Fluorocitrate inhibition of aconitate hydratase and the tricarboxylate carrier of rat liver mitochondria

Brand, Martin D.; Evans, Susan M.; Mendes-Mourão, J.; Chappell, Joseph

doi:10.1042/bj1340217

Cited by 37 publications

(17 citation statements)

References 13 publications

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“…We now report that aconitase is a sensitive target of hyperoxic damage in vitro and in vivo and demonstrate that inhibition of aconitase with fluoroacetate (or fluorocitrate) (29)(30)(31)(32)(33)(34) dehydrogenase, and porcine heart isocitrate dehydrogenase were from Sigma. Sodium (+)-fluorocitrate was prepared by titrating barium fluorocitrate with a slight excess of sulfuric acid, centrifuging to remove BaSO4, and then neutralizing the supernatant with NaOH.…”

Section: Introductionmentioning

confidence: 99%

Aconitase is a sensitive and critical target of oxygen poisoning in cultured mammalian cells and in rat lungs.

Gardner

Nguyen

White

1994

Proc. Natl. Acad. Sci. U.S.A.

387

234

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The effect of hyperoxia on activity of the superoxide-sensitive citric acid cycle enzyme aconitase was measured in cultured human epithelial-like A549 cells and in rat lungs. Rapid and progressive loss of >80% of the aconitase activity in A549 cells was seen during a 24-hr exposure to a Po2 of 600 mmHg (1 mmHg = 133 Pa). Inhibition of mitochondrial respiratory capacity correlated with loss of aconitase activity in A549 cells exposed to hyperoxia, and this effect could be mimicked by fluoroacetate (or fluorocitrate), a metabolic poison of aconitase. Exposure ofrats to an atmospheric Po2 of 760 mmHg or 635 mmHg for 24 hr caused respective 73% and 61% decreases in total lung aconitase activity. We propose that early inactivation of aconitase and inhibition ofthe energy-producing and biosynthetic reactions of the citric acid cycle contribute to the sequelae of lung damage and edema seen during exposure to hyperoxia. (1,4,(7)(8)(9)(10)(11)(12)(13). Morphological and biochemical alterations in the lung ultimately cause morbidity resulting from decreased blood oxygenation (4). Edema of the interstitial space and increased permeability of pulmonary microvasculature are early signs of pathology in the lungs of rats exposed to lethal levels of dioxygen (Po2 of 760 mmHg). This initial damage is followed by and amplified by the activation and infiltration of platelets, macrophages, and neutrophils and precedes the death of the animal or individual (3, 12). After sublethal exposures of rats to hyperoxia (Po2 of 456-650 mmHg), damage is marked by fibrosis, alveolar type II cell proliferation (11, 12), and mitochondrial structural deformities (7,8,11,12). The contribution of individual reactive oxygen intermediates and dioxygen to the morphological and biochemical alterations in the lungs, however, remains only vaguely defined (5, 6, 12).Mitochondrial respiration and energy production have been identified as sensitive and critical sites of hyperoxic damage in lung tissue (9, 10) and in cell culture models (14). In rats exposed to a Po2 of 760 mmHg for 24 hr, lung mitochondrial respiratory capacity and citric acid cycle activity are impaired (9, 10). Potential enzymatic sites for the poisoning action of hyperoxia have been described. The mitochondrial enzymes NADH dehydrogenase (14), succinate dehydrogenase (14, 15), and a-ketoglutarate dehydrogenase (14) have various sensitivities to hyperoxic exposure. However, evidence is lacking for a loss of dehydrogenase activities during the early impairment of rat lung oxidative metabolism by normobaric hyperoxia.The citric acid cycle enzyme aconitase is a member of a growing family of 02--sensitive [4Fe-4S]-containing (de)-hydratases that have been implicated to be important sites of 02 -/02 toxicity (16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26). The activity ofaconitase is sensitive to inactivation by 02- (16,22) and is modulated by changes in 02-levels in bacteria and mammalian cells (23, 24, 26).Thus, the ability of elevated levels of 02 to exacerbate mitochondrial productio...

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Section: Introductionmentioning

confidence: 99%

Aconitase is a sensitive and critical target of oxygen poisoning in cultured mammalian cells and in rat lungs.

Gardner

Nguyen

White

1994

Proc. Natl. Acad. Sci. U.S.A.

387

234

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“…Effects of alterations in ambient temperature, which is directly related to metabolic rate and inversely related to life span [19,20], on aconitase oxidative damage and enzymic activity were examined at different ages. To determine whether aconitase activity was essential for survival, the effect of administration of fluoroacetate, which is a competitive inhibitor of aconitase activity [21], on the median life span of the flies was examined. Oxidative damage to the proteins in D. melanogaster was found to be both highly selective and inversely proportional to the life span of the flies.…”

Section: Introductionmentioning

confidence: 99%

Selectivity of protein oxidative damage during aging in Drosophila melanogaster

DAS

Levine

Orr

et al. 2001

Biochemical Journal

102

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The purpose of the present study was to determine whether oxidation of various proteins during the aging process occurs selectively or randomly, and whether the same proteins are damaged in different species. Protein oxidative damage to the proteins, present in the matrix of mitochondria in the flight muscles of Drosophila melanogaster and manifested as carbonyl modifications, was detected immunochemically with anti-dinitrophenyl-group antibodies. Aconitase was found to be the only protein in the mitochondrial matrix that exhibited an age-associated increase in carbonylation. The accrual of oxidative damage was accompanied by an approx. 50% loss in aconitase activity. An increase in ambient temperature, which elevates the rate of metabolism and shortens the life span of flies, caused an elevation in the amount of aconitase carbonylation and an accelerated loss in its activity. Exposure to 100% ambient oxygen showed that aconitase was highly susceptible to undergo oxidative damage and loss of activity under oxidative stress. Administration of fluoroacetate, a competitive inhibitor of aconitase activity, resulted in a dose-dependent decrease in the life span of the flies. Results of the present study demonstrate that protein oxidative damage during aging is a selective phenomenon, and might constitute a mechanism by which oxidative stress causes age-associated losses in specific biochemical functions.

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“…Kinetic studies of this compound with aconitase have yielded a range of K I values and have demonstrated that depending on which substrate was used in the studies, inhibition may be either competitive or noncompetitive and that this competition is reversible (11)(12)(13). Villafranca and Platus (11) further showed that aconitase is rapidly inactivated by (Ϫ)-erythro-2-fluorocitrate.…”

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confidence: 99%

The reaction of fluorocitrate with aconitase and the crystal structure of the enzyme-inhibitor complex

Lauble

Kennedy

Emptage

et al. 1996

Proc. Natl. Acad. Sci. U.S.A.

114

121

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It has been known for many years that f luoroacetate and f luorocitrate when metabolized are highly toxic, and that at least one effect of f luorocitrate is to inactivate aconitase. In this paper we present evidence supporting the hypothesis that the (؊)-erythro diastereomer of 2-f luorocitrate acts as a mechanism based inhibitor of aconitase by first being converted to f luoro-cis-aconitate, followed by addition of hydroxide and with loss of f luoride to form 4-hydroxy-trans-aconitate (HTn), which binds very tightly, but not covalently, to the enzyme. Formation of HTn by these reactions is in accord with the working model for the enzyme mechanism. That HTn is the product of f luorocitrate inhibition is supported by the crystal structure of the enzymeinhibitor complex at 2.05-Å resolution, release of f luoride stoichiometric with total enzyme when (؊)-erythro-2-f luorocitrate is added, HPLC analysis of the product, slow displacement of HTn by 10 6 -fold excess of isocitrate, and previously published Mössbauer experiments. When (؉)-erythro-2-f luorocitrate is added to aconitase, the release of f luoride is stoichiometric with total substrate added, and HPLC analysis of the products indicates the formation of oxalosuccinate, and its derivative ␣-ketoglutarate. This is consistent with the proposed mechanism, as is the formation of HTn from (؊)-erythro-2-f luorocitrate. The structure of the inhibited complex reveals that HTn binds like the inhibitor trans-aconitate while providing all the interactions of the natural substrate, isocitrate. The structure exhibits four hydrogen bonds <2.7 Å in length involving HTn, H 2 O bound to the [4Fe-4S] cluster, Asp-165 and His-167, as well as low temperature factors for these moieties, consistent with the observed very tight binding of the inhibitor.The mechanism of the inhibitory effects of fluorocitrate on the enzyme aconitase [citrate(isocitrate)hydrolyase, EC 4.2.1.3] has been a long-standing problem in biochemistry. The toxic nature of fluoroacetate was discovered over 50 years ago (1, 2) and citrate was found to accumulate in tissues poisoned with compounds that could provide the fluoroacetyl residue. On this basis Peters (3) and Martius (4) proposed that the inhibitory substance was a fluorotricarboxylic acid. Subsequently it was shown that 2-fluorocitrate is indeed produced metabolically via the citrate synthase reaction (5) and that in the presence of this substance the enzyme aconitase is inhibited (6). Aconitase catalyzes the conversion of citrate to isocitrate (Iso) via the obligatory intermediate cis-aconitate (Scheme I).

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Fluorocitrate inhibition of aconitate hydratase and the tricarboxylate carrier of rat liver mitochondria

Cited by 37 publications

References 13 publications

Aconitase is a sensitive and critical target of oxygen poisoning in cultured mammalian cells and in rat lungs.

Aconitase is a sensitive and critical target of oxygen poisoning in cultured mammalian cells and in rat lungs.

Selectivity of protein oxidative damage during aging in Drosophila melanogaster

The reaction of fluorocitrate with aconitase and the crystal structure of the enzyme-inhibitor complex

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