Aconitase inhibition

NO has a cytostatic effect by inhibiting ATP synthesis [99] via Kreb s cycle (aconitase inhibition, [100]), glycolysis (GADPH inhibition) and mitochondrial respiration (NAD ubiquinone oxydoreductase and succinate ubiquinone oxydoreductase inhibitions, [101]). Another pathway is the ornithine decarboxylase inhibition. This enzyme is implicated in polyamine production necessary to cell proliferation and its activity is inhibited by NO in human colon cancer cells HT-29 and Caco-2 [102]. Furthermore NO directly inactivates ribonucleotide reductase [103] of TA3 cancer cells (murine breast cancer cells) [104]. This enzyme controlling DNA synthesis catalyses desoxyribonucleotides synthesis, and its inhibition blocks cells in S phase. This inhibition is rapid and reversible in K562 and TA3 cells [105]. 

A further way in which metabolic control may be exercised is the artificial deprivation of required ions and cofactors, for example aconitase must have ferrous ions for activity. Conversely, addition of toxic ions is possible, for example aconitase is inhibited by cupric ions. Finally the use of metabolic analogues is possible. If monofluoroacetate is added to cells then monofluorocitrate is produced by titrate synthase and this compound inhibits the activity of aconitase. Great care has to be taken when using metabolic analogues, however, they are often less than 100% specific and may have unexpected and unwanted serious side effects. 

The effects of copper ions on the process were elucidated as follows. It was known that copper ions inhibit aconitase activity. 

When induced in macrophages, iNOS produces large amounts of NO which represents a major cytotoxic principle of those cells. Due to its affinity to protein-bound iron, NO can inhibit a number of key enzymes that contain iron in their catalytic centers. These include ribonucleotide reductase (rate-limiting in DNA replication), iron-sulfur cluster-dependent enzymes (complex I and II) involved in mitochondrial electron transport and cis-aconitase in the citric acid cycle. In addition, higher concentrations of NO,... 

Citrate is isomerized to isocitrate by the enzyme aconitase (aconitate hydratase) the reaction occurs in two steps dehydration to r-aconitate, some of which remains bound to the enzyme and rehydration to isocitrate. Although citrate is a symmetric molecule, aconitase reacts with citrate asymmetrically, so that the two carbon atoms that are lost in subsequent reactions of the cycle are not those that were added from acetyl-CoA. This asymmetric behavior is due to channeling— transfer of the product of citrate synthase directly onto the active site of aconitase without entering free solution. This provides integration of citric acid cycle activity and the provision of citrate in the cytosol as a source of acetyl-CoA for fatty acid synthesis. The poison fluo-roacetate is toxic because fluoroacetyl-CoA condenses with oxaloacetate to form fluorocitrate, which inhibits aconitase, causing citrate to accumulate. 

The mechanism of fluoroacetate toxicity in mammals has been extensively examined and was originally thought to involve simply initial synthesis of fluorocitrate that inhibits aconitase and thereby the functioning of the TCA cycle (Peters 1952). Walsh (1982) has... 

There are several examples in which metabolites that toxify the organism responsible for their synthesis are produced. The classic example is fluoroacetate (Peters 1952), which enters the TCA cycle and is thereby converted into fluorocitrate. This effectively inhibits aconitase—the enzyme involved in the next metabolic step—so that cell metabolism itself is inhibited with the resulting death of the cell. Walsh (1982) has extensively reinvestigated the problan and revealed both the complexity of the mechanism of inhibition and the stereospecihcity of the formation of fluorocitrate from fluoroacetate (p. 239). It should be noted, however, that bacteria able to degrade fluoroacetate to fluoride exist so that some organisms have developed the capability for overcoming this toxicity (Meyer et al. 1990). 

The key to unraveling the toxicity of fluoracetate came from observations of Buffa and Peters (1949) that in animals treated with FAc, considerable quantities of citrate accumulated in some tissues. Oxygen uptake was also diminished. The citric acid cycle was thus implicated as the site of inhibition. Fluorcitrate was then isolated from the affected tissues. It was found to be a powerful competitive inhibitor of aconitase, thus blocking citrate oxidation. The suggestion was therefore made that fluoracetate was toxic not in itself, but because it was metabolized in cells via fluoracetyl CoA to give a toxic derivative, an example of lethal synthesis —the capacity of organisms to metabolize nontoxic compounds and convert them to potentially lethal products. 

Fluoroacetate undergoes a "lethal synthesis"(18) to 2-fluorocitrate which may reversibly inhibit aconitase and which irreversibly binds to a membrane-associated citrate transport protein(19,20). Insecticidal and other biocidal uses of fluoroacetate (or its metabolic precursors) received considerable attention twenty-five years ago( ) but most uses have been abandoned due to high nonspecific vertebrate toxicity of these compounds. Vfe have reported the use of o)-fluoro fatty acids and their derivatives as delayed-action toxicants for targeted... 

Potent metabolic inhibitors of the citric acid cycle. Fluo-roacetate (F-CH2COO ) must first be converted to flu-oroacetyl-S-CoA (by acetyl-CoA synthetase) and thence to fluorocitrate (by citrate synthase) before it can act as a potent metabohc inhibitor of the aconitase reaction as well as citrate transport. Submicromolar concentrations of ( )-erythro-Q iOTOcitTate can irreversibly inhibit citrate uptake by isolated brain mitochondria. 

Krebs cycle Inhibition of aconitase by superoxide and fluoroacetate, of succinate dehydrogenase by methamphetamine and mal-onate, of alpha-ketoglutarate dehydrogenase by salicylic add... 

A few natural organofluorine compounds exist, most notably in plants (Fig. 1c). These are generally noted for their toxicity most importantly, fluoroacetate enters the tricarboxylic acid (TCA) cycle and as fluorocitrate inhibits c/s-aconitase [4,106,107]. Of course, toxicity provides an opportunity to generate specific poisons and fluoroacetate is widely used as a rodenticide providing opportunities for NMR [108]. F NMR has been used for extensive studies of body fluids such as milk and urine with respect to xenobiotica [109-115]. 

The toxicity of fluoroacetic acid and of its derivatives has played an historical decisive role at the conceptual level. Indeed, it demonstrates that a fluorinated analogue of a natural substrate could have an activity profile that is far different from that of the nonfluorinated parent compound. The toxicity of fluoroacetic acid is due to its ability to block the citric acid cycle (Krebs cycle), which is an essential process of the respiratory chain. The fluoroacetate is transformed in vivo into 2-fluorocitrate by the citrate synthase. It is generally admitted that aconitase (the enzyme that performs the following step of the Krebs cycle) is inhibited by 2-fluorocitrate the formation of aconitate through elimination of the water molecule is a priori impossible from this substrate analogue (Figure 7.1). 

Although this interpretation can be found in most books, the reality is much more complex. Citrate synthase yields the sole (2/f,3/f)-2-fluorocitrate with a large stereoselectivity. This stereomer (and three other ones) does not inhibit aconitase, but it is a substrate of this enzyme. Aconitase cannot afford cw-aconitate by elimination since... 

Nevertheless, the toxicity of fluoroacetate seems to be only partially due to the inhibition of aconitase. The competitive nature of the inhibition, its Xj value (Xj = 20-60 pM)," and the time-dependent nature (but reversible) of the inhibition of aconitase seem to be poorly compatible with the sharp and irreversible toxicity of fluorocitrate. Thus, it has been suggested that fluorocitrate can covalently bind with the proteins that are involved in citrate transport through the mitochondrial membrane. ... 

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