The tricarboxylic acid (TCA) cycle, widely recognized as the Krebs cycle or citric acid cycle, stands as a pivotal metabolic hub within cells (Figure 1. Krebs Cycle). This cycle, crucial for energy production, is orchestrated by eight enzymes. Interestingly, seven of these enzymes are nestled within the mitochondrial matrix, the inner compartment of mitochondria, with succinate dehydrogenase being the exception as it is embedded in the inner mitochondrial membrane, closely linked to the respiratory chain. This cycle acts as a central gateway for aerobic metabolism, processing molecules that can be converted into acetyl groups or dicarboxylic acids. The regulation of the TCA cycle is meticulously controlled at three key enzymatic steps: citrate synthase, isocitrate dehydrogenase, and alpha-ketoglutarate dehydrogenase. Beyond energy generation, the Krebs cycle is also instrumental in providing precursors for the synthesis of essential biomolecules, including amino acids and cholesterol, vital building blocks for cellular functions and storage forms of energy-rich fuels.1
Understanding the Environment of the Krebs Cycle
It’s a common misconception that the Krebs cycle directly requires oxygen. In fact, oxygen is essential for the final stage of aerobic cellular respiration, known as oxidative phosphorylation, which occurs after the Krebs cycle. The Krebs cycle itself prepares energy-rich organic molecules (carbohydrates, lipids, and proteins) for this final, oxygen-dependent stage. Prior to entering the Krebs cycle, these molecules are broken down and transformed into acetyl-CoA. Acetyl-CoA is a crucial molecule composed of an acetyl group (CH3CO-) linked to coenzyme A, an acyl carrier.
While various fuel sources can feed into acetyl-CoA, glycolysis, the breakdown of glucose, is the primary and preferred pathway. Within the Krebs cycle, the acetyl group from acetyl-CoA is systematically oxidized. This oxidation process releases energy that is subsequently harnessed to synthesize ATP, the cell’s energy currency, in conjunction with oxidative phosphorylation. In eukaryotic cells, the Krebs cycle location is specifically within the mitochondrial matrix. This matrix is a dense, aqueous environment enclosed by the inner mitochondrial membrane. It’s packed with all the necessary components for the Krebs cycle to function: the enzymes that catalyze each biochemical reaction, essential coenzymes, and phosphate. The Krebs cycle’s activity is finely tuned by the availability of its substrates, NAD+ and FAD. Conversely, high concentrations of NADH, a product of the cycle, act as an inhibitor, preventing overproduction and maintaining metabolic balance.
Krebs Cycle at the Cellular Level: A Detailed Look
Glucose metabolism commences in the cytosol, the fluid-filled space outside the mitochondria, through a process called glycolysis. Glycolysis doesn’t require oxygen and yields a small amount of ATP along with pyruvate, a 3-carbon compound. Pyruvate then embarks on a journey into the mitochondria, where the pyruvate dehydrogenase complex (PDC) facilitates its conversion into acetyl-CoA and carbon dioxide (CO2). Each molecule of acetyl-CoA that enters the TCA cycle is capable of generating approximately 12 ATP molecules, highlighting the cycle’s significant energy-producing capacity. The PDC itself is a sophisticated assembly of three protein subunits and requires five cofactors to carry out its enzymatic function effectively. This cofactor requirement is not just structural; it ensures that the PDC can be precisely regulated to meet the cell’s energy demands.
When blood sugar levels are high, glucose becomes the primary source of acetyl-CoA via pyruvate. However, during periods of starvation or fasting, the body cleverly adapts, and beta-oxidation, the breakdown of fatty acids, becomes a significant contributor to acetyl-CoA production. Within the Krebs cycle, acetyl-CoA undergoes a complete oxidation to CO2 in a series of eight carefully orchestrated steps. The energy liberated during these oxidative reactions is not directly used to make ATP within the cycle itself. Instead, it’s captured and stored in the form of reduced electron carriers, NADH+H+, FADH2, and a high-energy nucleotide, GTP. These reduced carriers, NADH+H+ and FADH2, then proceed to the electron transport chain (also known as the mitochondrial respiratory chain), where they are oxidized in a series of reactions that ultimately culminate in the synthesis of ATP through oxidative phosphorylation. 2 Furthermore, intermediates generated within the TCA cycle are not solely confined to energy production; they serve as crucial precursors for both catabolic (breakdown) and anabolic (synthesis) processes. This remarkable versatility underscores the Krebs cycle’s role as a central hub, seamlessly connecting various metabolic pathways such as glycolysis, gluconeogenesis (glucose synthesis), ketogenesis (ketone body production), and lipogenesis (fatty acid synthesis). 3
The pyruvate dehydrogenase complex (PDC), which bridges glycolysis and the Krebs cycle, is subject to intricate regulation through three primary mechanisms: covalent modification, allosteric regulation, and transcriptional regulation. Covalent modification, particularly phosphorylation of the pyruvate decarboxylase subunit of PDC, is a key regulatory strategy. Phosphorylation reduces PDC activity, while dephosphorylation, promoted by phosphatase activity (which is upregulated by calcium ions), activates PDC. Allosteric regulation involves direct activation or inhibition by substrates and products. For example, excess acetyl-CoA or NADH directly inhibit PDC, whereas increased levels of CoASH (a precursor to acetyl-CoA) or NAD+ activate it. Transcriptional regulation, the third layer of control, dictates the amount of PDC enzyme produced, with insulin promoting enzyme production in the fed state and reduced production during fasting. 4
The Krebs Cycle’s Role in Development
Beyond its fundamental role in energy metabolism, the Krebs cycle is also essential during development. The energy generated by this metabolic pathway is indispensable for the proper growth of the endothelial system, which is crucial for the formation of blood and lymphatic vessels. Disruptions in the Krebs cycle during fetal development can have severe consequences. For instance, if the cycle’s different phases are impaired during the fetal period, newborns may experience heart problems at birth. Alterations in the Krebs cycle can lead to elevated cortisol levels, which, in turn, can disrupt placental metabolism and fetal development, potentially compromising the future child’s heart function and even leading to mortality.
Organ Systems and the Ubiquity of the Krebs Cycle
The Krebs cycle is not confined to specific organs; it is a ubiquitous metabolic pathway, operating in virtually every cell that utilizes oxygen to produce energy. Its presence across diverse cell types and organ systems highlights its fundamental importance in sustaining life. This metabolic pathway is employed not only as a central principle in anabolic cellular processes, building up complex molecules, but also in catabolic processes, breaking down molecules to release energy and building blocks.
Function: Step-by-Step through the Krebs Cycle
The Krebs cycle is a series of eight enzymatic reactions, each playing a specific role in oxidizing acetyl-CoA and generating energy carriers. Let’s delve into each step:
1. Citrate Synthesis: The cycle begins with the enzyme citrate synthase catalyzing the condensation of acetyl-CoA and oxaloacetate to form citrate. This initial step is highly exergonic (delta-G-prime of -7.7 Kcal/M) and essentially irreversible, strongly favoring citrate formation. Citrate synthase activity is regulated by substrate availability and product feedback. Citrate itself acts as an inhibitor, while oxaloacetate binding increases the enzyme’s affinity for acetyl-CoA. Interestingly, citrate, the product of this reaction, also inhibits phosphofructokinase-1 in glycolysis, while activating acetyl-CoA carboxylase for fatty acid synthesis, illustrating the intricate interconnectivity of metabolic pathways. 5
2. Isomerization of Citrate: Aconitase, an enzyme containing an iron-sulfur center, catalyzes the reversible isomerization of citrate to isocitrate, involving the intermediate cis-aconitate. This step essentially repositions a hydroxyl group, preparing the molecule for the next oxidative step. 6
3. Oxidative Decarboxylation of Isocitrate: Isocitrate dehydrogenase, a NAD+-dependent enzyme, catalyzes the oxidative decarboxylation of isocitrate to alpha-ketoglutarate. This reaction releases carbon dioxide (CO2), produces the first NADH and a proton (H+) of the cycle, and is considered the rate-limiting step of the TCA cycle. The irreversible nature of this reaction, due to CO2 production, commits substrates to continue through the cycle. Isocitrate dehydrogenase is allosterically regulated; ADP and calcium ions activate it, signaling low energy status, while ATP and NADH inhibit it, indicating energy sufficiency. 7
4. Oxidative Decarboxylation of Alpha-ketoglutarate: The alpha-ketoglutarate dehydrogenase complex, structurally and functionally similar to the pyruvate dehydrogenase complex, catalyzes the conversion of alpha-ketoglutarate to succinyl-CoA. This multi-enzyme complex also produces NADH, CO2, and H+. The reaction mechanism involves multiple cofactors, including thiamine pyrophosphate, lipoic acid, coenzyme A, NAD+, and FAD. Alpha-ketoglutarate dehydrogenase is inhibited by its products: succinyl-CoA, NADH, and ATP. 8, 9
5. Cleavage of Succinyl Coenzyme A: Succinate thiokinase (also known as succinyl-CoA synthetase) catalyzes the reversible cleavage of the thioester bond in succinyl-CoA, converting it to succinate. This reaction is coupled to the phosphorylation of GDP to GTP (or ADP to ATP in some tissues), a process called substrate-level phosphorylation, analogous to ATP production in glycolysis. 10
6. Oxidation of Succinate: Succinate dehydrogenase, uniquely located in the inner mitochondrial membrane and also known as complex II of the electron transport chain, catalyzes the oxidation of succinate to fumarate. In this reaction, FAD is reduced to FADH2, which remains bound to the enzyme. Ubiquinone is also reduced to ubiquinol by electrons passed from FADH2. 11
7. Hydration of Fumarate: Fumarase (or fumarate hydratase) catalyzes the reversible hydration of fumarate to malate. Interestingly, fumarate is also produced in the urea cycle, further highlighting the interconnectedness of metabolic pathways. 12
8. Oxidation of Malate: Malate dehydrogenase catalyzes the final step, the reversible oxidation of malate to oxaloacetate, regenerating the starting molecule of the cycle. This reaction produces the last NADH of the Krebs cycle. While the reaction has a positive delta-G-prime, favoring malate formation, the continuous consumption of oxaloacetate by citrate synthase drives the reaction forward, ensuring the cycle continues. 13
Cataplerotic and Anaplerotic Processes: Maintaining Cycle Intermediates
The Krebs cycle is not a closed loop; intermediates can be drawn off (cataplerotic processes) to participate in other biosynthetic pathways. For instance, citrate can be used for fatty acid synthesis, succinyl-CoA for heme synthesis, alpha-ketoglutarate and oxaloacetate for amino acid synthesis, and malate for gluconeogenesis. 14, 4 To replenish these intermediates and ensure the continued operation of the cycle, anaplerotic processes come into play. Pyruvate carboxylase, for example, can carboxylate pyruvate to oxaloacetate, feeding oxaloacetate back into the cycle. The liver can also generate alpha-ketoglutarate through the deamination or transamination of glutamate. 15, 4
Related Testing: Assessing Mitochondrial Function
Evaluating mitochondrial function often involves assessing the Krebs cycle. Mitochondrial dysfunction is a key feature in conditions like nonalcoholic liver disease (NAFLD). Measuring plasma levels of Krebs cycle intermediates like isocitrate and citrate can provide insights into mitochondrial health and function, offering a potential diagnostic approach.
Pathophysiology: When the Krebs Cycle Goes Awry
Mitochondrial dysfunction, and consequently, Krebs cycle impairment, can arise from various factors. Excessive calorie intake can overwhelm the cycle’s capacity to process molecules, leading to metabolic imbalances. Obesity is often associated with mitochondrial alterations, including increased oxidative stress, reactive oxygen species (ROS) production, inflammation, and apoptosis.
Mitochondrial dysfunction can also manifest as overactivity relative to normal needs, potentially due to increased metabolic demand or cellular stress. In animal models of Duchenne muscular dystrophy, elevated levels of mitochondrial metabolites have been observed in various tissues, possibly linked to increased oxidative stress.
Clinical Significance: Diseases Linked to Krebs Cycle Defects
Defects in Krebs cycle enzymes or related pathways can lead to a range of clinical disorders:
Pyruvate Dehydrogenase Complex Deficiency: This neurodegenerative disorder results from mutations affecting the pyruvate dehydrogenase complex (PDC), impairing the conversion of pyruvate to acetyl-CoA. The buildup of pyruvate leads to increased lactate production and potentially fatal metabolic acidosis. Symptoms can include lethargy, hypotonia, muscle spasticity, neurodegeneration, and early death. 16, 17, 18
Leigh Syndrome: Subacute necrotizing encephalomyelopathy, or Leigh syndrome, is a severe neurological disorder caused by genetic mutations affecting proteins of the PDC or other mitochondrial components. It typically presents in early childhood with loss of motor skills, poor feeding, vomiting, and neurological deterioration. 19, 20, 21
Thiamine Deficiency: Thiamine, or vitamin B1, is a crucial cofactor for PDC. Thiamine deficiency, similar to PDC deficiency, can shunt pyruvate metabolism towards lactate, causing metabolic acidosis. Thiamine deficiency manifests in various forms, including beriberi, characterized by neurological (dry beriberi) and cardiovascular (wet beriberi) symptoms. 22, 23
Fumarase Deficiency: This rare autosomal recessive disorder results from mutations in the FH gene, leading to fumarase deficiency and fumaric acid accumulation. It primarily affects the nervous system, causing developmental delay, microcephaly, hypotonia, encephalopathy, seizures, and failure to thrive. 24, 25, 26, 27
Mutations of Isocitrate Dehydrogenase: Mutations in isocitrate dehydrogenase (IDH) have been implicated in various cancers, including leukemia, gliomas, and sarcomas. Mutant IDH enzymes produce 2-hydroxyglutarate, an oncometabolite that can promote cancer development. 2-hydroxyglutarate serves as a biomarker and potential therapeutic target in these cancers. 28, 29, 7, 30
Review Questions
Figure: Krebs Cycle Illustration by K Humphreys
Figure 1: Krebs Cycle
Figure 1: An illustration depicting the Krebs Cycle, highlighting the sequence of reactions and key molecules involved. This diagram helps visualize the complexity and cyclical nature of this essential metabolic pathway.
References
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Disclosure: Tamim Alabduladhem declares no relevant financial relationships with ineligible companies.
Disclosure: Bruno Bordoni declares no relevant financial relationships with ineligible companies.
