Critical Role of Lys212 and Tyr140 in Porcine NADP-dependent Isocitrate Dehydrogenase

Kim, Tae Kang; Lee, Peychii; Colman, Roberta F.

doi:10.1074/jbc.m303781200

Cited by 34 publications

(51 citation statements)

References 31 publications

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“…As shown in Table 2, the V max and K m (ICT) values of the wild-type IDH1 are comparable to that of PmIDH and E. coli NADP-IDH (EcIDH) [22][23][24][25][26]. Compared with the wild-type IDH1, the homodimeric R132H mutant manifests a 200-fold increase in the K m whereas its specific activity is decreased to about 5%; similarly, the K m of the R132A mutant is also substantially increased by 106-fold while its specific activity is decreased to about 18% ( Table 2).…”

Section: Kinetic Studies Of the Wild-type And Mutant Idh1mentioning

confidence: 67%

“…Our structures strengthen this notion by showing that although ICT is bound with both subunits, the wild-type subunit of the heterodimer could not accomplish the conformational changes and concurrently, the two key structural segments exhibit remarkably increased flexibility. As in the wild-type enzyme, the conformational changes of the two segments and the inter-domain motion are induced by ICT binding, and a number of residues in these two segments are involved in ICT binding, metal ion coordination, and catalysis [14,22]; we speculate that the increased flexibility of these two regions rendered by the R132H mutation might interfere with the ICT binding and/or the overall conformational changes of the enzyme, leading to the impairment of the IDH activity. …”

Section: The R132h Mutation Renders An Increased Flexibility Of Two Kmentioning

confidence: 99%

“…There is discrepancy in the IDH activity of the R132H mutant as Zhao et al [12] showed a 2-fold decrease in the catalytic rate while Dang et al [13] reported a 1000-fold reduction, and our data are in better agreement with that reported by Zhao et al In addition, the kinetic parameters of the R132C, R132S, and R132L mutants are similar to that of the R132H mutant ( Table 2). The Y139A and K212A mutants display < 1% activity ( Table 2), indicating that Tyr139 and Lys212, like their counterparts in EcIDH and PmIDH [22,25,27], play critical roles in the catalytic reaction. Our data also show that mutations D252A and D275A have severe effects on the K m and/or k cat due to their involvements in both ICT binding and catalysis (Table 2).…”

Section: Kinetic Studies Of the Wild-type And Mutant Idh1mentioning

confidence: 99%

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Molecular mechanisms of “off-on switch” of activities of human IDH1 by tumor-associated mutation R132H

et al. 2010

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Human cytosolic NADP-IDH (IDH1) has recently been found to be involved in tumorigenesis. Notably, the tumorderived IDH1 mutations identified so far mainly occur at Arg132, and mutation R132H is the most prevalent one. This mutation impairs the oxidative IDH activity of the enzyme, but renders a new reduction function of converting α-ketoglutarate (αKG) to 2-hydroxyglutarate. Here, we report the structures of the R132H mutant IDH1 with and without isocitrate (ICT) bound. The structural data together with mutagenesis and biochemical data reveal a previously undefined initial ICT-binding state and demonstrate that IDH activity requires a conformational change to a closed pre-transition state. Arg132 plays multiple functional roles in the catalytic reaction; in particular, the R132H mutation hinders the conformational changes from the initial ICT-binding state to the pre-transition state, leading to the impairment of the IDH activity. Our results describe for the first time that there is an intermediate conformation that corresponds to an initial ICT-binding state and that the R132H mutation can trap the enzyme in this conformation, therefore shedding light on the molecular mechanism of the "off switch" of the potentially tumor-suppressive IDH activity. Furthermore, we proved the necessity of Tyr139 for the gained αKG reduction activity and propose that Tyr139 may play a vital role by compensating the increased negative charge on the C2 atom of αKG during the transfer of a hydride anion from NADPH to αKG, which provides new insights into the mechanism of the "on switch" of the hypothetically oncogenic reduction activity of IDH1 by this mutation.

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Section: Kinetic Studies Of the Wild-type And Mutant Idh1mentioning

confidence: 67%

Section: The R132h Mutation Renders An Increased Flexibility Of Two Kmentioning

confidence: 99%

Section: Kinetic Studies Of the Wild-type And Mutant Idh1mentioning

confidence: 99%

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Molecular mechanisms of “off-on switch” of activities of human IDH1 by tumor-associated mutation R132H

et al. 2010

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“…The porcine NADP-IDH is a dimer of identical subunits, as are the NADP-IDHs of E. coli (7) and B. subtilis (9). Each manganeseisocitrate bound to crystalline porcine NADP-IDH is located close to the interface between the two subunits (10), and amino acids from both subunits contribute to binding and catalysis (19,28). For manganese-isocitrate bound at the B subunit, the direct ligands of the hexacoordinate Mn 2ϩ are the carboxylate of Asp-252 from the A subunit and of Asp-275 from the B subunit, the ␣-hydroxyl and ␣-carboxylate of isocitrate, and two water molecules (10).…”

Section: Discussionmentioning

confidence: 99%

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

Soundar

O’Hagan

Fomulu

et al. 2006

Journal of Biological Chemistry

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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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“…To facilitate separation from the E. coli isocitrate dehydrogenase, the enzyme was expressed as a maltose-binding fusion protein with a thrombin cleavage site located between the maltose-binding protein and the porcine isocitrate dehydrogenase (15). Site-directed mutagenesis of pMALcIDP1 was performed using the QuikChange XL Kit to produce mutant DNA (11,12). The oligonucleotides (forward and reverse primers) used to generate the mutant enzymes by the QuikChange method are listed in Table I.…”

mentioning

confidence: 99%

Location of the Coenzyme Binding Site in the Porcine Mitochondrial NADP-dependent Isocitrate Dehydrogenase

Huang

Colman

2005

Journal of Biological Chemistry

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Critical Role of Lys212 and Tyr140 in Porcine NADP-dependent Isocitrate Dehydrogenase

Cited by 34 publications

References 31 publications

Molecular mechanisms of “off-on switch” of activities of human IDH1 by tumor-associated mutation R132H

Molecular mechanisms of “off-on switch” of activities of human IDH1 by tumor-associated mutation R132H

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

Location of the Coenzyme Binding Site in the Porcine Mitochondrial NADP-dependent Isocitrate Dehydrogenase

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