Reactions of Citric Acid Cycle

TRICARBOLYLIC ACID CYCLE (TCA) OR KREBS CYCLE OR CITRIC ACID CYCLE

Generally cyclic pathway refers to the repeated pathway in which the initiating compound or material is regenerated and is re-entered to the pathway again and again (For example: Oxaloacetate in TCA cycle).

TCA cycle is the complete enzymatic series of reactions that results in the oxidation of acetyl group i.e. (CH₃-CO-) of acetyl CoA to two molecules of carbon dioxide along with the yield of reduced electron carriers NADH and FADH₂ which on respiratory chain donate their electron with the subsequent yield of ATP. This cyclic pathway was proposed by Hans Adolf Krebs in 1937 and is also known by his name as "Krebs cycle"

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The TCA cycle is the common and universal oxidative pathway for the compounds derived from the breakdown macro biomolecules like carbohydrate, protein and fat are oxidized to CO₂ with the yield of temporarily energy holding molecules (electron carriers) FADH₂ and NADH. This pathway is considered as the central metabolic heart of the cell. The electron carriers, in the course take part in electron transport chain and transfer their electron to oxygen and the energy released during flow of energy is trapped and stored as ATP. About 70% of the net ATP produced in an aerobic cell is produced in this cyclic pathway. The fuel runner of this cycle which are derived after the breakdown of carbohydrates, proteins of fat enter to this cycle in the form of acetyl-CoA. This cycle is not only significant as it is highly energy yielding cyclic pathway but also as it is an important source of precursors of many molecules amino acids, glucose, cholesterol, etc. This cyclic metabolic pathway occurs in cytosol of prokaryotic cells whereas in matrix of mitochondria of eukaryotic cells. The gate way of this cycle is initiated by the substrate, acetyl-CoA that combines with the four carbon molecule, oxaloacetate thus giving the first six carbon molecule, citrate. Oxaloacetate is regenerated and reutilized time and again at the end of cycle remaining same the self but deforming the acetyl-CoA. Thus a single molecule of oxaloacetate is enough for the oxidation of many acetyl CoA molecules.

A complete cycle of TCA is observed as follows:

Stepwise reactions of TCA cycle:

Citric acid cycle is completed with the series of eight reactions. Starting with the formation of citrate and ending with the regeneration of oxaloacetate molecule this cyclic pathway gets one complete cycle and is repeated in same manner. In between these reactions, there is the oxidation of acetyl CoA and oxaloacetate to Co₂ and the overall reaction is irreversible in nature.

• Formation of Citrate:

The first reaction of TCA cycle initiates with an anabolic reaction. Here acetyl CoA (2 carbon) and oxaloacetate (4 carbon) condense to form a six carbon molecule called citric acid of simply citrate. In this reaction the acetyl group of acetyl CoA is fused to the carbonyl group of oxaloacetate. This reaction as catalyzed by the enzyme, citrate synthase. This reaction is hydrolysis lead by aldolcondensation first.

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[Remember: The enzyme synthase catalyzes the condensation reactions in absence of nucleoside triphosphates (ATP, GTP etc.) as energy sources whereas synthetase enzyme requires those energy sources in similar types of reactions (synthetic reactions)]

• Formation of Isocitrate:

This is the double step reaction in which an intermediate cis-aconitate is formed as an intermediate. The enzyme aconitase of aconitate hydratase catalyzes the reaction reversibly. There is the isomeric conversion of citrate to isocitrate with the formation of cis-aconitate as dehydrate intermediate.

First reaction is dehydration reaction in which there the formation of cis-aconitate by the loss of water molecule from citrate which is assisted by aconitate enzyme.

In second reaction is hydration reaction where there is the regain of the lost water molecule with the aid of same enzyme. The net effect of this reaction is the isomeric change in the structure of citrate to isocitrate.

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Oxidation of Isocitrate:

In this step of reaction α-ketoglutarate (5 carbon) is obtained as the oxidized product of isocitrate which is catalyzed by the enzyme, isocitrate dehydrogenase irreversibly. This reaction in overall view seems to be simple but is a complex reaction indeed.

This reaction in fact completes in two steps: initial step is the dehydrogenation reaction in which isocitrate is dehydrated to oxalosuccinate. In this reaction a molecule of NAD is reduced to NADH + H⁺ with the aid of Isocitrate dehydrogenase enzyme.

Secondly the intermediate oxalosuccinate losses carbonyl group in the form of CO₂ (decarboxylation) to give a five carbon molecule, α-ketoglutarate with the aid of enzyme, decarboxylase.

Here two enzymes act in the overall oxidation of Isocitrate to α-ketoglutarate and CO₂. Hence generally in overall reaction enzyme is named as "dehydrogenase complex"

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• Oxidation of α-ketoglutarate to succinyl-CoA and CO₂:

The next reaction is irreversible oxidative decarboxylation of α-ketoglutarate to succnyl CoA and CO₂ by the catalytic assistance of the enzyme, α-ketoglutarate dehydrogenase complex. This reaction is also a complex reaction where there is the occurrence of three main steps:

1. a) Loss of carboxylate group in the form of CO₂ from α-ketoglutarate,
2. b) Reduction of NAD to NADH+ H⁺ which results the oxidation of α-ketoglutarate and
3. c) Combination of coenzyme a with succinate to form succnyl CoA.

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(Note: H in NADH is not obtained from substrate i.e. α-ketoglutarate but from -SH of CoA-SH)

• Conversion of Succinyl-CoA to Succinate:

This is a reversible reaction in which succinyl CoA is chopped into two parts i.e. succinate and coenzyme A with an assistant catalysis of succinate thiokinase (also known as succinic acid CoA ligase). The cleavage of the bond between succinyl group and CoA group (thioester bond) is extremely exergonic and is used to synthesize a GTP from GDP and an ortho-phosphate (Pi).

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GTP can be converted to ATP by the aid of nucleotide kinase.

GTP + ADP → GDP + ATP

• Oxidation of succinate to Fumarate:

A flavoprotein, succinate dehydrogenase reversibly catalyzes the reversible oxidation of previously formed compound, succinate to fumarate. Here in this step FAD is reduced to FADH₂ which signifies the oxidation of succinate to fumarate. FADH₂ after involving in electron transfer chain yields two ATPs.

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• Hydration of Fumarate to Malate.

In this step the enzyme, fumarase or fumarase hydratase aids the reversible addition of water molecule to the double bond of fumarate to give malate.

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• Oxidation of malate to oxaloacetate:

This is the final step reaction in citric acid cycle in which malate dehydrogenase enzyme catalyzes the reversible oxidation of malate to oxaloacetate. As malate is oxidized, there is the reduction of NAD to NADH. The oxaloacetate thus formed is recycled in the cycle again by the condensation with acetyl CoA to form citrate.

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This way TCA cycle goes on repeating unless there is the deficiency of acetyl CoA in the cell.

One of the important part we need to remember:

There are 3 irreversible reactions in TCA cycle and rest of all are reversible. Step 1, 3 and 4 are only the irreversible reactions in overall TCA cycle. And the regulated enzymes are citrate synthetase, isocitrate dehydrogenase, α-ketoglutarate dehydrogenase complex and succinate dehydrogenase