:1␥. Based on the crystal structure of NADP-dependent isocitrate dehydrogenases from Escherichia coli, Bacillus subtilis, and pig heart, and a comparison of their amino acid sequences, ␣-Arg 88 , ␤-Arg 99 , and ␥-Arg 97 of human NADdependent isocitrate dehydrogenase were chosen as candidates for mutagenesis to test their roles in catalytic activity and ADP activation. A plasmid harboring cDNA that encodes ␣, ␤, and ␥ subunits of the human isocitrate dehydrogenase (Kim, Y. O., Koh, H. J., Kim, S. H., Jo, S. H., Huh, J. W., Jeong, K. S., Lee, I. J., Song, B. J., and Huh, T. L. (1999) J. Biol. Chem. 274, 36866 -36875) was used to express the enzyme in isocitrate dehydrogenase-deficient E. coli. Wild type (WT) and mutant enzymes (each containing 2 normal subunits plus a mutant subunit with ␣-R88Q, ␤-R99Q, or ␥-R97Q) were purified to homogeneity yielding enzymes with 2␣:1␤:1␥ subunit composition and a native molecular mass of 315 kDa. Specific activities of 22, 14, and 2 mol of NADH/ min/mg were measured, respectively, for WT, ␤-R99Q, and ␥-R97Q enzymes. In contrast, mutant enzymes with normal ␤ and ␥ subunits and ␣-R88Q mutant subunit has no detectable activity, demonstrating that, although ␤-Arg 99 and ␥-Arg 97 contribute to activity, ␣-Arg 88 is essential for catalysis. For WT enzyme, the K m for isocitrate is 2.2 mM, decreasing to 0.3 mM with added ADP. In contrast, for ␤-R99Q and ␥-R97Q enzymes, the K m for isocitrate is the same in the absence or presence of ADP, although all the enzymes bind ADP. These results suggest that ␤-Arg 99 and ␥-Arg 97 are needed for normal ADP activation. In addition, the ␥-R97Q enzyme has a K m for NAD 10 times that of WT enzyme. This study indicates that a normal ␣ subunit is required for catalytic activity and ␣-Arg 88 likely participates in the isocitrate site, whereas the ␤ and ␥ subunits have roles in the nucleotide functions of this allosteric enzyme.Mammalian NAD-dependent isocitrate dehydrogenase (threo-D s -isocitrate:NAD ϩ oxidoreductase (decarboxylating), EC 1.1.1.41) is an allosteric mitochondrial enzyme regulated by ADP, which activates by lowering the K m for the substrate isocitrate without changing the V max (1). It is a hetero-oligomeric enzyme, composed of three distinct types of subunits, present in the ratio 2␣:1␤:1␥. The subunits have molecular weights of 37,000, 39,000, and 39,000 for ␣, ␤, and ␥, respectively, whereas their isoelectric points are distinctive (2, 3).The pig heart NAD-dependent isocitrate dehydrogenase has been shown to bind tightly, per mole of enzyme tetramer, 2 mol of every ligand (isocitrate, Mn 2ϩ , NAD, ADP, NADH, and NADPH), indicating that there are half as many sites as subunits (4, 5). These observations have raised the question as to whether the subunits have specialized functions for particular ligands or if there is a functional similarity among the different subunits, and two subunits cont...

Pig heart mitochondrial NADP-dependent isocitrate dehydrogenase is the most extensively studied among the mammalian isocitrate dehydrogenases. ; however, the marked decrease in k cat suggests a role for Arg 101 in catalysis. The V max of wild type enzyme depends on the ionized form of an enzymic group of pK 5.5, and this pK aes is similar for the R101Q and R120Q enzymes. In contrast, the pK aes for R110Q and R133Q enzymes increases to 6.4 and 7.4, respectively, indicating that the positive charges of Arg 110 and Arg 133 normally lower the pK of the nearby catalytic base to facilitate its ionization. These results may be understood in terms of the structure of the porcine NADP-specific isocitrate dehydrogenase generated by the Insight II Modeler Program, based on the x-ray coordinates of the E. coli enzyme.The mitochondrial NADP-specific pig heart isocitrate dehydrogenase (EC 1.1.1.42) catalyzes the divalent metal ion-dependent oxidative decarboxylation of isocitrate to ␣-ketoglutarate, and it is considered that the metal-tribasic isocitrate complex is the preferred substrate (1). The enzyme is a homodimer (2, 3), with a subunit mass of 46,600 Da consisting of 413 amino acids of determined sequence (4). A 13 C-NMR study using specifically enriched isocitrate demonstrated that all three carboxyls of the substrate remain fully ionized from pH 5.5 to 7.5 when bound to the enzyme, although the carboxylates of free isocitrate become protonated over this pH range (5). This result could be due to the presence of positively charged groups in the region of the substrate binding site. The first evidence of the importance of arginines in the function of NADP-dependent isocitrate dehydrogenase came from the inactivation of the pig heart enzyme by 2,3-butanedione (6). A maximum of four arginines were implicated in catalytic activity and, because isocitrate markedly decreased the inactivation rate, it was suggested that at least some of these residues were at or near the isocitrate binding site.

The human NAD-dependent isocitrate dehydrogenase (IDH), with three types of subunits present in the ratio of 2␣:1␤:1␥, 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 ␣, ␤, or ␥ 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 ␣ subunit and those corresponding to ␣-Asp-206: 217 of the ␤ subunit and 215 of the ␥ subunit. Each expressed, purified mutant enzyme has two wild-type subunits and one subunit with a single mutation. Specific activities of WT, ␣-D206N, ␣-D230C, ␣-D234C, ␤-D217N, and ␥-D215N enzymes are 22, 29, 1.4, 0.2, 7.3 and 3.7 mol of NADH/min/mg, respectively, whereas ␣-D230N and ␣-D234N enzymes showed no activity. The K m,Mn 2؉ for ␣-D230C and ␥-D215N are increased 32-and 100-fold, respectively, along with elevations in K m,isocitrate . The K m,NAD of ␣-D230C is increased 16-fold, whereas that of ␤-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 ␣-Asp-230 and ␣-Asp-234 are critical for catalytic activity, but ␣-Asp-206 is not needed; ␣-Asp-230 and ␥-Asp-215 may interact directly with the Mn 2؉ ; and ␣-Asp-230 and ␤-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 ␣ and ␥ subunits and between ␣ and ␤ subunits.Mammalian NAD-dependent isocitrate dehydrogenase (threo-D s -isocitrate:NAD ϩ oxidoreductase (decarboxylating), EC 1.1.1.41) is a citric acid cycle enzyme that catalyzes the metaldependent oxidative decarboxylation of isocitrate to form ␣-ketoglutarate and CO 2 . This allosteric mitochondrial enzyme is regulated by ADP (1). The enzyme has a heterooligomeric structure with subunits, present in the ratio 2␣:1␤: 1␥, of molecular masses 37,000, 39,000, and 39,000 Da, respectively, and distinctive isoelectric points (2, 3).Our previous work on pig heart NAD-dependent isocitrate dehydrogenase showed that this enzyme has two binding sites per tetramer for each of its ligands: isocitrate, Mn 2ϩ , NAD, ADP, NADH, and NADPH (4, 5). These binding studies either indicate that these distinctive subunits have specialized functions for particular ligand sites or that the binding site for each ligand is shared between two subunits (4, 5). The three types of subunits of the pig heart enzyme can be separated by chromatofocusing in the presence of urea (6). Isolated ␣, ␤, and ␥ subunits are either inactive or exhibit very low activity, but recombination of isolated ␣ with either ␤ or ␥ results in formation of either ␣␤ or ␣␥, which have substantial catalytic activity (6). These observations suggest that dimers may be the minimal functional subunits. The active...

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