ARTICLE
`
`cros
`
`Molecular mechanisms of isocitrate dehydrogenase
`1 (IDH1) mutations identified in tumors: The role of size
`and hydrophobicity at residue 132 on catalytic efficiency
`
`Received for publication, January 10, 2017, and in revised form, March 16, 2017 Published, Papers in Press, March 22, 2017, DOI 10.1074/jbc.M117.776179
`Diego Avellaneda Matteo‡1, Adam J. Grunseth‡1, Eric R. Gonzalez‡, Stacy L. Anselmo‡, Madison A. Kennedy‡,
`Precious Moman‡, David A. Scott§, An Hoang‡, and Christal D. Sohl‡2
`From the ‡Department of Chemistry and Biochemistry, San Diego State University, San Diego, California 92182 and the §Sanford
`Burnham Prebys Medical Discovery Institute, La Jolla, California 92037
`
`Edited by John M. Denu
`
`Isocitrate dehydrogenase 1 (IDH1) catalyzes the reversible
`NADPⴙ-dependent conversion of isocitrate (ICT) to ␣-ketogl-
`utarate (␣KG) in the cytosol and peroxisomes. Mutations in
`IDH1 have been implicated in >80% of lower grade gliomas and
`secondary glioblastomas and primarily affect residue 132, which
`helps coordinate substrate binding. However, other mutations
`found in the active site have also been identified in tumors.
`IDH1 mutations typically result in a loss of catalytic activity, but
`many also can catalyze a new reaction, the NADPH-dependent
`reduction of ␣KG to D-2-hydroxyglutarate (D2HG). D2HG is
`a proposed oncometabolite that can competitively inhibit
`␣KG-dependent enzymes. Some kinetic parameters have been
`reported for several IDH1 mutations, and there is evidence that
`mutant IDH1 enzymes vary widely in their ability to produce
`D2HG. We report that most IDH1 mutations identified in
`tumors are severely deficient in catalyzing the normal oxidation
`reaction, but that D2HG production efficiency varies among
`mutant enzymes up to ⬃640-fold. Common IDH1 mutations
`have moderate catalytic efficiencies for D2HG production,
`whereas rarer mutations exhibit either very low or very high
`efficiencies. We then designed a series of experimental IDH1
`mutants to understand the features that support D2HG produc-
`tion. We show that this new catalytic activity observed in tumors
`is supported by mutations at residue 132 that have a smaller van
`der Waals volume and are more hydrophobic. We report that
`one mutation can support both the normal and neomorphic
`reactions. These studies illuminate catalytic features of muta-
`tions found in the majority of patients with lower grade gliomas.
`
`Metabolic changes in tumors have been described for nearly
`a century (1–3), but only relatively recently have enzymes
`
`This work was supported by National Institute of Health Grants K99 CA187594
`(to C. D. S.), R00 CA187594 (to C. D. S.), 5T34 GM008303 (to E. R. G. and
`M. A. K.), and P30 CA030199 (to D. A. S.), a Summer Undergraduate
`Research Program Grant from San Diego State University (to M. A. K.), and
`San Diego State University startup funds (to C. D. S.). The authors declare
`that they have no conflicts of interest with the contents of this article. The
`content is solely the responsibility of the authors and does not necessarily
`represent the official views of the National Institutes of Health.
`This article contains supplemental Figs. S1–S6.
`1 Both authors contributed equally to this work.
`2 To whom correspondence should be addressed: CSL 328, MC1030, 5500
`Campanile Dr., San Diego, CA 92182. Tel.: 619-594-2053; Fax: 619-594-
`4634; E-mail: csohl@mail.sdsu.edu.
`
`involved in metabolic processes been established as tumor sup-
`pressors or oncoproteins. One of the more striking examples of
`metabolic enzymes playing a role in tumorigenesis includes
`isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2).3 These
`homodimeric enzymes are responsible for the reversible
`NADP⫹- and Mg2⫹-dependent conversion of ICT to ␣KG
`(Fig. 1A) in the cytosol and peroxisomes (IDH1), or mito-
`chondria (IDH2). IDH3 is responsible for the same reaction
`within the context of the TCA cycle, although the oxidative
`decarboxylation catalyzed by this enzyme is non-reversible
`and NAD⫹-dependent.
`Mutations in IDH1 and IDH2 were identified in glioblastoma
`multiforme in a large sequencing effort (4), and soon ⬎80% of
`adult grade II/III gliomas and secondary glioblastomas were
`found to have IDH1 mutations, commonly R132H or R132C
`IDH1 (5, 6) (reviewed in Refs. 7–9). Subsequently ⬃10–20% of
`acute myeloid leukemias were shown to have primarily IDH2
`mutations, typically R140Q or R172K IDH2 (10). Early mecha-
`nisms of tumorigenesis focused on deficient conversion of ICT
`to ␣KG (11), suggesting that IDH serves as a tumor suppressor,
`in part through altering levels of hypoxia-inducible transcrip-
`tion factor-1␣ (12). However, IDH1 and IDH2 mutations
`appeared heterozygously in tumors, an unusual feature of a
`tumor suppressor. In landmark studies (13–15), the most com-
`mon IDH1 and IDH2 mutations were shown to catalyze a neo-
`morphic reaction: the Mg2⫹- and NADPH-dependent reduc-
`tion of ␣KG to D2HG (Fig. 1B). This suggested IDH1 and IDH2
`likely encode for oncoproteins. D2HG is proposed to be an
`oncometabolite; it competitively inhibits ␣KG-dependent en-
`zymes including the TET family of 5-methylcytosine hydroxy-
`lases and the JmjC family of histone lysine demethylases,
`resulting in cell de-differentiation (16, 17). Indeed, cancer
`patients with IDH mutations display hypermethylated pheno-
`types (18–20) resulting from D2HG-mediated inhibition of
`histone and DNA demethylation. The proposed oncometabo-
`lite D2HG alone can recapitulate tumorigenic phenotypes in
`cancer models (21, 22), but studies measuring global metabo-
`lomics changes between mutant IDH1 expression and D2HG
`treatment show some differences (23, 24), indicating loss of the
`
`3 The abbreviations used are: IDH, isocitrate dehydrogenase; ICT, isocitrate;
`␣KG, ␣-ketoglutarate; D2HG, D-2-hydroxyglutarate; AML, acute myeloid
`leukemia.
`
`J. Biol. Chem. (2017) 292(19) 7971–7983 7971
`
`© 2017 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.
`
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`Catalytic efficiency of IDH1 mutants
`
`Figure 1. WT and mutant IDH1 catalytic activities. Shown are the: A, nor-
`mal oxidative decarboxylation, and B, the neomorphic reduction.
`
`normal reaction and/or altered NADPH levels may also play an
`important role. Development of selected targeted therapy
`against oncogenic IDH1 and IDH2 mutations is underway and
`results are promising (8).
`A point mutation conferring a new catalytic activity suggests
`important mechanistic features, and many have used kinetics
`and structural methods to explore mutant IDH1 and IDH2
`activity (13, 25–30). There is evidence that IDH1 mutations
`produce varying concentrations of D2HG (30), with somewhat
`subtle but interesting alterations in conformational changes as
`shown in crystal structures of R132H IDH1 (26, 27, 29). In gen-
`eral, reported kinetic parameters of IDH1 mutants explored to
`date vary widely, making comparisons difficult. Interestingly,
`some mutations identified in tumors do not appear to generate
`D2HG (31, 32), suggesting that loss of the normal reaction itself
`has important consequences, or perhaps that they are simply
`passenger mutations.
`Here we report a thorough catalytic study of a wide spectrum
`of IDH1 mutations, including many identified in tumors and
`several mutants designed to clarify catalytic features. We show
`that IDH1 mutants vary widely in catalytic efficiency, with more
`polar and larger residues at position 132 supporting the normal
`reaction, and more hydrophobic and smaller residues driving
`the neomorphic reaction. These findings provide significant
`insight into the types of mutations that may be accommodated
`at residue 132 for efficient D2HG production. By determining
`the catalytic features of IDH1 mutations, we reveal features of
`driver mutations present in the majority of patients with lower
`grade gliomas and secondary glioblastomas.
`
`Results
`Structural modeling and thermal stability of IDH1 mutations
`For the mutations explored in this work, only structures of
`WT and R132H IDH1 in complex with both substrates (ICT
`and NADP⫹, or ␣KG and NADP⫹) have been reported (13, 27,
`29, 33), although a recent high resolution cryo-EM structure
`shows R132C IDH1 in complex with NADPH (34). To help
`inform the structural consequences of R132C, R132G, R100Q,
`A134D, and H133Q IDH1, these mutants were modeled in pre-
`viously solved structures of R132H IDH1 in complex with ␣KG,
`NADP⫹, and Ca2⫹ (Protein Data Bank (PDB) 4KZO (27)) and
`WT IDH1 in complex with ICT, NADP⫹, and Ca2⫹ (PDB 1T0L
`(33)) using the geometry minimization package in Phenix (35).
`These models were then aligned to the original structures using
`PyMOL (36) (Fig. 2). In both models, minimal global changes
`
`7972 J. Biol. Chem. (2017) 292(19) 7971–7983
`
`Figure 2. Structural modeling of IDH1 mutations identified in tumors. A,
`the structure of WT IDH1 complexed with ICT, NADP⫹, and Ca2⫹ (PDB 1T0L
`(33)) and B, R132H IDH1 complexed with ␣KG, NADP⫹, and Ca2⫹ (PDB 4KZO
`(27)) were used to model additional mutations. In both panels, WT IDH1 is
`shown in green, A134D in cyan, H133Q in black, R100Q in dark blue, R132H in
`orange, R132C in yellow, and R132G in gray. Substrates and residues that are
`mutated are highlighted in stick format, as well as catalytic residue Tyr-139.
`Ca2⫹ is shown as a sphere. Ligand restraint generation and optimization of
`provided cif files were generated using eLBOW in the Phenix software suite
`(35), and mutations were made using Coot (54). Geometry Minimization (Phe-
`nix software suite) (35) was used to regularize geometries of the models, with
`500 iterations and 5 macro cycles.
`
`were identified, consistent with previous structural work on
`R132H IDH1. The presence of the C3 carboxylate in ICT
`requires some adjustment of the catalytic residue Tyr-139,
`whereas less movement of this residue is seen in the models
`containing ␣KG.
`To explore the mechanistic features of these IDH1 mutations
`identified in tumors, cDNA constructs were generated for het-
`erologous expression and purification in Escherichia coli. WT,
`R132H, R132C, R132G, R100Q, A134D, and H133Q IDH1
`homodimers were expressed and purified to ⬎95% purity
`
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`(supplemental Fig. S1A). Thermal stability was assessed for
`each enzyme using circular dichroism to perform thermal shift
`assays. The melting temperature (Tm) of each of the IDH1
`mutants varied little compared with WT IDH1 (supplemental
`Fig. S2A). R132C IDH1 had the lowest Tm (46.8 °C), but this
`represents only a 5% change from WT IDH1 (Tm ⫽ 49.1 °C).
`R100Q IDH1 had the highest Tm (51.9 °C), again signifying only
`a 5% change from WT IDH1.
`
`Efficiency of reactions catalyzed by IDH1 mutants found in
`tumors
`The most common IDH1 mutations found in gliomas are
`R132H followed by R132C (37). R132G, which has been identi-
`fied with higher frequency in chondrosarcomas (38, 39), is less
`frequently seen in gliomas and is a known D2HG producer (30).
`R100Q IDH1, a long predicted D2HG producer based on the
`R140Q IDH2 mutation affecting the identical residue, is rela-
`tively rare (40). A134D and H133Q IDH1 are rare mutations
`found in thyroid cancers and are predicted to only be deficient
`in the normal reaction (31, 40). Steady-state kinetic assays were
`used to determine the catalytic efficiency of the conversion of
`ICT to ␣KG (normal reaction) by monitoring the production of
`NADPH at A340 nm, or ␣KG to D2HG (neomorphic reaction) by
`monitoring the consumption of NADPH, at both 21 and 37 °C.
`All mutants were deficient in the normal reaction, ranging from
`a relatively minor 3.5-fold loss of catalytic efficiency (kcat/Km)
`for H133Q IDH1, whereas the other mutations exhibited more
`severe ⬃300- to 1,340-fold losses in efficiency (Fig. 3, supple-
`mental Fig. S3, Table 1). The observed changes in catalytic effi-
`ciency are driven both by decreases in kcat and increases in Km.
`Mutants varied widely in their relative catalytic efficiency of
`D2HG production (Fig. 4, supplemental Fig. S4, Table 2). Only
`rate-saturating concentrations of substrates generated rates
`of D2HG production above the signal-to-noise threshold for
`A134D IDH1 and H133Q IDH1. Thus only upper limits of kcat
`values are reported as kobs (Fig. 4, B and D) because Km values
`could not be obtained. R132G IDH1 is the most efficient pro-
`ducer of D2HG (⬃125-fold more efficient than WT IDH1),
`driven primarily by low Km values but also by a high kcat. R132C
`and R132H IDH1 are ranked next in catalytic efficiency, with a
`low Km value reported for R132C IDH1 (Table 2). This suggests
`that production of D2HG in tumors by R132G and R132C IDH1
`may be more significant than R132H IDH1 when cytosolic con-
`centration of ␣KG is considered. This trend is supported by
`D2HG measurements in glioma tissue (30). Due to its high Km,
`R100Q IDH1 was one of the least efficient producers of D2HG
`(Table 2).
`
`GC/MS analysis confirms D2HG production by IDH1 tumor
`mutants
`Although the normal reaction is reversible (Fig. 1A), a lower
`pH and source of CO2 (typically NaHCO3) are required to favor
`the reverse reaction in vitro, and work by Leonardi et al. (25)
`have shown that the reverse reaction is deficient in IDH1
`mutants. Regardless, we desired to confirm that the less well
`characterized IDH1 mutants, namely R100Q and R132G, favor
`D2HG production over ICT when incubated with ␣KG and
`NADPH. R132H and R132C IDH1 are well established to pro-
`
`Catalytic efficiency of IDH1 mutants
`
`duce D2HG (first reported in Ref. 13, but confirmed by many
`groups). Gas chromatography/mass spectrometry (GC/MS)
`was used to identify and quantify the amount of D2HG as well
`as ␣KG and ICT (not shown) produced in these incubations
`(Fig. 5) (20). R132G IDH1 gave robust production of D2HG
`consistent with kinetic data, whereas an incubation with R100Q
`IDH1 showed D2HG production levels near the lower limit of
`detection, again consistent with kinetic findings (Figs. 4 and 5).
`Levels of ICT for both mutants were ⬍0.1 nmol, based on limits
`of detection. This indicates that NADPH oxidation in the pres-
`ence of ␣KG is preferably coupled to D2HG production under
`these reaction conditions. This also supports previous findings
`that both mutants generate D2HG in in vitro assays (30). These
`experiments do not necessarily indicate that the reverse of the
`normal reaction is ablated, however, as pH ⬍ 7 and CO2 are
`required for this reaction in vitro (25).
`
`Generation of IDH1 mutants engineered to explore
`mechanistic features of D2HG production
`In addition to R132H/R132C/R132G IDH1, several rarer
`IDH1 mutations in gliomas have been identified, including
`R132S/R132L/R132V IDH1 (5, 6, 41, 42). In vitro kinetic assays
`have shown that R132S and R132L IDH1 catalyze production of
`D2HG at rates similar to the more common R132H and R132C
`IDH1 mutations (13, 25). Similarly, ectopic expression of R132S
`and R132L IDH1 in HEK293T cells indicate D2HG production
`levels are comparable with cell lines expressing R132C and
`R132H IDH1 (30). R132H/R132C/R132G/R132S/R132L/
`R132V IDH1 all vary in the degree of hydrophobicity at residue
`132, and all have a smaller van der Waals volume than the
`wild-type arginine. Although these clues illuminate interesting
`mechanistic characteristics of R132H IDH1, the features that
`allow IDH1 mutants to generate D2HG with varying catalytic
`efficiency are not fully clear.
`We designed several IDH1 mutations to serve as tools to
`probe the limits of hydrophobicity (43) and van der Waals vol-
`ume (44) at residue 132 that support D2HG production. R132A
`IDH1 is truly an engineered mutation, as to our knowledge it
`has not been identified in tumors. This residue serves as an
`example of a more hydrophobic and smaller residue at position
`132, similar to R132G IDH1. R132A IDH1 has been shown to be
`deficient in the normal reaction (29), but its ability to catalyze
`the neomorphic reaction has not yet been explored. R132N
`IDH1 also has not been identified in tumors to date. Asparagine
`has a much smaller van der Waals volume than arginine,
`although the ranked polarities of these two amino acids are
`similar. R132Q IDH1 plays an important role in driving chon-
`drosarcomas and a small number of gliomas, and mouse
`mR132Q IDH1 generates D2HG about 20-fold more efficiently
`than human R132H IDH1 in vitro (39, 45). This mutation has
`the most similar ranking in hydrophobicity as compared with
`WT, but a smaller van der Waals volume. R132K IDH1 is ho-
`mologous to R172K IDH2, one of the most common D2HG-
`producing mutations seen in acute myeloid leukemia (11, 41).
`However, R132K IDH1 has not been reported in tumors, and
`the activity of this enzyme has not been assessed. R132K IDH1
`is most comparable with WT IDH1 when considering both van
`der Waals volume and polarity ranking of residue 132. Finally,
`
`J. Biol. Chem. (2017) 292(19) 7971–7983 7973
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`Catalytic efficiency of IDH1 mutants
`
`Figure 3. Concentration dependence of the ICT concentration on the observed rate of NADPH production in the normal reaction (37 °C). The deter-
`mined kobs values were obtained from two different enzyme preparations to ensure reproducibility. The kobs values resulting from each of the two enzyme
`preparations are distinguished by using either a circle or an ⫻ in the plots. The observed rate constants (kobs) were calculated from the linear range of the slopes
`of plots of concentration versus time using GraphPad Prism software (GraphPad, San Diego, CA). These kobs values were then fit to a hyperbolic equation to
`generate kcat and Km values, and the standard error listed in Table 1 results from the deviance from these hyperbolic fits is indicated. The determined kobs values
`were obtained from two different enzyme preparations to ensure reproducibility. Results from assays at 21 °C are shown in supplemental Fig. S3. A, WT IDH1.
`B, H133Q IDH1. C, A134D IDH1. D, R100Q IDH1. E, R132H IDH1. F, R132C IDH1. G, R132G IDH1.
`R132H IDH1 in complex with ␣KG, NADP⫹, and Ca2⫹ (PDB
`R132W IDH1 is another example of an engineered mutation in
`that it has not been identified in tumors. It was selected to
`code 4KZO (27)) using the geometry minimization package in
`represent the most extreme case of a large van der Waals vol-
`Phenix (35) followed by alignment in PyMOL (36) (Fig. 6). Few
`ume coupled with high hydrophobicity.
`changes are observed globally or within the active site. The size
`of the amino acid at position 132 does necessitate some local
`adjustments to avoid steric hindrance, but overall, changes in
`the models are minimal.
`All five IDH1 mutants were successfully heterologously
`expressed and purified to ⬎95% purity (supplemental Fig. S1B).
`
`Structural modeling and thermal stability of engineered IDH1
`mutations
`Because no crystal structures of these mutants are currently
`available, each was modeled in a previously solved structure of
`
`7974 J. Biol. Chem. (2017) 292(19) 7971–7983
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`Catalytic efficiency of IDH1 mutants
`
`Thermal stability was assessed using circular dichroism in ther-
`mal shift assays. Again, minimal changes were observed in Tm
`(supplemental Fig. S2B). R132K IDH1 had the highest Tm
`(49.8 °C), which varied from WT IDH1 by only 2%.
`
`Kinetic analysis of engineered IDH1 mutants
`The catalytic efficiency of the normal reaction was measured
`for all mutants, and efficiencies were plotted against relative
`hydrophobicity according to Monera et al. (43) (Fig. 7A), and
`against van der Waals volume (44) (Fig. 7B). All mutants were
`significantly deficient in converting ICT to ␣KG, driven both by
`a decrease in kcat as well as an increase in Km (Table 3, supple-
`mental Fig. S5). Two IDH1 mutations maintained moderate
`oxidative decarboxylation activity; R132Q and R132K IDH1
`had 33- and 56-fold losses of ␣KG production efficiency relative
`to WT IDH1, respectively. All other mutations had ⱖ220-fold
`decreases in catalytic efficiency.
`IDH1 mutants were also incubated with ␣KG and NADPH to
`measure presumptive D2HG production efficiency (Fig. 7,
`Table 4, supplemental Fig. S6). R132Q IDH1 was the most effi-
`cient D2HG producer of the mutants explored in this work,
`with 4-fold higher efficiency than the next most efficient
`mutant, R132G IDH1. There was a notable decrease in effi-
`ciency in all other mutants, with R132A IDH1 having similar
`catalytic efficiencies as R132G/R132C/R132H IDH1. R132N/
`R132K/R132W and WT IDH1 were all very poor at producing
`D2HG. The severely deficient catalytic efficiency seen in R132N
`IDH1 was primarily driven by a very high Km value (Table 4).
`This suggests that like R100Q IDH1, D2HG production by
`R132N IDH1 may not be physiologically relevant when the
`cytosolic concentration of ␣KG is considered. Relative efficien-
`cies of the other mutants were driven both by changes in kcat
`and Km, with a low Km value driving R132A IDH1 production
`(Table 4).
`
`GC/MS analysis confirms D2HG production by engineered
`IDH1 mutants
`To confirm that an incubation of the engineered IDH1
`mutants with ␣KG and NADPH favors D2HG production
`rather than the reverse of the normal reaction (i.e. ICT produc-
`tion), GC/MS was used to quantify levels of D2HG, ICT, and
`␣KG of the engineered IDH1 mutants. Measured amounts of
`D2HG were as expected under the incubation lengths at exper-
`imentally measured kinetic efficiency. R132Q and R132G IDH1
`generated the highest levels of D2HG (Fig. 5), followed by
`R132A IDH1. Again, levels of ICT were difficult to measure due
`to their very low concentrations (⬍0.1 nmol, based on limits of
`detection). This suggests that NADPH oxidation is coupled
`primarily to D2HG production, rather than ICT, under these
`experimental conditions.
`
`Discussion
`Here we report the first in-depth, simultaneous catalytic
`characterization of 11 IDH1 mutations and WT IDH1, includ-
`ing mutations identified in tumors (R132H/R132C/R132G/
`R132Q, R100Q, A134D, and H133Q IDH1) and additional
`mutations (R132A/R132K/R132N/R132W IDH1) designed to
`measure the effects of hydrophobicity (43) and van der Waals
`
`J. Biol. Chem. (2017) 292(19) 7971–7983 7975
`
`0.28⫾0.05
`0.30⫾0.05
`0.020⫾0.003
`0.16⫾0.02
`0.074⫾0.003
`35⫾9
`7.3⫾1.3⫻102
`mM⫺1s⫺1
`
`0.14⫾0.02
`0.58⫾0.08
`1.0⫾0.9
`0.070⫾0.006
`0.7⫾0.1
`0.101⫾0.008
`0.03⫾0.01
`
`mM
`
`3.6⫾0.6
`5.3⫾0.8
`6⫾1
`9⫾1
`2.7⫾0.3
`0.28⫾0.07
`0.015⫾0.003
`
`1.0⫾0.06
`1.61⫾0.08
`0.120⫾0.006
`1.40⫾0.06
`0.200⫾0.008
`9.4⫾0.6
`11.0⫾0.4
`s⫺1
`
`1.3⫾0.2
`0.54⫾0.05
`0.57⫾0.08
`0.7⫾0.2
`0.29⫾0.08
`1.1⫾0.2⫻102
`3.9⫾0.4⫻102
`mM⫺1s⫺1
`
`0.067⫾0.007
`0.75⫾0.07
`1.6⫾0.5
`0.18⫾0.02
`1.2⫾0.3
`0.16⫾0.02
`0.08⫾0.03
`
`mM
`
`7⫾1
`8.2⫾0.8
`4.2⫾0.6
`8⫾2
`8⫾2
`0.40⫾0.08
`0.22⫾0.02
`
`9.3⫾0.6
`4.4⫾0.1
`2.4⫾0.1
`5.6⫾0.4
`2.3⫾0.2
`45⫾2
`85⫾4
`s⫺1
`
`48(Gly)
`86(Cys)
`118(His)
`148(Arg)
`148(Arg)
`148(Arg)
`148(Arg)
`
`0(Gly)
`49(Cys)
`8(His)
`⫺14(Arg)
`⫺14(Arg)
`⫺14(Arg)
`⫺14(R)
`
`R132G
`R132C
`R132H
`R100Q
`A134D
`H133Q
`WT
`
`bFromRef.44.
`aFromRef.43.
`
`(kcat/Km,ICT,21°C)
`Efficiency,mMⴚ1sⴚ1
`
`(21°C)
`Km,ICT
`
`(21°C)
`kcat
`
`(kcat/Km,ICT,37°C)
`
`Efficiency,
`
`(37°C)
`Km,NADPⴙ
`
`(37°C)
`Km,ICT
`
`(37°C)
`kcat
`
`vanderWaalsvolume
`
`residue132,Å3b
`ofsidechainat
`
`(21°C)
`Km,NADPⴙ
`Valuesresultfromfitsofkineticdatausingtwodifferentenzymepreparations.Thestandarderrorisdeterminedfromthedeviancefromthesehyperbolicfits(Fig.3,supplementalFig.S3).
`Kineticparametersforthenormalreaction,conversionofICTto␣KG,catalyzedbyIDH1
`Table1
`
`hydrophobicityof
`
`residue132a
`
`Relative
`
`IDH1
`
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`
`

`

`Catalytic efficiency of IDH1 mutants
`
`Figure 4. Concentration dependence of ␣KG concentration on the observed rate of NADPH depletion in the neomorphic reaction (37 °C). The
`determined kobs values were obtained from two different enzyme preparations to ensure reproducibility. The kobs values resulting from each of the two enzyme
`preparations are distinguished by using either a circle or an ⫻ in the plots. The observed rate constants (kobs) were calculated from the linear range of the slopes
`of plots of concentration versus time using GraphPad Prism software (GraphPad). These kobs values were then fit to a hyperbolic equation to generate kcat and
`Km values, and the S.E. results from the deviance from these hyperbolic fits is indicated. Km values and efficiency are in terms of [␣KG]. Due to limits of detection,
`Km values could not be obtained for low efficiency IDH1 enzymes because only saturating kobs rates could be detected. In this case, kobs rates are reported,
`which approximate kcat rates. Results from assays at 21 °C are shown in supplemental Fig. S4. A, WT IDH1. B, H133Q IDH1. D, R100Q IDH1. E, R132H IDH1. F, R132C
`IDH1. G, R132G IDH1.
`volume (44) at residue 132 on catalytic efficiency. To date, a
`relatively wide range of catalytic rates have been reported for
`WT and mutant IDH1. For WT IDH1, kcat values for the normal
`reaction typically range from ⬃9 to 12 s⫺1 (26, 27, 29) at ambi-
`ent temperature. This is in good agreement with our findings
`(Table 1), although there are reports of much higher rates (13,
`15). Km values for both ICT and NADP⫹ typically range from 5
`to 65 ␮M at ambient temperature (13, 15, 25–27, 29), again
`
`consistent with our values (Table 1). Plasma levels of ␣KG have
`been reported to be ⬃23 ␮M (46), which is in line with measured
`Km values.
`H133Q IDH1 displays a relatively minor change in catalytic
`efficiency for ␣KG production compared with WT IDH1, and
`D2HG production was extremely slow, indicating that this may
`be a passenger mutation (Tables 1 and 2). R100Q IDH1 shows
`drastic increases in Km values both for ICT and ␣KG, resulting
`
`7976 J. Biol. Chem. (2017) 292(19) 7971–7983
`
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`

`Catalytic efficiency of IDH1 mutants
`
`Figure 5. Absolute quantitation of D2HG present in an incubation of
`IDH1 mutants with ␣KG and NADPH. Measurements are reported as a cal-
`culated mean ⫾ S.E. Only trace amounts of ICT (⬍0.1 nmol, based on limits of
`detection) were generated under these experimental conditions, indicating
`that NADPH oxidation was coupled to D2HG production, rather than ICT
`production.
`
`Figure 6. Structural models of experimental IDH1 mutants. The structure
`of R132H IDH1 complexed with ␣KG, NADP⫹, and Ca2⫹ (PDB 4KZO (27)) was
`used to model mutations of the tool IDH1 mutations. R132H IDH1 is shown in
`orange, R132Q in magenta, R132N in cyan, R132A in dark blue, R132K in black,
`and R132W in purple. Substrates and residues that are mutated are high-
`lighted in stick format, as well as catalytic residue Tyr-139. Ca2⫹ is shown as a
`sphere. Ligand restraint generation and optimization of provided cif files were
`generated using eLBOW in the Phenix software suite (35), and mutations
`were made using Coot (54). Geometry Minimization (Phenix software suite)
`(35) was used to regularize geometries of the models, with 500 iterations and
`5 macro cycles.
`
`in significant decreases in catalytic efficiency for both reactions
`studied (Tables 1 and 2). This was surprising because the ho-
`mologous mutation in IDH2, R140Q, is the most common IDH
`mutation found in AML (47). However, kinetic characteriza-
`tions of IDH2 mutants are limited (28, 48). Kinetic and struc-
`
`J. Biol. Chem. (2017) 292(19) 7971–7983 7977
`
`mM⫺1s⫺1
`
`1.5⫾0.2
`2.3⫾0.4
`0.24⫾0.08
`0.013⫾0.001
`ND
`ND
`ND
`
`(kcat/Km,␣KG,21°C)
`
`Efficiency
`
`ⱕ0.025
`ⱕ0.025
`ⱕ0.005
`ⱕ0.0025
`ND
`ND
`ND
`
`(21°C)
`Km,NADPH
`
`mM
`
`0.30⫾0.05
`0.36⫾0.06
`1.8⫾0.6
`10⫾1
`ND
`ND
`NDc
`
`(21°C)
`Km,␣KG
`
`0.45⫾0.02
`0.84⫾0.03
`0.43⫾0.04
`0.128⫾0.006
`ⱕ0.020
`ⱕ0.034
`ⱕ0.017
`s⫺1
`
`sⴚ1(21°C)
`
`kcat,
`
`mM⫺1s⫺1
`
`5⫾1
`4.4⫾0.6
`3.8⫾0.9
`0.028⫾0.005
`ND
`ND
`0.04⫾0.02
`
`⬍0.025
`0.010⫾0.009
`ⱕ0.025
`0.005⫾0.003
`ND
`ND
`ⱕ0.010
`
`mM
`
`0.34⫾0.08
`0.36⫾0.05
`1.1⫾0.3
`12⫾2
`ND
`ND
`0.5⫾0.3
`
`(kcat/Km,␣KG,37°C)
`
`Efficiency
`
`(37°C)
`Km,NADPH
`
`(37°C)
`Km,␣KG
`
`1.59⫾0.09
`1.60⫾0.07
`4.2⫾0.3
`0.34⫾0.02
`ⱕ0.019
`ⱕ0.016
`0.019⫾0.001
`s⫺1
`
`kcat(37°C)
`
`cND,notdetermined.
`bFromRef.44.
`aFromRef.43.
`
`R132G
`R132C
`R132H
`R100Q
`A134D
`H133Q
`WT
`
`IDH1
`
`Valuesresultfromfitsofkineticdatausingtwodifferentenzymepreparations.Thestandarderrorisdeterminedfromthedeviancefromthesehyperbolicfits(Fig.4,supplementalFig.S4).
`Kineticparametersfortheneomorphicreaction,conversionof␣KGtoD2HG,catalyzedbyIDH1
`Table2
`
`48(Gly)
`86(Cys)
`118(His)
`148(Arg)
`148(Arg)
`148(Arg)
`148(Arg)
`
`0(Gly)
`49(Cys)
`8(His)
`⫺14(Arg)
`⫺14(Arg)
`⫺14(Arg)
`⫺14(Arg)
`
`atresidue132,Å3b
`volumeofsidechain
`
`vanderWaals
`
`hydrophobicityof
`
`residue132a
`
`Relative
`
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`
`

`

`Catalytic efficiency of IDH1 mutants
`
`Figure 7. Comparisons of catalytic efficiency by IDH1 with mutations at residue 132. The observed rate constants (kobs) were calculated from the linear
`range of the slopes of plots of concentration versus time, and then fit to a hyperbolic equation to generate kcat and Km values. All experiments were performed
`at 37 °C. These catalytic parameters result from fits of kinetic data resulting from two different enzyme preparations to ensure reproducibility. A, relative
`catalytic efficiencies (kcat/Km) of the conversion of ICT to ␣KG using Km values for ICT are plotted against relative hydrophobicity (43). B, relative catalytic
`efficiencies (kcat/Km) of the conversion of ICT to ␣KG using Km values for ICT are plotted against van der Waals volume (44). C, relative catalytic efficiencies
`(kcat/Km) of the conversion of ␣KG to D2HG using Km values for ␣KG are plotted against relative hydrophobicity (43). D, relative catalytic efficiencies (kcat/Km) of
`the conversion of ␣KG to D2HG using Km values for ␣KG are plotted against van der Waals volume (44).
`
`Table 3
`Kinetic parameters for the normal reaction, conversion of ICT to ␣KG, catalyzed by IDH1
`Values result from fits of kinetic data using two different enzyme preparations. The standard error is determined from the deviance from these hyperbolic fits (supplemental
`Fig. S5).
`Km,ICT
`kcat
`(37 °C)
`(37 °C)
`sⴚ1
`mM
`1.21 ⫾ 0.08
`3.6 ⫾ 0.6
`10.4 ⫾ 0.2
`5.7 ⫾ 0.4
`9.2 ⫾ 0.3
`0.8 ⫾ 0.2
`7.2 ⫾ 0.4
`1.1 ⫾ 0.2
`0.047 ⫾ 0.001
`1.5 ⫾ 0.1
`
`Relative hydrophobicity
`of residue 132a
`
`van der Waals volume of side
`chain at residue 132, Å3 b
`
`97 (Trp)
`41 (Ala)
`⫺10 (Gln)
`⫺23 (Lys)
`⫺28 (Asn)
`
`163 (Trp)
`67 (Ala)
`114 (Gln)
`135 (Lys)
`96 (Asn)
`
`IDH1
`
`R132W
`R132A
`R132Q
`R132K
`R132N
`a From Ref. 43.
`b From Ref. 44.
`
`Efficiency
`(kcat/Km,ICT, 37 °C)
`mMⴚ1 sⴚ1
`0.34 ⫾ 0.06
`1.8 ⫾ 0.1
`12 ⫾ 3
`7 ⫾ 1
`0.031 ⫾ 0.008
`
`Table 4
`Kinetic parameters for the neomorphic reaction, conversion of ␣KG to D2HG, catalyzed by IDH1
`Values result from fits of kinetic data using two different enzyme preparations. The standard error is determined from the deviance from these hyperbolic fits (supplemental
`Fig. S6).
`Km,␣KG
`kcat
`(37 °C)
`(37 °C)
`sⴚ1
`mM
`0.54 ⫾ 0.01
`0.82 ⫾ 0.08
`0.37 ⫾ 0.01
`0.11 ⫾ 0.02
`4.7 ⫾ 0.2
`0.26 ⫾ 0.04
`0.57 ⫾ 0.02
`0.61 ⫾ 0.07
`0.79 ⫾ 0.06
`10 ⫾ 2
`
`Relative hydrophobicity
`of residue 132a
`
`van der Waals volume of side
`chain at residue 132, Å3 b
`
`97 (Trp)
`41 (Ala)
`⫺10 (Gln)
`⫺23 (Lys)
`⫺28 (Asn)
`
`163 (Trp)
`67 (Ala)
`114 (Gln)
`135 (Lys)
`96 (Asn)
`
`IDH1
`
`R132W
`R132A
`R132Q
`R132K
`R132N
`a From Ref. 43.
`b From Ref. 44.
`
`Efficiency
`(kcat/Km,␣KG, 37 °C)
`mMⴚ1 sⴚ1
`0.659 ⫾ 0.007
`3.4 ⫾ 0.6
`18 ⫾ 3
`0.9 ⫾ 0.1
`0.08 ⫾ 0.02
`
`tural comparisons for R100Q IDH1 and R140Q IDH2 will be
`important for characterizing any mechanistic differences
`between these homologous mutations. Currently, crystal struc-
`tures of R140Q IDH2 are limited to complexes with inhibitors
`(49). R100Q IDH1 has been characterized as a D2HG-producer,
`but our data suggests this mutant does so only weakly.
`Similarly, R132K IDH1 is homologous to R172K IDH2, the
`second most common IDH2 mutation identified in AML (50).
`Thus the relative catalytic inefficiency of D2HG production by
`R132K IDH1 (Table 4) was also surprising. Kinetic and struc-
`
`tural analysis of R132K IDH1 and R172K IDH2 will also be
`critical for understanding any functional differences between
`these homologous mutations. Nearly negligible in vitro D2HG
`production by R132K and R100Q IDH1 may explain why these
`mutations are rare (or not identified) in gliomas, despite being
`frequently observed as R172K and R140Q IDH2 in AML.
`As noted, Km values for D2HG production for some IDH1
`mutants are higher than physiologically relevant ␣KG concen-
`trations. However, IDH1 mutations are found heterozygously
`in tumors, and a caveat to this work in that mutant IDH1
`
`7978 J. Biol. Chem. (2017) 292(19) 7971–7983
`
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`

`homodimers were studied. For IDH1 mutant/WT hetero-
`dimers, local concentration of ␣KG may be much higher
`due to production of this metabolite at the WT IDH1 mono-
`mer, particularly if substrate channeling occurs. Furthermore,
`Km values may be lower overall due to favorable substrate bind-
`ing at the WT monomer of the heterodimer. Ward et al. (28)
`have shown that D2HG production by IDH1 mutations in cells
`is increased if WT IDH1 activity is retained. However, in cases
`of very high Km values such as those observed for R100Q IDH1,
`it is possible that the reaction measured is not physiologically
`relevant, even in heterodimer