Aconitase fluorocitrate

Fluorocitrate is an inhibitor of the citric acid cycle enzyme, aconitase. Fluorocitrate is produced by catalytic combination of fluoroacetyl-CoA (see fluoroacetate) with oxaloacetate by the enzyme citrate synthase. 

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). 

Aconitase, an unstable enzyme,4 is concerned with the reversible conversion of cis-aconitate to either citric acid or isocitric acid. It may be noted that the entire system of tricarboxylic cycle enzymes are present in the mitochondria separated from cells, and, furthermore, it has been found that the mitochondrial enzymes differ from the isolated enzymes in that the former require no addition of D.P.N. (co-enzyme I) or T.P.N. (co-enzyme II) for activity. Peters suggests that the citrate accumulation is caused by the competitive reaction of the fluorocitrate with aconitase required for the conversion of citrate to isocitrate. This interference with the tricarboxylic acid... 

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. 

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. ... 

In contrast, selective inhibition of enzyme activity involves highly specific interactions between the protein and chemical groups on the xenobiotic. An excellent example of this type of inhibition is seen in the toxic effect of fluoroacetate, which is used as a rodenticide. Although fluoroacetate is not directly toxic, it is metabolized to fluoroacetyl-CoA, which enters the citric acid cycle due to its structural similarity to acetyl-CoA (Scheme 3.5). Within the cycle, fluoroacetyl-CoA combines with oxalo-acetate to form fluorocitrate, which inhibits the next enzyme, aconitase, in the cycle [42]. The enzyme is unable to catalyze the dehydration to cis-aconitate, as a consequence of the stronger C-F bond compared with the C-H bond. Therefore, fluorocitrate acts as a pseudosubstrate, which blocks the citric acid cycle and, subsequently, impairs ATP synthesis. 

Scheme 3.5 Metabolism of fluoroacetate to fluorocitrate, a specific inhibitor of aconitase. Modified from [42].

This naturally occurring toxicant is an analogue of acetate and is incorporated into acetyl CoA (fluoroacetate) and hence into Krebs cycle (TCA cycle) as fluorocitrate. This blocks the enzyme aconitase, as the fluorine atom cannot be removed. The TCA cycle is blocked, and citrate accumulates. The mitochondrial energy supply is disrupted, hence cardiac damage occurs. Lack of oxaloacetate will allow ammonia to accumulate leading to convulsions. 

Lauble H, Kennedy MC, Emptage MH, et al. The reaction of fluorocitrate with aconitase and the crystal structure of the enzyme inhibitor complex. Proc Natl Acad Sci U S A 1996 93 13699-13703. 

Fluoroacetate causes inhibition of aconitase, an enzyme in the tricarboxylic acid cycle. This is due to the formation of fluorocitrate, which binds to aconitase and inhibits the enzyme. This is because the fluorine atom cannot be removed from the fluorocitrate unlike the hydrogen atom in the normal substrate, citrate. The result is complete blockade of the cycle and this means tissues become starved of ATP and other vital metabolic intermediates. This causes adverse effects in the heart as the organ is particularly sensitive to deficiency of ATP. 

Citrate is isomerized to isocitrate by aconitase (see Figure 9.5). [Note Aconitase is inhibited by fluoroacetate, a compound that is used as a rat poison. Fluoroacetate is converted to fluoroacetyl CoA, which condenses with oxaloacetate to form fluorocitrate—a potent inhibitor of aconitase—resulting in citrate accumulation.]... 

Among the most deadly of simple compounds is sodium fluoroacetate. The LD50 (the dose lethal for 50% of animals receiving it) is only 0.2 mg/kg for rats, over tenfold less than that of the nerve poison diisopropylphosphofluoridate (Chapter 12).a b Popular, but controversial, as the rodent poison "1080," fluoroacetate is also found in the leaves of several poisonous plants in Africa, Australia, and South America. Surprisingly, difluoroacetate HCF2-COO is nontoxic and biochemical studies reveal that monofluoroacetate has no toxic effect on cells until it is converted metabolically in a "lethal synthesis" to 2R,3R-2-fluorocitrate, which is a competitive inhibitor of aconitase (aconitate hydratase, Eq. 13-17).b This fact was difficult to understand since citrate formed by the reaction of fluorooxalo-acetate and acetyl-CoA has only weak inhibitory activity toward the same enzyme. Yet, it is the fluorocitrate formed from fluorooxaloacetate that contains a fluorine atom at a site that is attacked by aconitase in the citric acid cycle. 

Other mechanisms have been considered. Clarke has proposed that the acute toxicity of fluorocitrate may be caused by irreversible inhibition of aconitase by (2is,4R)-4-fluoro-aconitate, formed by the dehydration of fluorocitrate by aconitase59. Hornfeldt and Larson reported evidence that seizures accompanying fluorocitrate toxicity may result from Ca+2 chelation in the spinal cord60. 

Answer Oxidation of co-fluorooleate in the /3-oxidation pathway forms fluoroacetyl-CoA in the last pass through the sequence. Entry of fluoroacetyl-CoA into the citric acid cycle produces fluorocitrate, a powerful inhibitor of the enzyme aconitase. As a result of this inhibition, the citric acid cycle shuts down and the flow of reducing equivalents to oxidative phosphorylation is fatally impaired. 

Fluoroacetyl-CoA competes with acetyl-CoA as a substrate for citrate synthase and is converted into 4-fluorocitrate. It is 4-fluorocitrate that blocks the citric acid cycle by being a potent inhibitor of aconitase (Fig. 12-13) and in this lies its toxicity. 

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