Gas chromatographic separation of esters of fluoro analogs of citric acid cycle intermediates

Dummel, Robert J.; Kun, Ernest

doi:10.1016/s0021-9673(01)80254-5

Cited by 9 publications

(5 citation statements)

References 11 publications

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“…This isotope effect was determined to be 1.0522 ( 0.0001 at pH 7.5, which is identical to the value reported for the dianion of oxaloacetate (12). The decarboxylation of fluorooxaloacetate is ∼2.5 times faster than the decarboxylation of oxaloacetate at this pH (14). …”

Section: Comparative Initial Velocity Studies With L-malate and (2r3supporting

confidence: 81%

Determination of the Kinetic and Chemical Mechanism of Malic Enzyme Using (2R,3R)-erythro-Fluoromalate as a Slow Alternate Substrate

1998

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(2R,3R)-erythro-Fluoromalate, but not the threo isomer, is a slow substrate for chicken liver malic enzyme with either NADP or 3-acetylpyridine-NADP (APADP) as the other substrate. The Km for erythro-fluoromalate is similar to that of malate, but the turnover number with NADP is 3300-fold slower, although 5.5-fold faster with APADP than with NADP. Deuteration of fluoromalate at C-2 gave an isotope effect on V/K of 1.39 with NADP and 3.32 with APADP. With NADP, the 13C isotope effects at C-4 were 1.0490 with unlabeled and 1.0364 with deuterated fluoromalate. With APADP, the corresponding values were 1.0138 and 1.0087. These data show that the mechanism is stepwise with both nucleotide substrates, in contrast to the reaction of malate and APADP, which was postulated to be concerted by Karsten et al. [Karsten, W. E., and Cook, P. F. (1994) Biochemistry 33, 2096-2103], a conclusion recently shown to be correct by Edens et al. [Edens, W. A., Urbauer, J. L., and Cleland, W. W. (1997) Biochemistry 36, 1141-1147]. To explain the effect of deuteration on the 13C isotope effect with APADP, it is necessary to assume a secondary 13C isotope effect at C-4 on the hydride transfer step of approximately 1.0064 (assuming 5.7 as the intrinsic primary deuterium isotope effect and 1.054 as the product of the 13C equilibrium isotope effect on hydride transfer and the intrinsic 13C isotope effect on decarboxylation). The secondary 13C isotope effect on hydride transfer is thought to result from hyperconjugation between the carbonyl group and C-4 of the enzyme-bound fluorooxaloacetate intermediate.

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Section: Comparative Initial Velocity Studies With L-malate and (2r3supporting

confidence: 81%

Determination of the Kinetic and Chemical Mechanism of Malic Enzyme Using (2R,3R)-erythro-Fluoromalate as a Slow Alternate Substrate

1998

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“…The concentration of (Z)-F-P-enolpyruvate was measured by using the coupled pyruvate kinase-lactate dehydrogenase assay with a limiting amount of this substrate. 3-Fluorooxalacetate was synthesized by acid hydrolysis of diethyl fluorooxalacetate (Kun et al, 1958) and recrystallized from ether-chloroform (Dummel et al, 1971). Diethyl fluorooxalacetate was obtained by condensing diethyl oxalate and ethyl fluoroacetate in a suspension of sodium ethoxide in benzene, followed by washing with anhydrous ether (Stubbe & Kenyon, 1972).…”

Section: Methodsmentioning

confidence: 99%

Stereochemistry of phosphoenolpyruvate carboxylation catalyzed by phosphoenolpyruvate carboxykinase

Hwang¹,

Nowak²

1986

Biochemistry

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The stereochemistry of the carboxylation of phosphoenolpyruvate to yield oxalacetate, catalyzed by chicken liver phosphoenolpyruvate carboxykinase and by Ascaris muscle phosphoenolpyruvate carboxykinase, was determined. The substrate (Z)-3-fluorophosphoenolpyruvate was used for the stereochemical analysis. The carboxylation reaction was coupled to malate dehydrogenase to yield 3-fluoromalate, and the stereochemistry of the products was identified by 19F NMR. In separate experiments, the enantiomeric tautomers of 3-fluorooxalacetate were shown to be utilized by malate dehydrogenase to yield (2R,3R)- and (2R,3S)-3-fluoromalate in nearly identical amounts. The products were identified by 19F NMR. When (Z)-3-fluorophosphoenolpyruvate was used as a substrate for phosphoenolpyruvate carboxykinase from avian liver and from Ascaris, and malate dehydrogenase was used to trap the product, only a single diastereomer was observed. This product was shown to be (2R,3R)-3-fluoromalate in each case. The assignments were based on coupling constants taken from Keck et al. [Keck, R., Hess, H., & Rétey, J. (1980) FEBS Lett. 114, 287]. These results indicate that the stereochemistry of carboxylation, catalyzed by chicken phosphoenolpyruvate carboxykinase and by Ascaris phosphoenolpyruvate carboxykinase, is identical and takes place from the si side of the enzyme-bound phosphoenolpyruvate. The carboxylation reaction was run both in H2O and in D2O. No deuterium incorporation into fluoromalate was shown to occur. The product 3-fluorooxalacetate is thus released from phosphoenolpyruvate carboxykinase as the keto form and is reduced more rapidly by reduced nicotinamide adenine dinucleotide with malate dehydrogenase than by the occurrence of tautomerization.

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“…In spite of this precaution, our material appeared to contain about 10% fluoropyruvic acid (see Results). After crystallization (Dummel et al, 1971), the product melted at 82-85 °C (decomposition) (lit., Dummel et al, 1971: 86-87 °C): UV 265 nm (400 M-1 cm-1) in 1 M NaOH (lit.. NMR in D20, 5.25 ppm (d, J = 46 Hz); upon raising the pH to ~1 3 with NaOD, the doublet disappeared rapidly. We determined the maximal velocity of MDH-catalyzed reduction of FOAA by NADH (50 mM potassium phosphate, pH 7.5); the rate is about 14 µ min-1 mg-1.…”

Section: Methodsmentioning

confidence: 99%

Fluorinated substrate analogs as stereochemical probes of enzymic reaction mechanisms

Goldstein¹,

Cheung²,

Marletta³

et al. 1978

Biochemistry

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Gas chromatographic separation of esters of fluoro analogs of citric acid cycle intermediates

Cited by 9 publications

References 11 publications

Determination of the Kinetic and Chemical Mechanism of Malic Enzyme Using (2R,3R)-erythro-Fluoromalate as a Slow Alternate Substrate

Determination of the Kinetic and Chemical Mechanism of Malic Enzyme Using (2R,3R)-erythro-Fluoromalate as a Slow Alternate Substrate

Stereochemistry of phosphoenolpyruvate carboxylation catalyzed by phosphoenolpyruvate carboxykinase

Fluorinated substrate analogs as stereochemical probes of enzymic reaction mechanisms

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