Anaplerotic Reactions and Their Role in the Citric Acid Cycle

Anaplerotic reactions are metabolic pathways used to replenish oxaloacetate in the citric acid cycle after it has been consumed. The purpose of these reactions is to maintain adequate levels of ATP so that cellular respiration can carry on uninterrupted.

What is an Anaplerotic Reaction?

The anaplerotic reaction is the anabolic reaction that helps to generate the intermediate compounds of the biochemical; pathways. The intermediate reaction step of such a reaction is known as anaplerotic routes. Anaplerotic reactions are an important part of the metabolism; that is, they are an important part of the biochemical pathways like citric acid pathways, lipid biosynthesis. In this article, we mainly focus on the anaplerotic reaction, anaplerotic routes for anaplerotic pathways. It focuses on the physiological role of anaplerosis.

In the citric acid cycle, amino acid metabolism and synthesis of triglyceride in adipose tissue, which is also known as lipid biosynthesis. 

What is Anaplerosis?

Anaplerosis can be defined as the reaction that can replenish the intermediates of the pathway. In simpler terms, anaplerotic reaction maintains the dynamic balance of an anaplerotic route in such a way that the concentration of the crucial but depleted intermediate has remained as a constant. 

Anaplerotic routes are the reaction steps that are followed to generate the intermediates of the biochemical pathways. 

Details of Anaplerotic Reaction

1. Pyruvate Carboxylase Pathway 

This pathway produces oxaloacetate from two molecules of pyruvate. It is activated by high levels of ATP and inhibited by high levels of ADP. The pathway uses the enzyme pyruvate carboxylase to convert pyruvate into oxaloacetate.

2. PEP Carboxykinase Pathway 

This pathway produces oxaloacetate from one molecule of pyruvate and one molecule of PEP. It is activated by high levels of ATP and inhibited by high levels of ADP. The pathway uses the enzyme PEP carboxykinase to convert pyruvate and PEP into oxaloacetate.

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3. The Citric Acid Cycle 

The citric acid cycle also called the tricarboxylic acid cycle or the Krebs cycle is a series of enzymatic reactions that convert the energy in food molecules into ATP. The cycle begins with the oxidation of pyruvate, a product of glycolysis, to acetyl CoA. Acetyl CoA is then oxidized by the citric acid cycle, which results in the oxidation of NADH and FADH to NAD and FAD, respectively. In turn, these coenzymes are oxidized by oxidative phosphorylation to produce ATP.

The Physiological Role of Anaplerosis in the Citric Acid Cycle

Anaplerotic reactions are a very important part of the citric acid cycle, also known as the TCA cycle. The citric acid cycle is an amphibolic pathway. Amphibolic pathways are those pathways that can perform both anabolic reactions as well as catabolic reactions. The anaplerotic reaction is also known as the anaplerotic pathways of the citric acid cycle. They are responsible for the anabolic part of the cycle. The primary role of the citric acid cycle is the oxidation of acetyl-CoA to carbon dioxide.

It is important to understand that TCA cycle intermediates are sufficient to sustain the

oxidative carbon flux during high energy consumption like individuals performing exercise or during lower energy consumption like fasting. It is important to note that there is not a large change in the pool size of TCA intermediates. In several physiological states, there is a large influx of intermediates like 4- and 5-carbon intermediates into the TCA cycle. It is notable that even with the change in the intermediate concentrations, the citric acid cycle can not act as a carbon sink, so it maintains a dynamic balance between incoming and outgoing intermediates by anaplerosis and cataplerosis. 

Anaplerotic Reactions of the Citric Acid Cycle

 There are four major anaplerotic reactions in the TCA cycle.

1. Pyruvate to oxaloacetate

2. Phosphoenolpyruvate to oxaloacetate

3. Phosphophenol pyruvate to oxaloacetate using PEP carboxykinase.

4. Pyruvate to malate

1. Pyruvate to Oxaloacetate

This reaction takes place in the cells of the liver and kidney. The enzyme required to convert pyruvate to oxaloacetate is pyruvate carboxylase. This reaction is a reversible reaction. Oxaloacetate is the intermediate of the TCA cycle that undergoes condensation reaction by citrate synthase to yield citrate. The chemical reaction can be written as 

\[Pyruvate + HCO_{3}^{-} + ATP \rightarrow  oxaloacetate + ADP + Pi\]

This reaction is catalyzed by pyruvate carboxylase. The enzyme catalyzes a reversible reaction. This is the most important part of the anaplerotic route or anaplerotic pathway.

Pyruvate Carboxylase- It is a mitochondrial enzyme that catalyzes carboxylation reactions. It is considered the regulatory enzyme of the citric acid cycle. It requires an allosteric activator for its activity. Acetyl CoA acts as the positive allosteric modulator of this enzyme. Pyruvate carboxylase has two major roles one in anaplerosis and second in gluconeogenesis.

Biotin acts as a prosthetic group for this enzyme. Biotin acts as a carrier of a one-carbon group in its oxidized state. Biotin is an essential part of the human diet and is abundant in many food sources. 

Reaction Steps- There are the following steps in which pyruvate carboxylase catalyzes the reaction.

ATP binds to the pyruvate carboxylase; the carboxylation of ATP produces carbonic phosphoric anhydride

↓

Phosphoric anhydride forms carboxy phosphate

↓

Carboxy phosphate carboxylates the biotin, prosthetic group of carboxylase enzyme

↓

This carboxylation of the reaction requires a positive modulator like Acetyl CoA.

↓ 

The activated carbon is transferred into the second catalytic site.

↓

Pyruvate then accepts the carbon dioxide from the second catalytic site.

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Addition of carbon dioxide to pyruvate yields oxaloacetate

↓

Oxaloacetate is removed from the active site and enters the citric acid cycle.

2. Phosphoenolpyruvate to Oxaloacetate

Phosphoenolpyruvate is converted into oxaloacetate to maintain the steady flow of intermediate by the anaplerotic reaction. This reaction takes place in the heart and skeletal cells. This reaction is catalyzed by the PEP carboxylase. It is important to note that the production of GTP is associated with this reaction. The reaction can be written as,

Phosphophenolpyruvate + carbon dioxide + GDP ---------> oxaloacetate + GTP

The enzyme PEP carboxylase mechanism of action is widely studied among enzymes. The enzymes require cofactors such as \[Co^{2+}, Mg^{2+}, or Mn^{2+}\]. These are metallic cofactors that bind to substrate allosteric sites. The reaction is an exothermic reaction, thus rendering it a reversible enzyme.

Reaction Mechanism

The reaction mechanism of the enzyme can be defined in two main steps as follows.

Nucleophilic attack to the phosphate group of the PEP is mediated by the bicarbonate.

↓

PEP splits to form carboxyl phosphate and pyruvate enolate (a reactive form of the pyruvate)

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Carboxy phosphate mediates the proton transfer by a histidine residue. 

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Carboxy phosphate then undergoes decomposition to form carbon dioxide and inorganic phosphate.

↓

Pyruvate enolate bound to the metallic factor reacts with carbon dioxide to form oxaloacetate.

3. Pyruvate to Malate

This conversion of pyruvate to malate is catalyzed by the enzyme malic enzyme. The enzyme performs reductive carboxylation; it uses NADP as the cofactor. This reaction is also a reversible reaction. The site of this anaplerotic pathway is widely distributed in eukaryotes and bacteria. 

The reaction can be written as:

\[Pyruvate + HCO_{3} + NADPH \rightarrow malate + NADP \]

4. Phosphoenolpyruvate to Oxaloacetate Using PEP Carboxykinase

There is an important distinction between the oxaloacetate produced by PEP carboxylase and PEP carboxykinase. In this reaction, oxaloacetate formation is accompanied by the formation of the GTP. This reaction takes place in the higher plant taxa, yeasts, and bacteria. It acts as a junction between glycolysis and the TCA cycle. The chemical reaction can be written as:

\[Phosphophenolpyruvate + HCO_{3} \rightarrow oxaloacetate + Pi\]

Metabolic Fate of Amino Acid of TCA Cycle

It is important to note that one of the anaplerotic pathways, the intermediates of the TCA, leads to the production of amino acids from phosphoenolpyruvate. Amino acids such as serine, glycine, cysteine, phenylalanine, tyrosine, and tryptophan have been generated from this pathway. 

The metabolic fate of the amino acids are as follows- 

1. Amino acids converted to pyruvate. Examples of such amino acids include alanine, serine, glycine, threonine, cysteine, tryptophan.

2. Amino acids are converted to oxaloacetate. Examples of such amino acids include aspartate, asparagine.

3. Amino acids are converted to -ketoglutarate. Example of such amino acids includes glutamate, glutamine, proline, histidine, arginine.

4. Amino acids are converted to fumarate. Examples of such amino acids include phenylalanine, tyrosine.

5. Amino acids converted to succinyl-CoA. Example of such amino acids includes methionine, isoleucine, valine.

6. Amino acids converted to acetyl-CoA. Examples of such amino acids include leucine, isoleucine, lysine, phenylalanine, tyrosine, tryptophan, threonine.

Anaplerotic Reaction in Lipid Biosynthesis

The anaplerotic route followed during lipid biosynthesis is of the fatty acid. The anaplerotic reaction in the beta-oxidation of the fatty acid to provide succinyl CoA. The oxidation of the fatty acyl CoA with odd numbers of carbon chain leads to the formation of the final product Succinyl CoA. The final product can enter the TCA cycle directly or can undergo conversion to form acetyl CoA to enter the citric acid cycle. 

Conclusion

Anaplerotic reactions are pathways used to replenish oxaloacetate in the citric acid cycle after it has been consumed. The purpose of these reactions is to maintain adequate levels of ATP so that cellular respiration can carry on uninterrupted. There are two main pathways for replenishing oxaloacetate: the pyruvate carboxylase pathway and the PEP carboxykinase pathway. Both of these pathways are activated by high levels of ATP and inhibited by high levels of ADP.