Question

Mode of Action of the Rodenticide Fluoroacetate Fluoroacetate, prepared commercially for rodent control, is also produced by a South African plant. After entering a cell, fluoroacetate is converted to fluoroacetyl-CoA in a reaction catalyzed by the enzyme acetate thiokinase: You perform a perfusion experiment to study the toxic effect of fluoroacetate using intact isolated rat heart. After perfusing the heart with \(0.22 \mathrm{~mm}\) fluoroacetate, you see a decrease in the measured rate of glucose uptake and glycolysis as well as an accumulation of glucose 6-phosphate and fructose 6-phosphate. Examination of the citric acid cycle intermediates reveals that their concentrations are below normal, except for citrate, which has a concentration 10 times higher than normal. a. Where did the block in the citric acid cycle occur? What causcd citrate to accumulate and the other cycle intermediates to be depleted? b. Fluoroacetyl-CoA is enzymatically transformed in the citric acid cycle. What is the structure of the end product of fluoroacetate metabolism? Why does it block the citric acid cycle? How might the inhibition be overcome? c. In the heart perfusion experiments, why did glucose uptake and glycolysis decrease? Why did hexose monophosphates accumulate? d. Why is fluoroacetate poisoning fatal?

Short answer

a: The block is after citrate formation. b: Fluorocitrate is the end product; it blocks aconitase. c: Inhibition of ATP production lowers uptake/glycolysis. d: It disrupts cellular respiration, leading to fatality.

Step-by-step solution

Identify the Block in the Citric Acid Cycle

The accumulation of citrate and the depletion of other intermediates suggest a block after citrate formation. The fluoroacetyl group from fluoroacetyl-CoA is incorporated into the citric acid cycle to form fluorocitrate via citrate synthase, which then inhibits aconitase, preventing the conversion of citrate to isocitrate.

Understand Fluoroacetate Metabolism

Fluoroacetate is converted into fluoroacetyl-CoA, which enters the citric acid cycle, forming fluorocitrate. Fluorocitrate inhibits aconitase, blocking the cycle and causing citrate accumulation. The inhibition is due to the structural similarity of fluorocitrate to substrates of aconitase, and it's difficult to overcome this inhibition unless the inhibitor is removed.

Analyze the Impact on Glycolysis and Glucose Uptake

The block in the citric acid cycle causes a reduction in NAD+ and FAD regeneration, leading to lower ATP production, which hinders glucose uptake and glycolysis. Hexose monophosphates accumulate due to feedback inhibition from glucose 6-phosphate and fructose 6-phosphate buildup as a result of slowed glycolysis.

Examine the Fatality of Fluoroacetate Poisoning

Fluoroacetate poisoning is fatal because it disrupts cellular respiration by blocking the citric acid cycle, leading to decreased ATP production. This affects vital organs, particularly the heart and central nervous system, ultimately causing organ failure and death.

Key concepts

Fluoroacetate Metabolism

Fluoroacetate is a potent toxin, commonly used as a rodenticide. Upon entering an organism's cell, fluoroacetate is enzymatically transformed by acetate thiokinase into fluoroacetyl-CoA. This fluoroacetyl-CoA then enters the citric acid cycle, a pivotal pathway for energy production in cells. Here, it reacts with oxaloacetate to form fluorocitrate via citrate synthase. Consequently, the production of fluorocitrate disrupts the cycle. This conversion underscores the toxic nature of fluoroacetate, which leads to the subsequent inhibition effects within the citric acid cycle.
The metabolic transformation effectively converts a seemingly benign compound into a highly disruptive agent within cellular metabolism. The ultimate end product, fluorocitrate, is structurally analogous to normal substrates processed by aconitase, leading to its role as an effective inhibitor.

Aconitase Inhibition

Aconitase is a key enzyme within the citric acid cycle, responsible for the isomerization of citrate to isocitrate. This step is crucial for the continuous flow of the cycle, which is essential for energy production. When fluorocitrate forms, due to fluoroacetate metabolism, it specifically inhibits aconitase. This inhibition halts the conversion of citrate to isocitrate, causing citrate to accumulate rapidly. Consequently, other intermediates within the cycle become depleted.
The structural similarity between fluorocitrate and the substrates of aconitase is central to its inhibitory potential. The inhibition effectively places a roadblock within the cycle. It disrupts the overall flow of the citric acid cycle, thus reducing the cell's ability to produce energy. Overcoming this inhibition is challenging and primarily involves removing fluoroacetate from the system, which is difficult once it has entered the cells.

• This block causes an imbalance in the cycle, leading to energy production troubles.
• Normal cell functions become impaired, demonstrating the potency of this inhibition.

Glycolysis Feedback Inhibition

During fluoroacetate exposure, the inhibition of the citric acid cycle affects glycolysis, which is the initial stage of glucose metabolism. The reduced activity within the citric acid cycle leads to a decrease in the regeneration of electron carriers, NAD⁺ and FAD, essential for further ATP production. This connection causes an energetic crisis in the cell, resulting in decreased glucose uptake and glycolysis.
As a chain reaction, the accumulation of citrate also causes an increase in glucose 6-phosphate and fructose 6-phosphate due to feedback inhibition. These hexose monophosphates build up because their conversion and further processing in glycolysis is slowed or inhibited.

• With inadequate ATP production, cells cannot effectively bring in more glucose.
• Feedback inhibition from the accumulated glucose intermediates adds more hurdles, stalling the glycolytic pathway.

This buildup further contributes to the overall reduction in cellular energy efficiency, magnifying the toxic effects initiated by fluoroacetate.