THE JOURNAL OF BIOLOGICAL CHEMISTRY
`Vol. 250, No. 16, Issue of August 25, pp. 6351-6354, 1975
`Printed in U.S.A.
`
`a-Methylisocitrate
`
`A SELECTIVE INHIBITOR OF TPN-LINKED ISOCITRATE DEHYDROGENASE FROM BOVINE
`HEART AND RAT LIVER*
`
`GERHARD w. E. PLAUT, RICHARD L. BEACH,:j: AND TADASHI AOGAICHI
`From the Department of Biochemistry, Temple University School of Medicine, Philadelphia, Pennsylva(cid:173)
`nia 19140, and the Chemistry Department, Rider College, Trenton, New Jersey 08602
`
`(Received for publication, February 24, 1975)
`
`a-Methylisocitrate (3-hydroxy-1,2,3-butanetricarboxylate) is a potent inhibitor, competitive with
`isocitrate ( l-hydroxy-1,2,3-propanetricarboxylate), of the TPN-linked isocitrate dehydrogenase from
`bovine heart and rat liver; it does not inhibit the DPN-specific enzyme from these tissues. In the presence
`of magnesium ion, values of K;s for DL-a-methylisocitrate for purified bovine heart enzyme, rat liver
`cytosol, and rat liver mitochondrial extract were in the range of 0.1 µM to 0.3 µM. This compared to values.
`of apparent Km for DL-isocitrate for the same tissue preparations of 14 µM to 20 µM. One of the DL isomer
`pairs of a-methylisocitrate was inactive; the observations suggest that it is threo-a-methylisocitrate
`which inhibits TPN-linked isocitrate dehydrogenase. A method of synthesis of DL-threo-a-methylisocitric
`lactone (2-methyl-5-oxo-2,3-furandicarboxylic acid) from dimethyl trans-epoxymethylsuccinate and
`dimethylmalonate is described.
`
`In a study of the substrate activities of a-hydroxy-a,,6-dicar(cid:173)
`boxylic acid derivatives for
`isocitrate dehydrogenases (1)
`attention was focused on the effect of substituents at the ,6
`carbon of these derivatives. It was found that addition of a
`methylene group to isocitrate, resulting in the higher homo(cid:173)
`logue homoisocitrate, led to retention of substrate activity for
`the DPN-specific isocitrate dehydrogenase, but not the TPN(cid:173)
`linked enzyme from bovine heart. o-Garcinia acid (2R,3R-2-
`hydroxycitric acid), in which the ,6-hydrogen of o-threo-isocit(cid:173)
`rate is substituted by a hydroxyl group was a substrate for
`both isocitrate dehydrogenases.
`Substitution of the a-hydrogen of isocitrate would abolish
`the substrate activity of such analogues but might not prevent
`binding to the enzyme. Such analogues might be useful for
`elucidating the mechanism of action and the metabolic role of
`the isocitrate dehydrogenases. Formation of a-methylisocitrate
`and methylcitrate from a-methyl-cis-aconitate by the action of
`aconitase had been reported by Gawron and Mahajan (2).
`a-Methylisocitrate prepared in this manner was cleaved to
`pyruvat,e and succinate by isocitrate lyase from several orga(cid:173)
`nisms (3) The differential inhibition of the DPN-linked and
`TPN-specific isocitrate dehydrogenases from heart and liver by
`chemically synthesized preparations of a-methylisocitrate is
`the subject of the present report.
`
`* This work was supp,irted in part by Grant AM 15404 from the
`National Institute of Arthritis, Metabolic' and Digestive Diseases,
`National Institutes of Health.
`:j: This work was performed at Temple University in 1973 to 1974 dur(cid:173)
`ing a period of sabbatical leave of R. L. B. from Rider College.
`
`EXPERIMENTAL PROCEDURE
`
`Materials
`DPN-linked isocitrate dehydrogenase was purified to homogeneity
`from lyophilized bovine heart mitochondria (4). TPN-linked isocitrate
`dehydrogenase from the extramitochondrial fraction of bovine heart
`homogenate was purified by a modification of the method of Rose (5).
`The final preparation had a specific activity of 8 µmo! of TPNH-formed
`min- 1 mg- 1 protein at 25° under the conditions of assay described by
`Siebert et al. (6). Rat liver mitochondria were prepared by the method
`of Schneider and Hogeboom (7), but in a medium containing 225 mM
`mannitol, 75 mM sucrose, and 0.1 mM EDTA. The cytosol fraction of the
`liver homogenate was clarified by centrifugation at 38,000 x g for 60
`min before assay of TPN-specific isocitrate dehydrogenase activity.
`Extracts of rat liver mitochondria were prepared by sonication at 5-8°
`in a medium containing 225 mM mannitol, 75 mM sucrose, and 0.05
`mM EDTA at pH 7.0, followed by centrifugation at 150,000 x g for 30
`min to remove submitochondrial particles.
`
`Assays
`DPN-linked isocitrate dehydrogenase activity was determined spec(cid:173)
`trophotometrically at 25° as described previously (4), in a medium
`containing 166 mM Na-Hepes 1 at pH 7.4, 0.3 mM ADP, and 0.33 mM
`DPN+; the concentrations of DL-threo-isocitrate, divalent metal ion
`activators, and a-methylisocitrate were as reported in the text and
`table. TPN-linked isocitrate dehydrogenase in soluble enzyme prepa(cid:173)
`rations was determined at 25°, as described by Siebert et al. (6) in a
`medium containing 167 mM Na-Hepes at pH 7.4 and 0.10 mM TPN +;
`the concentrations of DL-threo-isocitrate, divalent metal ions, and
`a-methylisocitrate are reported in the legends of tables and figures.
`TPN-linked isocitrate dehydrogenase of intact liver mitochondria was
`determined under the conditions of Hogeboom and Schneider (8).
`
`1 The abbreviation used is: Hepes, N-2-hydroxyethylpiperazine-N'-
`2-ethanesulfonic acid.
`
`This is an Open Access article under the CC BY license.
`
`6351
`
`Rigel Exhibit 1036
`Page 1 of 4
`
`

`

`6352
`
`Kinetic parameters for the soluble enzyme preparations were
`obtained by fitting the experimental initial velocities to the appropri(cid:173)
`ate equation using the computer programs developed by Cleland (9).
`a-Methylisocitric lactone and isocitric lactone were hydrolyzed
`under alkaline conditions before assay, as described previously (10).
`
`Preparation of a-Methylisocitrate
`DL-a-Methylisocitric lactone (2-methyl-5-oxo-2,3-furandicarboxylic
`acid) was prepared from dimethyl trans-epoxymethylsuccinate and
`dimethylmalonate by an adaptation of the procedure of Gawron et al.
`(11) for the preparation of isocitric lactone. This synthesis, described
`below, should favor the formation of the DL-threo compound, and the
`product showed a single component by electrophoresis on Whatman
`No. 3MM paper (or ChromAR) in 0.06 M pyridine 0.79 M acetate buffer
`at pH 3.5 (1.5 hours at 1900 volts). a-Methylisocitric lactone syn(cid:173)
`thesized by the procedure of Rach (12) exhibited two components by
`electrophoresis and could be separated into two fractions (Fraction A
`and Fraction B) by fractional crystallization from ethyl acetate. The
`elementary analysis and neutralization equivalents of either fraction
`corresponded to the composition expected for a-methylisocitric lac(cid:173)
`tone. However, the single component found by paper electrophoresis
`of Fraction B corresponded in migration to the compound synthe(cid:173)
`sized by the method described below, whereas Fraction A migrated
`more rapidly toward the anode in buffers between pH 3 and pH 4. Fur(cid:173)
`thermore, the NMR spectra of the newly synthesized compound and
`Fraction B were identical, but differed from Fraction A.
`
`2-Methyl-5-oxo-2,3-furandicarboxylic Acid (a-Methylisocitric--y(cid:173)
`lactone)
`trans-epoxymethylsuccinate-Diazomethane generated
`Dimethyl
`from Diazald is distilled into a 500-ml Erlenmeyer flask containing
`7.21 g of trans-epoxymethylsuccinic acid,, t9 mmol) (13) suspended in
`20 ml of anhydrous diethyl ether. The reaction mixture is cooled in an
`ice bath and stirred with a magnetic stirrer throughout the addition.
`Diazomethane addition is stopped when the reaction reaches comple(cid:173)
`tion, as indicated by the yellow color of diazomethane in the reaction
`flask. The excess diazomethane is destroyed by the addition of 2 to 3
`drops of glacial acetic acid, and the ether is removed by distillation,
`yielding 8.85 g of a clear syrup. The product (5.6 g, 67%) may be
`distilled at 80° 0.9 torr.
`
`C,H 100,
`Calculated: C 48.28, H 5.79
`Found
`C 48.26, H 5.69
`
`2-M e thy l-5-oxo-2, 3, 4-tric arbom et hoxy te trahydrof uran-To a
`stirred solution of sodium methoxide at 5° (prepared from 0.7 g of
`sodium metal (0.03 g atom) and 15 ml of dry methanol) are added 4.01 g
`(30.4 mmol) of redistilled dimethylmalonate. After a white precipitate
`forms, 5.3 g (30.4 mmol) of dimethyl trans-epoxymethylsuccinate are
`added, and the mixture is stirred for 3 days at room temperature.
`Concentrated HCl (75 ml) is then added to the reaction mixture, and
`stirring is continued for an additional 2 hours. The NaCl precipitate is
`removed by filtration, and the reaction mixture is concentrated to a
`syrup at 45° under reduced pressure. The product (4.3 g, 51.5%) distills
`at 160-170° at 1. 7 torr.
`
`C 11H,.O,
`Calculated: C 48.18, H 5.15
`Found:
`C 48.15, H 4.67
`
`2-Methyl-5-oxo-tetrahydrofuran-2,3-dicarboxylic acid-One gram
`(3.65 mmol) of 2-methyl-5-oxo-2,3,4-tricarbomethoxytetrahydrofuran
`is refluxed in 40 ml of 6 N HCl for 3 hours. The solvent is evaporated in
`a vacuum at 50°, and residual HCl is removed completely from the
`product by repeated vacuum distillations following addition of 10-ml
`portions of water to the residue. The solid is sublimed at 180° under
`reduced pressure (1.5 torr). A solution of the solid in acetone is treated
`with Norit A, filtered, and concentrated to dryness. The residue is
`heated for 1 hour at 100° under reduced pressure (1 torr). The
`substance is crystallized from a mixture of 0. 7 ml of acetone and 7 ml of
`benzene. The crystalline compound was recovered in a yield of 242 mg
`(35%).
`The following analytical data were obtained for the a-methylisocit(cid:173)
`ric lactone preparations.
`
`C,H,O,
`Calculated: C 44.69, H 4.29
`
`(Equivalent weight calculated cold: 94.1 calculated heated excess base,
`62.7).
`
`Present preparation
`Found: C 44.40, H 4.32
`
`Melting point 183-185° decomposition; equivalent weight cold, 95.9;
`equivalent weight heated excess base, 65.5. NMR (acetone d,) ppm
`1.88 (singlet, :m), 2.88-3.19 (multiplet, 2H), 3.50-3.97 (multiplet, lH).
`
`Rach preparation
`Found: C 44.67, H 4.32
`
`Melting point, 168°-171 ° (decomposition), literature 168°. Equiva(cid:173)
`lent weight (cold) 94.1; equivalent weight (heated) 67.9.
`
`Fraction A
`Found: C 44.97, H 4.38
`
`Melting point 196-202° (decomposition); equivalent weight (cold)
`94.5; equivalent weight (heated) 64.7. NMR (acetone d,) ppm 1.70
`(singlet, 3H), 2.93-3.07 (doublet, 2H, J ~ 6 cps), 3.77-3.99 (triplet, 1H
`J ~ 6 cps).
`
`Fraction B: melting point 183-186° (decomposition) equivalent
`weight (cold) 96.6; equivalent weight (heated) 64.1. NMR identical to
`preparation synthesized by new procedure.
`Unless specified otherwise, DL-a-methylisocitrate prepared by the
`new method was used in these studies.
`
`RESULTS AND DISCUSSIONS
`
`TPN-linked Isocitrate Dehydrogenase-a-Methylisocitrate
`was an effective inhibitor for the dehydrogenase from rat liver
`cytosol and for enzyme purified from the extramitochondrial
`fraction of bovine heart. The inhibition was competitive with
`respect to isocitrate (Fig. 1). When activated by magnesium
`ion, apparent Km for DL-isocitrate ranged from 13.9 µM to 20 µM
`and K;s for DL-a-methylisocitrate was between 0.23 µMand 0.30
`µM for the heart enzyme; the corresponding values for liver
`cytosol were 14.7 µM to 18.5µMfor Km and about0.lOµMfor K;,
`(Table I).
`a-Methylisocitrate also inhibited the TPN-specific isocitrate
`dehydrogenase activity from rat liver mitochondria, and under
`identical assay conditions, the values of apparent Km for
`isocitrate and of K;, for a-methylisocitrate were nearly identi(cid:173)
`cal for enzyme activities of extracts of sonicated mitochondria
`and of the extramitochondrial fraction from rat liver homoge(cid:173)
`nate. In the presence of 4.0 mM magnesium ion, the values were
`15.9 µMand 14.7 µM for Km and 0.10 µMand 0.10 µM for K; with
`the mitochondrial extract and the supernatant fraction, re(cid:173)
`spectively (Table I). Inhibition by a-methylisocitrate of the
`TPN-linked isocitrate dehydrogenase activity of intact rat liver
`mitochondria was observed when assayed by the procedure of
`Hogeboom and Schneider (8). However, measurements with
`the suspensions were not precise enough for kinetic comparison
`with activities of the rat liver extract preparations.
`a-Methylisocitrate is a very potent inhibitor of TPN-linked
`isocitrate dehydrogenase; depending on incubating conditions,
`Km!Kts ratios between 10 and 185 were observed (Table I). The
`values of apparent Km for isocitrate were lower with Mn 2 + than
`Mgz+, in accord with previous results that Mn 2+ is a more
`effective activator of the heart enzyme than Mgz+ (14);
`however, K;, for a-methylisocitrate was lower when Mgz+ was
`used instead of Mn2+ for activation (Table I). A similar,
`although less pronounced, effect of the divalent cations on
`inhibition constants was obsnved with the enzyme activity
`
`Rigel Exhibit 1036
`Page 2 of 4
`
`

`

`from liver cytosol. The chelate of isocitrate with divalent metal
`ion activators has been reported to be the actual substrate for
`TPN-linked isocitrate dehydrogenase from porcine heart (15).
`It is possible that the chelates of a-methylisocitrate are
`likewise the actual inhibitors of the enzyme. The stability
`constants of divalent metal complexes of a-methylisocitrate
`
`I
`V
`
`'
`
`C
`E
`0 ...
`....
`<I
`<l
`
`140
`
`130
`
`120
`
`110
`
`100
`
`90
`
`80
`
`70
`
`40
`
`30
`
`20
`
`10
`
`0
`0
`
`30
`
`20
`10
`1/DL-ISOCITRATE, 14 M-I
`Fm. 1. Inhibition of TPN-linked isocitrate dehydrogenase. Condi(cid:173)
`tions of incubation are described under "Experimental Procedure."
`Data are shown as double reciprocal plots of velocity versus DL-isocit(cid:173)
`rate, and the fixed concentrations of DL-a-methylisocitrate (µM) are
`shown above the lines. - -, purified enzyme from bovine heart
`incubated in the presence of 6.67 mM magnesium ion. Calculated
`values of apparent Km for DL-isocitrate and K,. for DL-a-methylisocit(cid:173)
`rate were 20.0 ± 2.0 µM and 0.30 ± 0.05 µM, respectively. - - -,
`supernatant fraction from rat liver cytosol incubated in the presence of
`ion. Calculated values of apparent Km for
`4.0 mM magnesium
`DL-isocitrate and K,, for DL-a-methylisocitrate were 14.7 ± 1.47 µMand
`0.096 ± 0.006 µM, respectively.
`
`6353
`
`are unknown; however, the large excess of divalent cations over
`a-methylisocitrate and isocitrate in the assay media would
`the metal
`favor most of the a-methylisocitrate being in
`complex form under the incubation conditions used (Table I).
`DPN-linked Isocitrate Dehydrogenase-In contrast to the
`to a(cid:173)
`marked sensitivity of the TPN-specific enzyme
`methylisocitrate, the DPN-linked enzyme was essentially un(cid:173)
`affected . It is difficult to demonstrate complete inactivity of
`the analogue, since it competes with isocitrate for activating
`divalent cations. Kinetic studies have suggested that magne(cid:173)
`sium isocitrate- is the actual substrate for the DPN-specific
`enzyme from bovine heart, and that free Mg 2 + causes inhibi(cid:173)
`tion which is competitive with respect to magnesium isocit(cid:173)
`relatively high concentrations of a(cid:173)
`the
`(16). At
`rate
`methylisocitrate necessary to demonstrate inhibition, decrease
`of activity could be due to depletion of the substrate isocitrate
`complex . In the experiments shown (Table II), the ratios of
`concentrations of total isocitrate to total divalent metal ions
`were adjusted to maintain relatively low constant concentra(cid:173)
`tions of inhibiting free divalent cations as the concentrations of
`substrate and a-methylisocitrate varied . Under these condi(cid:173)
`tions, a-methylisocitrate appeared inert as an inhibitor of the
`enzyme from rat liver mitochondria and from bovine heart with
`either Mg or Mn as activator.
`Activity of Isomers of a-Methylisocitrate-Preparation of
`a-methylisocitrate by the cyanohydrin synthesis of Rach (12)
`should yield DL-threo-a-methylisocitrate and DL-erythro-a(cid:173)
`methylisocitrate (Scheme I) in about equal amounts. a-Methyl(cid:173)
`isocitrate synthesized by the method of Rach was about one(cid:173)
`half as active as an inhibitor of TPN-linked isocitrate dehy(cid:173)
`drogenase as the newly synthesized compound (Table III).
`a-Methylisocitric lactone synthesized by the Rach procedure
`was separated into Fraction A and Fraction B by fractional
`crystallization. Fraction A did not inhibit TPN-specific isocit(cid:173)
`rate dehydrogenase, whereas Fraction B and the present
`preparation showed equal inhibition at equivalent concentra(cid:173)
`tions (Table III). The following observations suggest that
`threo-a-methylisocitrate is the active inhibitor. (a) The new
`method of synthesis should favor formation of the threo isomer.
`( b) In paper electrophoresis at pH 3 to pH 4, the rate of
`migration of Fraction A toward the anode was identical with
`that of DL-erythro-isocitric lactone, but more rapid than the
`migration of Fraction B (or the newly synthesized compound)
`
`TABLE I
`Inhibition of TPN-linked isocitrate dehydrogenase preparations from heart and liver
`The assay conditions are described under "Experimental Procedure."
`
`Eznyme preparation
`
`Activa ting
`divalent cation
`
`DL-lsocitrate
`
`DL-ar-Methylisocitrate
`
`Purified bovine heart enzyme
`Purified bovine heart enzyme
`Purified bovine heart enzyme
`Purified bovine heart enzyme
`Rat liver cytosol (supernatant)
`Rat liver cytosol (supernatant)
`Rat liver cytosol (supernatant)
`Rat liver cytosol (supernatant)
`Rat liver mitochondrial extract
`
`MH
`
`Mg"
`Mg"
`Mn•
`Mn•
`Mgc
`Mgc
`Mgc
`Mnc
`Mg<
`
`Cone.
`
`mM
`1.33
`6.67
`0.27
`1.33
`1.33
`4.0
`6.67
`1.3
`4.0
`
`Km(app.)
`
`µM
`13.9 ± 2.1
`20.0 ± 2.0
`6.1 ± 1.9
`5.7 ± 1.2
`18.5 ± 1.7
`14.7 ± 1.5
`15.5 ± 2.0
`10.1 ± 1.1
`15.9 ± 1.8
`
`K i,
`
`µM
`0.23 ± 0.03
`0.30 ± 0.05
`0.60 ± 0.18
`0.52 ± 0.11
`0.10 ± 0.01
`0.10 ± 0.01
`0.11 ± 0.01
`0.23 ± 0.03
`0.10 ± 0.02
`
`a The concentrations of DL-isocitrate and DL-a-methylisocitrate were from 20 to 165 µMand from 0.1 to 0.4 µM, respectively.
`• The concentrations of DL-isocitrate and DL-a-methylisocitrate were from 18 to 105 µMand from 0.9 to 3.5 µM, respectively.
`c The concentrations of DL-isocitrate and DL-a-methylisocitrate were from 16 to 72 µMand from 0.05 to 0.30 µM, respectively.
`
`Km!K,,
`
`ratio
`60
`67
`10
`11
`185
`147
`141
`43
`159
`
`Rigel Exhibit 1036
`Page 3 of 4
`
`

`

`6354
`
`TABLE II
`Effect of DL-a-methylisocitrate on DPN-linked isocitrate
`dehydrogenase from heart and liver
`The general conditions of assay are described under "Experimental
`Procedure." The concentrations of free divalent cations were main(cid:173)
`tained by adjusting the concentrations of total divalent cations as
`described previously ( 16). For purposes of these approximations, it was
`assumed that the stability constants of the divalent metal chelates of
`isocitrate and a-methylisocitrate were equal.
`
`Enzyme
`
`Free divalent
`cation
`
`DL-a-
`methyl-
`isocitrate
`
`DL-iso-
`citrate
`
`Activity"
`
`mM
`Purified from Mg2+ (0.04-0.05)
`bovine heart
`Purified from Mn 2+ (0.04-0.06)
`bovine heart
`Rat liver
`mitochondrial
`extract
`
`Mg2+ (0.04-0.05)
`
`mM
`1.5
`
`1.5
`
`1.5
`
`mM
`2.0
`
`(%±8.D.)
`99.8 ± 3.7 (9)
`
`0.9
`
`102.1 ± 9.5 (12)
`
`2.9
`
`88.7 ± 8.3 (4)
`
`0 Velocities measured at each concentration of isocitrate and a(cid:173)
`methylisocitrate indicated were compared to the reaction rates in the
`absence of a-methylisocitrate. The numbers in parentheses refer to
`numbers of experiments. Additional experiments have been done at
`other levels of substrate and inhibitor; only the results obtained at the
`lowest levels of isocitrate (Km(app.) total DL-isocitrate ~ 10 mM) and
`the highest concentrations of a-methylisocitrate used are reported
`here.
`
`COOH
`I
`CH 3-C-OH
`I
`HOOC-C-H
`CH 2
`I
`COOH
`
`I
`
`COOH
`I
`CH 3-C-OH
`I
`H-C-COOH
`I
`CH 2
`I
`COOH
`
`D-<t-Methyl - threo -
`isocitric acid
`
`D-«- Methyl-erythro(cid:173)
`isocitric acid--
`
`SCHEME 1
`
`which corresponded to that of DL-threo-isocitric lactone. The
`relative rates of migration in electrophoresis of the erythro and
`threo-
`isomers would be in agreement with the finding of
`Gawron and Glaid ( 17) that the values of pK' a, and pK' a 2 of
`DL-erythro-isocitric lactone were lower than those of DL-threo(cid:173)
`isocitric lactone. (c) In preliminary experiments with aconitase
`from bovine heart, Fraction A was inert, whereas Fraction B
`and the newly synthesized compound had substrate activity.
`Gawron and Mahajan (2) had shown that a-methyl-cis-aconi(cid:173)
`tate was a substrate for aconitase and, on the basis of the
`stereospecificity of the enzyme, suggested that the isocitrate
`formed had the D-threo configuration. Presumably, the same
`isomer of threo-a-methylisocitrate in the synthetic prepara(cid:173)
`tions was active for aconitase. Although a resolution of the D
`and L
`isomers has not been accomplished, the substrate
`specificity of isocitrate dehydrogenase (18) makes it likely that
`D-threo-a-methylisocitrate is the inhibitor of the TPN-specific
`enzyme.
`Ando et al. ( 19) have observed urinary excretion of methylcit(cid:173)
`ric acid in patients with propionic aciduria and methylma-
`
`TABLE III
`Inhibition of TPN-linked isocitrate dehydrogenase by various
`preparations of DL-a-methylisocitrate
`Assays were done as described under "Experimental Procedure"
`1 with purified enzyme from bovine heart in the presence of 1.33 mM
`MnS0,, 70 µM DL-isocitrate, and a-methylisocitrate at the concentra(cid:173)
`tions indicated. The samples of a-methylisocitrate lactone were
`hydrolyzed before use, as described under "Experimental Procedure."
`
`Method of preparation
`of a-methylisocitrate
`
`a-Methylisocitrate
`
`Inhibition
`
`New synthesis
`New synthesis
`New synthesis
`Rach
`Rach
`Rach
`Fraction A
`Fraction B
`
`µM
`1.75
`3.5
`7.0
`1.75
`3.50
`7.0
`7.0
`7.0
`
`%
`20
`35
`52
`8
`14
`25
`0
`54
`
`ionic aciduria. They have suggested that methylcitrate or a
`metabolite of this compound may lead to increased ketone
`body formation in such patients by blocking the citric acid
`cycle. If the isomer of methylcitrate formed in such patients
`were convertible to threo-a-methylisocitrate by the action of
`aconitase, the inhibition of TPN-linked isocitrate dehydrogen(cid:173)
`ase by a-methylisocitrate could contribute to increased diver(cid:173)
`sion of acetyl-CoA from the citric acid cycle to ketone body
`formation.
`
`Acknowledgments-The authors wish to thank Dr. Colleen
`M. Smith for preparations of rat liver homogenate fractions.
`
`REFERENCES
`1. Plaut, G. W. E., Beach, R. L. & Aogaichi, T. (1975) Biochemistry
`14, 2581-2588
`2. Gawron, 0. & Mahajan, K. P. (1966) Biochemistry 5, 2343-2350
`3. McFadden, B. A., Rose, I. A. & Williams, J. 0. (1972) Arch.
`Biochem. Biophys. 148, 84-88
`4. Giorgio, N. A., Jr., Yip, A. T., Fleming, J. & Plaut, G. W. E. (1970)
`J. Biol. Chem. 245, 5469-5477
`5. Rose, Z. B. (1960) J. Biol. Chem. 235, 928-933
`6. Siebert, G., Dubuc, J., Warner, R. C. & Plaut, G. W. E. (1957)
`J. Biol. Chem. 226, 965-975
`7. Schneider, W. C. & Hogeboom, G. H. (1950) J. Biol. Chem. 183,
`123-128
`8. Hogeboom, G. H. & Schneider, W. C. (1950) J. Biol. Chem. 186,
`417-427
`9. Cleland, W. W. (1963) Nature 198, 463-465
`10. Chen, R. F. & Plaut, G. W. E. (1963) Biochemistry 2, 1023-1032
`11. Gawron, 0., Glaid, A. J., III, LoMonte, A. & Gary, S. (1958) J. Am.
`Chem. Soc. 80, 5856-5860
`12. Rach, C. (1886) Ann. Chem. 234, 35-43
`13. Liwschitz, Y., Rabinsohn, Y. & Perera, D. (1962) J. Chem. Soc.
`1116-1119
`14. Siebert, G., Carsiotis, M. & Plaut, G. W. E. (1957J J. Biol. Chem.
`226, 977-991
`15. Colman, R. F. (1972) J. Biol. Chem. 247, 215-223
`16. Plaut, G. W. E., Schramm, V. L. & Aogaichi, T. (1974) J. Biol.
`Chem. 249, 1848-1856
`17. Gawron, 0. & Glaid, A. J., III (1955) J. Am. Chem. Soc. 77,
`6638-6640
`18. Englard, S. & Colowick, S. (1957) J. Biol. Chem. 226, 1047-1058
`19. Ando, T., Rasmussen, K., Wright, J. M. & Nyhan, W. (1972) J.
`Biol. Chem. 247, 2200-2204
`
`Rigel Exhibit 1036
`Page 4 of 4
`
`