Decoding Krebs' Citric Acid Cycle: An Essential Guide for all type of Medical Professionals Exams and Tests

Mastering Krebs' Citric Acid Cycle (Tricarboxylic Acid Cycle): A Must-Read Guide for Medical Professional Exams Preparation

    Get ready to embark on a journey of discovery as we delve into the world of the Tricarboxylic Acid Cycle, the cornerstone of cellular metabolism. Whether you're a medical student preparing for your MBBS, BS Nursing (BSN), Paramedic, or BS Nutrition exams, or just looking to brush up on your biochemistry knowledge for entry tests, this article is for you. We'll cover all the important aspects of the TCA Cycle, from its introduction to sources of Acetyl Co-A, key steps of the Citric Acid Cycle, and the factors that regulate it. We'll also discuss the diseases caused by interruptions or disturbances in Krebs' Cycle. Get ready to learn in an easy, self-explanatory, and comprehensive manner and score higher in all your exams!"

A comprehensive guide on Kreb's Citric Acid Cycle 
From Biochemistry Library of H.E.S (Health, Education, and Skills)

Introduction to Kreb's Citric Acid Cycle | Tricarboxylic Acid Cycle

• The Krebs or Citric Acid Cycle, also known as the Tricarboxylic Acid Cycle (TCA Cycle), is a series of chemical reactions in the cellular respiration process that generates energy for the cells in the body. 
• This cycle, first discovered by Sir Hans Adolf Krebs in 1937, plays a crucial role in the production of ATP, the main energy source for cellular processes. 
• The cycle takes place in the mitochondria (in close proximity to the respiratory chain) of cells and involves the oxidation of acetyl-CoA to carbon dioxide, releasing energy in the form of ATP and other high-energy compounds. 
• The Krebs cycle is a continuous process that runs in conjunction with other metabolic pathways to ensure a constant supply of energy for the cells i.e. TCA Cycle is not exclusively a Carbohydrate metabolic process.
• It is an amphibolic process i.e. anabolism and catabolism are taking place simultaneously.
• No ATP is utilized in this cycle.
• The cycle starts with Acetyl Co-A and Oxaloacetate.
• 3 NADH2, 1 FADH2, and 1 GTP are produced in this cycle.

Significance of Krebs' Cycle

    It has several significant points, including:

• Energy Generation: The TCA Cycle is a central source of energy production in the body, generating high-energy molecules that can be used to fuel cellular processes.
• Waste Reduction: The cycle plays a role in reducing waste and preventing the buildup of toxic byproducts by breaking down molecules into CO2.
• Synthesis of Important Molecules: The TCA Cycle also contributes to the synthesis of important molecules such as amino acids, nucleotides, and fatty acids.
• Maintenance of Acid-Base Balance: The cycle helps maintain the body's acid-base balance by producing and eliminating bicarbonate.
• Regulation of Metabolism: The TCA Cycle is regulated by enzymes and hormones, allowing it to adjust its activity in response to the body's energy needs.

Sources of Acetyl Co-A

    Acetyl-CoA is produced from a variety of organic compounds and serves as the starting point for the Krebs Cycle. The main sources of acetyl-CoA are:

a. Pyruvate Oxidation: Pyruvate, the end product of glucose metabolism in glycolysis, is converted to acetyl-CoA by the pyruvate dehydrogenase complex in the mitochondria.

b. Beta-oxidation of Fatty acids: Long-chain Fatty acids are broken down into smaller units by a process called beta-oxidation, producing acetyl-CoA as a byproduct.

c. Amino acid degradation: The breakdown of Amino acids, either through catabolism or transamination, also produces acetyl-CoA.

d. Ketone body metabolism: In times of low glucose availability, such as during fasting or low carbohydrate diets, the liver produces ketone bodies from fatty acids, one of which is acetoacetate which can be converted to acetyl-CoA.

Important steps of the TCA cycle (Kreb's Cycle)

    The steps of the TCA cycle are as follows:

I. Conversion of Oxaloacetate (OA) + Acetyl Co-A to Citrate

• The cycle starts when OA (Oxaloacetate) combines with acetyl Co-A and forms Citrate.

        Oxaloacetate (OA) + Acetyl Co-A → Citrate

• The reaction is catalyzed by Citrate Synthase.
• Citrate inhibits Phosphofructo kinase (the rate-limiting enzyme of glycolysis).
• Citrate synthase is inhibited by ATP, NADH2, and Succinyl Co-A (Negative feedback).

II.  Conversion of citrate to isocitrate

• Citrate is converted to isocitrate by the action of the enzyme Aconitase.

        Citrate + H2O -> Isocitrate + CO2

• Aconitase catalyzes this reaction by facilitating the transfer of an HCO3- ion from citrate to water, resulting in the formation of isocitrate and CO2.

III. Oxidation of Isocitrate to Alpha-Ketoglutarate 

• This irreversible reaction is catalyzed by Isocitrate Dehydrogenase.
• It is the rate-limiting reaction.

        Isocitrate -> Alpha-Ketoglutarate + CO2

• 1st NADH2 is produced here, which provides 3 ATPs through the respiratory chain.
• The enzyme is activated by ADP and NAD and inhibited by ATP and NADH2.

IV. Decarboxylation of Alpha-ketoglutarate to Succinyl-CoA

• Alpha-ketoglutarate is decarboxylated by the enzyme alpha-ketoglutarate dehydrogenase, producing succinyl-CoA.

        Alpha-Ketoglutarate + CoA + NAD+ -> Succinyl-CoA + CO2 + NADH

• Dehydrogenase complex is the complex of three enzymes (same as that of pyruvate dehydrogenase complex with a slight difference) i.e. alpha-ketoglutarate decarboxylase, dihydrolipoyl, and dihydrolipoyl dehydrogenase. 
• The reaction requires five coenzymes i.e. FAD, NAD, TPP, Co-A, and Lipoic Acid.
• 2nd NADH2 is produced here, which provides 3 ATPs through the respiratory chain.
• The enzyme is inhibited by ATP, GTP, NDH2, and Succinyl Co-A (negative feedback).

V. Conversion of Succinyl-CoA to Succinate

• Succinyl-CoA is converted to succinate by the enzyme succinyl-CoA Thiokinase.

        Succinyl-CoA + GDP + Pi → Succinate + CoA + GTP

• One phosphate is released which is taken up by GDP and becomes GTP.
• ADP then takes up one Pi (inorganic phosphate) from GTP and becomes ATP.
• Therefore 1 ATP is produced at the substrate level.   

VI. Oxidation of Succinate to Fumarate

• Succinate is oxidized by the enzyme succinate dehydrogenase, producing fumarate.
• 1 FADH2 is produced which provides 2 ATPs through the respiratory chain.

        Succinate + FAD --> Fumarate + FADH2

VII. Hydration of Fumarate to Malate

• Fumarate is hydrated by the enzyme Fumarase, producing malate.
• It involves the addition of a water molecule to fumarate, leading to the formation of malate.

        Fumarate + H2O --> Malate

VIII. Oxidation of Malate to Oxaloacetate

• Malate is oxidized by the enzyme Malate Dehydrogenase, producing oxaloacetate.

        Malate + NAD+ --> Oxaloacetate + NADH + H+    

• 3rd NADH2 is produced here which provides 3 ATPs through the respiratory chain.
• This reaction is responsible for the fermentation of alcohol by yeast and some bacteria including intestinal flora.
• Oxaloacetate (produced in this step) is converted back to citrate by the enzyme citrate synthase, starting a new cycle.

Factors regulating TCA cycle | Kreb's Cycle

    Here are some of the key regulatory factors of the TCA Cycle:

1. Enzymes: 

a. Citrate synthase:

• Citrate synthase catalyzes the formation of Citrate from Acetyl-CoA and Oxaloacetate (the first reaction of the TCA cycle).
• Citrate synthase plays a critical role in the regulation of the TCA cycle by modulating its rate in response to changes in the energy demands of the cell. By controlling the rate of the cycle, citrate synthase helps to coordinate energy production with the needs of the cell, ensuring that energy is produced when it is needed and conserved when it is not.
• Here are a few ways in which citrate synthase regulates the TCA cycle:

i. Allosteric regulation: The activity of Citrate Synthase is influenced by the presence of other molecules. For example, the accumulation of Citrate in the cell can stimulate the activity of Citrate Synthase, increasing the rate of the TCA cycle.

ii. Substrate availability: In case of short supply of subtrates of Citrate Synthase (i.e Oxaloacetate and Acetyl CoA), the activity of citrate synthase will be inhibited, slowing down the TCA cycle.

iii. Feedback inhibition: The accumulation of certain intermediates in the TCA cycle can inhibit its activity. For example, the accumulation of succinyl-CoA can inhibit the activity of citrate synthase, slowing down the TCA cycle.

b. Isocitrate Dehydrogenase (IDH)

• It catalyzes the conversion of isocitrate to alpha-ketoglutarate, the first committed step in the TCA Cycle. 
• As a regulatory enzyme, IDH has the ability to control the rate of the TCA Cycle by modulating its activity in response to various signals within the cell.
• IDH is subject to feedback inhibition by its end product, alpha-ketoglutarate, which can bind to the enzyme and reduce its activity. 
• IDH is also subject to allosteric regulation by various metabolites, including NADH and ADP. An increase in NADH levels, for example, can stimulate IDH activity and increase the rate of the TCA Cycle.
• Hormones, such as insulin increases IDH activity, which can lead to an increase in the rate of glucose oxidation and energy production.

c. Alpha-Ketoglutarate Dehydrogenase

• Alpha-Ketoglutarate Dehydrogenase (α-KGDH) complex catalyzes the conversion of alpha-ketoglutarate to succinyl-CoA, the second to last step in the TCA Cycle. 
• α-KGDH has the ability to control the rate of the TCA Cycle by modulating its activity in response to various signals within the cell.
• Thus α-KGDH serves as a crucial regulatory factor in the TCA Cycle, allowing it to adjust its activity in response to changes in cellular energy demand and nutrient availability.

d. ADP availability 

• The amount of ATP produced in the TCA Cycle is directly related to the availability of ADP. 
• When the amount of ATP in the cell decreases, the concentration of ADP increases, stimulating the TCA Cycle to produce more ATP. This is because the rate of TCA Cycle reactions is determined by the availability of substrate, such as ADP, that the enzymes involved in the cycle used to produce ATP.
• On the other hand, when the amount of ATP in the cell increases, the concentration of ADP decreases, slowing down the TCA Cycle and preventing the overproduction of ATP. This helps to regulate the energy balance in the cell, ensuring that energy is produced only when it is needed.
• In addition, the availability of other substrates, such as citrate, can also play a role in regulating the TCA Cycle. For example, when citrate levels are high, the TCA Cycle slows down, preventing the overproduction of ATP and conserving energy.

Diseases caused by abnormalities in TCA Cycle | Clinical Note

    Abnormalities in the TCA Cycle can result in various diseases and disorders i.e.

a. Mitochondrial disorders 

• Mitochondrial disorders are a group of diseases caused by mutations in genes involved in Tricarboxylic Acid (TCA). These mutations can affect the function of the mitochondria.
• For example, mutations in genes encoding enzymes involved in the TCA Cycle can result in a deficiency of these enzymes, leading to a reduction in energy production and a buildup of toxic compounds. This can cause damage to the mitochondria, leading to oxidative stress, inflammation, and cell death.
• Mitochondrial disorders can also result from mutations in genes involved in the regulation of the TCA Cycle. For example, mutations in genes that regulate the activity of enzymes involved in the cycle can cause these enzymes to become overactive or underactive, leading to disruptions in energy production and other cellular processes.

b. Leigh syndrome 

• Leigh syndrome is a rare, inherited neurological disorder that affects the central nervous system. 
• It is caused by mutations in genes involved in the TCA Cycle, leading to a decrease in energy production in the brain and other tissues.
• The specific mechanisms by which these mutations cause Leigh syndrome vary, but they generally lead to a decrease in energy production and oxidative stress, which can damage the mitochondria and other cellular structures. 
• This can cause symptoms such as muscle weakness, loss of muscle coordination, developmental delays, and vision and hearing loss.
• In Leigh syndrome, the symptoms usually start in infancy or early childhood and progressively worsen over time. 
• The disease primarily affects the nervous system, but can also affect other organs and systems, including the heart, liver, and kidneys.

c. Glutaric acidemia type I 

• Glutaric acidemia type I is a rare genetic disorder that affects the metabolism of certain amino acids. 
• GA-I is caused by mutations in the gene encoding glutaryl-CoA dehydrogenase, an enzyme involved in the TCA Cycle that is responsible for breaking down the amino acid lysine and the organic acid glutaric acid. In GA-I, the mutated enzyme is not functional, leading to a buildup of these toxic compounds in the body and a deficiency in energy production.

d. MELAS syndrome

• Mitochondrial Encephalomyopathy, Lactic Acidosis, and Stroke-like episodes (MELAS) syndrome is a rare genetic disorder caused by abnormalities in the mitochondria. 
• MELAS is associated with mutations in the mitochondrial DNA, leading to a reduction in the efficiency of the Tricarboxylic Acid (TCA) Cycle.
• The reduction in energy production leads to an increase in a lactic acid buildup in the body, as the mitochondria are not able to effectively produce ATP.
• The accumulation of lactic acid and the reduction in energy production can lead to oxidative stress, which can damage the cells and lead to the production of free radicals.

e. Multiple acyl-CoA dehydrogenase deficiency (MADD)

• MADD is a rare genetic disorder that affects the metabolism of certain fatty acids. 
• It is caused by a deficiency in enzymes involved in the TCA Cycle, leading to a buildup of toxic compounds and a decrease in energy production.

ATPs produced in the TCA cycle

• 3 NADH2 = 9 ATPs
• 1 FADH2 = 2 ATPs
• 1 GTP = 1 ATP
• Net ATP produced = 12 ATPs/ Acetyl co-A oxidized.

Conclusion

    The Krebs or Citric Acid Cycle, also known as the Tricarboxylic Acid Cycle (TCA Cycle), is a key process in cellular respiration that generates energy for cells. This cycle was discovered by Sir Hans Adolf Krebs in 1937 and occurs in the mitochondria of cells. It involves the oxidation of acetyl-CoA to carbon dioxide, releasing energy in the form of ATP and other high-energy compounds. The cycle is a continuous process that runs alongside other metabolic pathways to provide a constant source of energy to cells. It is an amphibolic process, meaning that both anabolism and catabolism occur simultaneously.

    Acetyl-CoA, the starting point of the Krebs Cycle, is produced from several sources including pyruvate oxidation, beta-oxidation of fatty acids, amino acid degradation, and ketone body metabolism. The steps of the TCA cycle include the conversion of oxaloacetate and acetyl-CoA to citrate, conversion of citrate to isocitrate, oxidation of isocitrate to alpha-ketoglutarate, and decarboxylation of alpha-ketoglutarate to succinyl-CoA.

    The TCA Cycle is important for several reasons, including energy generation, waste reduction, synthesis of important molecules, maintenance of acid-base balance, and regulation of metabolism. The cycle is regulated by enzymes and hormones, allowing it to adjust its activity in response to the body's energy needs. In conclusion, the Krebs Cycle plays a vital role in cellular respiration and energy production, contributing to the overall functioning of cells and the body.