Krebs cycle - Steps, Energetics, Significance, Practice problems and FAQs

You must be aware that there are many microbes which can breathe in the absence of oxygen. But can higher organisms like the plants or animals afford to do that? No, we cannot. Do you know why? This is because we have greater energy requirements than the microbes and respiration in the presence of oxygen yields more energy than the one without oxygen. 

You must be wondering, how does the presence and absence of oxygen affect energy production so drastically? Well, in the absence of oxygen, respiration occurs by the process of fermentation in which the glucose is first broken down to pyruvate in the cytoplasm by the process of glycolysis and then the pyruvate is incompletely oxidised either to ethanol or lactic acid. This method generates only two molecules of ATP.

However, in presence of oxygen pyruvate undergoes complete oxidation in the mitochondria to form carbon dioxide and water and yield a total of 38 ATP molecules. So now do you think that the mitochondria is justly called the ‘powerhouse of the cell’? 

The respiratory cycle which takes place in the mitochondria, in the presence of oxygen is known as the Krebs cycle. So, what happens during the Krebs cycle that end up generating such a huge amount of energy in the cell? Come let us see.

Table of contents

• Introduction to aerobic respiration
• Oxidative Decarboxylation
• Krebs cycle or TCA cycle 
• Overview of Krebs cycle 
• Summary of link reaction and TCA 
• Significance of TCA
• Practice problems
• FAQs

Introduction to aerobic respiration

The stepwise breakdown of respiratory substrates to CO₂ and H₂O in the presence of oxygen is termed as ‘aerobic respiration’. This kind of respiration involves at least 4 major steps-

• Glycolytic breakdown of glucose to pyruvic acid in the cytoplasm.
• Oxidative decarboxylation of pyruvic acid to acetyl CoA
• Krebs cycle
• Terminal oxidation and phosphorylation in the electron transport chain (ETS).

Thus, the product of glycolysis, pyruvic acid is first oxidatively decarboxylated to acetyl CoA in the mitochondrial matrix and then it enters the Krebs cycle for aerobic respiration.

                                        Fig: Aerobic respiration

Oxidative decarboxylation

The first step of aerobic respiration, taking place inside the mitochondrial matrix, is called oxidative decarboxylation. The pyruvic acid,which was generated in glycolysis, enters into the mitochondrial matrix where it undergoes decarboxylation and oxidation to form the final product called acetyl CoA. Oxidative decarboxylation is said to be the link reaction between glycolysis and Krebs’ cycle. Acetyl CoA produced in this reaction acts as a substrate for TCA (tricarboxylic acid) or Krebs cycle. The enzyme complex involved in this process is called pyruvate dehydrogenase complex. This enzyme complex functions only in the presence of the following cofactors ^- NAD⁺ and coenzyme A and also Mg²⁺.

This process involves -

1. Removal of a carboxyl group from pyruvate to form CO^{2 -} Decarboxylation
2. Loss of electrons from pyruvate which are taken up by NAD+ to NADH + H+ ^- Oxidation 
3. The two carbon acetyl group, formed due to decarboxylation, reacts with coenzyme A to form acetyl CoA

                                          Fig: Oxidative decarboxylation

Krebs cycle or TCA cycle 

The full form of the TCA cycle is the ‘TriCarboxylic Acid’ cycle. The reason for such a name is that the first stable product formed in the cycle is Citric Acid which has three carboxylic acid groups.

Discovery

Krebs cycle has been named after the scientist Hans A. Krebs, who discovered its steps in 1937-1940 and won a Nobel prize for it in 1953.

Site 

This cycle takes place inside the mitochondrial matrix where all the relevant enzymes are present.

Steps of TCA cycle

Step-1

The very first step of the TCA cycle is condensation of acetyl coA (two carbon or 2C compound) with oxaloacetic acid or OAA (four carbon or 4C compound) and water to form citric acid (six carbon or 6C compound). The enzyme used is citrate synthase and a molecule of CoA is released.

                                   Fig: Conversion of OAA to Citrate

Step-2

In this step, citrate is isomerised to isocitrate in the presence of enzyme aconitase.

                                 Fig: Conversion of citrate to aconitase

Step-3

In this step isocitrate (6C compound) undergoes oxidative decarboxylation to form ∝-ketoglutarate (5C compound). The enzyme used here is isocitrate dehydrogenase which acts in presence of Mn₂⁺. Oxidation of isocitrate results in the formation of an intermediate known as oxalosuccinate which undergoes decarboxylation to form ∝-ketoglutarate.

In this process, NAD+ is reduced to NADH + H+ and CO₂ is released.

                        Fig: Oxidative decarboxylation of isocitrate to ∝-ketoglutarate

Step-4 

In this step ∝-ketoglutarate (5C compound) undergoes oxidative decarboxylation to form succinyl CoA (2 C compound) in the presence of acetyl coA and NAD+. The enzyme ∝-ketoglutarate dehydrogenase catalyses the reaction.

In this process as well, NAD+ undergoes reduction to form NADH + H+ and CO₂ is released.

                                       Fig: Conversion of ∝-ketoglutarate to succinyl coA

Step-5 

Succinyl CoA synthetase catalyses the conversion of succinyl CoA to succinate and Coenzyme A is released in the process. The Coenzyme A of succinyl CoA is replaced by a phosphate group and this phosphate group in succinyl CoA is transferred to GDP to convert it into GTP. Thus, GTP is generated through substrate level phosphorylation and is later converted to ATP. 

                                      Fig: Conversion of succinyl CoA to succinate

Step-6 

In this step Succinate dehydrogenase (SDH) enzyme is found to be important. It is present attached to the inner mitochondrial membrane that is an integral component of the mitochondrial respiratory chain.

In the reaction succinate gets converted into fumarate in which two hydrogen atoms are removed from the succinate and added to FAD+ to form FADH₂.

                                         Fig: Conversion of succinate to fumarate

Step-7

Water is added to fumarate to form another four-carbon compound known as malate with the help of the enzyme fumarase.

                                      Fig: Conversion of fumarate to malate

Step-8

Malate dehydrogenase enzyme catalyses the conversion of malate to oxaloacetate. In this process, NAD+ is reduced to NADH + H+ and malate is oxidised to oxaloacetate.

Thus, oxaloacetate is regenerated which again takes part in the TCA cycle as a substrate.

                                                 Fig: Conversion of malate to oxaloacetate

Overview of Krebs cycle 

                                               Fig: Overview of Krebs cycle

Summary of Link reaction and TCA cycle

For each molecule of glucose, there are two molecules of pyruvate produced and thus, there are two rounds of the Krebs cycle required for the complete oxidation of glucose to carbon dioxide and water.

Carbon dioxide production

Sites of Coenzyme reduction

Total number of ATP molecules generated

A total of 30 ATP molecules are produced in the TCA cycle.

During the oxidation of each NADH molecule that is generated, transport of electrons from one molecule of NADH + H+ via the electron transport chain helps in the transport of three pairs of protons from the outer mitochondrial chamber to the mitochondrial matrix. This helps in the generation of three ATP molecules.

Similarly, transport of electrons from one molecule of FADH2 helps in the transport of two pairs of protons and hence results in formation of two ATP molecules.

Significance of TCA cycle

• TCA is the oxidative breakdown of respiratory substrates like carbohydrates, fats and amino acids. It releases energy in the form of ATP. ATP is utilised in most of the metabolic activities of the body.
• TCA cycle is the major pathway for energy production as 30 molecules of ATP are generated by this process.
• Various TCA intermediates serve as raw material (carbon skeleton or building blocks) for various anabolic processes, for example, acetyl CoA is used as a raw material for synthesis of fatty acids, aromatic compounds, etc., α keto glutaric acid is used in the synthesis of glutamic acid, etc.
• The TCA cycle is an amphibolic pathway, i.e., it serves as a catabolic as well as an anabolic pathway.

Practice problems

Q 1. How many molecules of acetyl CoA are generated by a single molecule of glucose , which enters the Krebs cycle?

a. 4
b. 3
c. 2

d. 1

Solution: During glycolysis, one molecule of glucose is converted to two molecules of pyruvic acid. Each molecule of pyruvic acid undergoes decarboxylation and oxidation to form the final product called acetyl CoA. Thus, from each molecule of glucose, two acetyl CoA molecules are produced.

Oxidative decarboxylation is the connecting link between glycolysis and Krebs’ cycle. Two Acetyl CoA produced in this reaction acts as a substrate for TCA or Krebs cycle. 

Thus, the correct option is c.

Q 2. Which Krebs cycle intermediate is utilised in the formation of amino acids?

a. Citric acid
b. Malic acid
c. Isocitric acid
d. 𝛼-ketoglutaric acid

Solution: In TCA cycle, isocitrate undergoes oxidative decarboxylation to form ∝-ketoglutarate. The enzyme used here is  isocitrate dehydrogenase. This  ∝-ketoglutarate is used in the formation of amino acids.

Thus, the correct option is d.

Q 3. Which of the following is not formed during the Krebs cycle?

a. Lactate
b. Isocitrate
c. Succinate
d. Both (a) and (b)

Solution: During Krebs cycle, Cis aconitate is converted to isocitrate with the help of the aconitase enzyme and succinyl CoA is converted to succinate with the help of succinyl CoA synthetase. 

Lactate is not formed during Krebs cycle. It is formed when pyruvate formed at the end of glycolysis undergoes lactic acid fermentation under anaerobic conditions and in the presence of the lactate dehydrogenase enzyme.

Thus, the correct option is a.

Q 4. FAD is reduced in which of the reactions of the Krebs cycle?

a. Isocitrate to oxaloacetate
b. Succinyl CoA to Succinate
c. Fumarate to malate
d. Succinate to fumarate

Solution: In the sixth step of TCA cycle, Succinate dehydrogenase (SDH) helps to oxidise succinate into fumarate by the removal of two hydrogen atoms from the succinate. This oxidation is facilitated by the reduction of FAD+ which accepts the hydrogen atoms to form FADH2.

Thus, the correct option is d.

FAQs 

Q 1. Why is oxygen needed for the TCA cycle?
Answer: Oxygen is not directly involved in the TCA cycle but it acts as the terminal accept for the H+ ions and electrons that are released due to oxidation of NADH + H+ and FADH₂ and passed through an electron transport chain. Oxygen accepts the H+ ions and electrons to finally form water molecules.

Q 2. Where are most of the enzymes required for the TCA cycle located? 
Answer: Most of the enzymes participating in the TCA cycle are located in the mitochondrial matrix except for succinate dehydrogenase which is located in the inner mitochondrial membrane.

Q 3. Which Krebs cycle intermediate can be used for the synthesis of aspartic acid?
Answer: The oxaloacetic acid generated as a result of oxidation of fumarate during the Krebs cycle is used for the synthesis of aspartic acid. 

Q 4. What are the 3 regulatory enzymes of the TCA cycle?
Answer: The three regulatory enzymes of the TCA cycle are-

1) citrate synthase
2) isocitrate dehydrogenase
3) α-ketoglutarate dehydrogenase. 

These enzymes catalyse the irreversible steps of the TCA cycle, which are the main regulatory reactions.

Youtube link- https://www.youtube.com/watch?v=MJ_0yHhvQ5g

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