Fluoroacetate aconitase inhibition

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. 

Differential outcomes are evident depending on the preparation used to study the in vitro effects of sodium fluoroacetate. Mitochondria-free preparations exhibit a Ki value of 22-45 pM for inhibition of aconitase by fluoroacetate. Aconitase bound to mitochondria appears to be much more sensitive to the effects of fluoroacetate fluoroacetate inhibits aconitase in these preparations in the picomolar concentration range. Inhibition of the tricarboxylic acid cycle in actrocytes results in a depletion of ATP and a... 

According to the present concepts of j3-oxidation and the formation of acetyl CoA, fluoroacetyl CoA should be formed from w-fluoroacetates of an even number of carbons. It has been suggested that fluoro-acetate condenses with another compound, the condensation product being toxic. Since fluorocitrate accumulates in systems poisoned with fluoroacetate, it may be concluded that fluoroacetyl CoA is formed and that it then condenses with oxalacetate. The fact that citrate also accumulates " indicates that either fluoroacetate or fluorocitrate is inhibitory for some step subsequent to the formation of citrate. Cfs-aconitate oxidation is not inhibited therefore, the inhibition appears to be exerted upon the conversion of citrate to cis-aconitate by aconitase. Experimental evidence, as well as theoretical considerations, indicates that fluoroacetate also inhibits fatty acid oxidation by forming complexes with magnesium ions. The apparent association constants of Mg(FAc)+(C i) and Mg(FAc)2(C s) were determined by Gillette and Kalnitsky ... 

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). 

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... 

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). 

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. 

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.]... 

Citrate is synthesized from oxaloacetate (OAA) and acetyl CoA by citrate synthase. This enzyme is allosterically activated by ADP, and inhibited by ATP, NADH, succinyl CoA, and fatty acyl CoA derivatives. Citrate is isomerized to isocitrate by aconitase, an enzyme that is targeted by the rat poison, fluoroacetate. 

There are two Krebs cycle inhibitors that are worth mentioning. Malonate inhibits succinate dehydrogenase because of its very similar structure. Fluoro-acetate inhibits cis-aconitase, which is an Fe-S enzyme. The fluoroacetate replaces acetate as a substrate in the citrate synthase reaction when this combines with cis-aconitase, however, no further reaction becomes possible. 

Fluoroacetate. Another metabolic inhibitor is fluoroacetic acid, a compound of high mammalian toxicity which has been widely used as a rodenticide. In 1952 Peters (99) discovered that fluoroacetic acid is converted in the body to fluorocitrate by acetyl CoA and oxaloacetate transacetase. The fluorocitrate proved by competition to be powerfully inhibiting to the metabolism of citric acid by aconitase. Thus the natural cycle is blocked and citrate accumulates in the tissues. Treatment with glycerol monoacetate has been used (24). 

Fluoroacetate interferes with the citric acid cycle by conversion to fluorocitrate, which by competition inhibits the metabolism of citric acid by aconitase. 

E-9) Fluoroacetate (rat poison) acts by converting to fluorocitrate and then inhibiting aconitase at this step. 

The activity of an enzyme may be inhibited by the presence of a toxic metabolite. Sodium fluoroacetate, known as rat poison 1080, is extremely toxic to animals. The toxic action, however, is not due to sodium fluoroacetate itself but to a metabolic conversion product, flu-orocitrate, formed through a reaction commonly known as "lethal synthesis," as shown in Figure 5.3. The resulting fluorocitrate is toxic because it is inhibitory to aconitase, the enzyme responsible for the conversion of citrate into czs-aconitate and then into isocitrate in the tricarboxylic acid cycle. Inhibition of aconitase results in citrate accumulation. The cycle stops for lack of metabolites, leading to disruption of energy metabolism. 

Synthesis of fluorocitrate from fluoroacetate through lethal synthesis." Inhibition of aconitase shuts down TCA Cycle. 

Fluoroacetic acid has been identifled as the toxic component of the South African plant gijblaar (Dichapetalum cymosum) [34]. Its mechanism of action is based on inhibition of the citric acid cycle, the main source of metabolic energy in all animals [35]. In this cyde, fluoroacetate can replace acetate as a substrate of aconi-tase, an enzyme complex which usually forms dtrate by addition of acetate to a-oxoglutarate. The resulting fluorocitrate is binds tightly to the enzyme, but cannot be further converted to ds-aconitate and isocitrate [36], thus inhibiting aconitase. 

MFA. Examined as a possible CW agent but humans are less susceptible than laboratory animals. The fluoroacetates inhibit the mitochondrial enzyme aconitase and disrupt the citric acid cycle. The first signs of poisoning are nausea and apprehension, followed by convulsions. Death is due to ventricular fibrillation. 

Fluoroacetate is converted to fluoroacetyl-CoA. This substance then reacts with oxaloacetate to produce fluorocitrate. Fluorocitrate is toxic because it inhibits aconitase, the enzyme that normally converts citrate to isocitrate, hence the buildup of citrate. In the plant fluoroacetate is stored, in vacuoles away from the mitochondria. 

Cells readily convert fluoroacetate to fluoroacetyl-CoA in a reaction catalyzed by the enzyme acetate thiokinase (reaction diagram). Fluoroacetyl-CoA can combine with oxaloacetate to form fluorocitrate in a reaction catalyzed by the citric acid cycle enzyme, citrate synthase. Fluorocitrate is toxic to cells because it inhibits aconitase. 

The enzyme will also act on fluoroacetate in the same manner, converting it to fluoroacetyl-CoA, which, after combination with oxaloacetate to form fluorocitrate, inhibits the citric acid cycle enzyme aconitase. Thus, fluoroacetate can be very deadly to cells. 

It exerts its effect by being an inhibitor of the Krebs cycle. Fluoroacetate substitutes for acetate and is converted to fluoroacetyl CoA. This is condensed with oxaloacetate to produce fluorocitrate in other words citrate synthase uses fluoroacetate as a substrate and forms fluorocitrate that inhibits aconitase (reaction 2 in Fig. 10-9A and B). If the Krebs cycle is not functioning correctly, the rate of NADH production will be insufficient to maintain the mitochondrial proton gradient, and the ATP concentration will decline. A further complication is that processes such as the Krebs cycle have an important role in regenerating moiety carrier molecules. If the cycle is inhibited, then vital carriers such as oxaloacetate and coenzyme A become depleted. 

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