🧬Biochemistry Unit 13 – Citric Acid Cycle

Key Concepts and Overview

• Citric Acid Cycle (CAC), also known as the Tricarboxylic Acid (TCA) cycle or Krebs cycle, is a central metabolic pathway in aerobic organisms
• Occurs in the mitochondrial matrix and plays a crucial role in energy production by oxidizing acetyl-CoA derived from carbohydrates, fats, and proteins
• Generates high-energy molecules (NADH and FADH2) used in the electron transport chain for ATP production
• Provides precursors for biosynthesis of amino acids, nucleotides, and other important biomolecules
• Highly regulated process that responds to cellular energy demands and nutrient availability
• Consists of a series of enzymatic reactions that form a cyclic pathway, with each turn of the cycle oxidizing one acetyl-CoA molecule
• Produces two molecules of CO2 per acetyl-CoA molecule oxidized, which are released as waste products

Biochemical Reactions and Steps

• The cycle begins with the condensation of acetyl-CoA and oxaloacetate to form citrate, catalyzed by citrate synthase
• Citrate undergoes isomerization to form isocitrate via the enzyme aconitase
• Isocitrate dehydrogenase catalyzes the oxidative decarboxylation of isocitrate to form α-ketoglutarate, releasing CO2 and reducing NAD+ to NADH
• α-Ketoglutarate dehydrogenase complex catalyzes another oxidative decarboxylation reaction, converting α-ketoglutarate to succinyl-CoA, releasing CO2, and reducing NAD+ to NADH
• Succinyl-CoA synthetase catalyzes the substrate-level phosphorylation of GDP or ADP to form GTP or ATP, respectively, while converting succinyl-CoA to succinate
• Succinate dehydrogenase, a component of the electron transport chain (Complex II), oxidizes succinate to fumarate, reducing FAD to FADH2
• Fumarase catalyzes the hydration of fumarate to form malate
• Malate dehydrogenase oxidizes malate to oxaloacetate, reducing NAD+ to NADH, completing the cycle

Enzymes and Cofactors Involved

• Citrate synthase: Condensation of acetyl-CoA and oxaloacetate to form citrate
• Aconitase: Isomerization of citrate to isocitrate via cis-aconitate intermediate
• Isocitrate dehydrogenase: Oxidative decarboxylation of isocitrate to α-ketoglutarate, requiring NAD+ as a cofactor
• α-Ketoglutarate dehydrogenase complex: Oxidative decarboxylation of α-ketoglutarate to succinyl-CoA, requiring NAD+ and thiamine pyrophosphate (TPP) as cofactors
• Succinyl-CoA synthetase: Substrate-level phosphorylation of GDP or ADP to form GTP or ATP, respectively, while converting succinyl-CoA to succinate
• Succinate dehydrogenase (Complex II): Oxidation of succinate to fumarate, requiring FAD as a cofactor
• Fumarase: Hydration of fumarate to form malate
• Malate dehydrogenase: Oxidation of malate to oxaloacetate, requiring NAD+ as a cofactor

Energy Production and Regulation

• The citric acid cycle generates high-energy molecules (NADH and FADH2) that are used in the electron transport chain to produce ATP through oxidative phosphorylation
• Each turn of the cycle produces 3 NADH, 1 FADH2, and 1 GTP (or ATP) directly
• The NADH and FADH2 generated in the cycle are oxidized in the electron transport chain, driving the production of a proton gradient across the inner mitochondrial membrane
• ATP synthase uses the proton gradient to generate ATP from ADP and inorganic phosphate (Pi)
• The citric acid cycle is tightly regulated to maintain cellular energy homeostasis and respond to changing metabolic demands
• Key regulatory enzymes include citrate synthase, isocitrate dehydrogenase, and α-ketoglutarate dehydrogenase complex
• Allosteric regulation by ATP, NADH, and calcium ions (Ca2+) modulates the activity of these enzymes
• Substrate availability (acetyl-CoA, NAD+, and FAD) also plays a crucial role in regulating the cycle's activity

Integration with Other Metabolic Pathways

• The citric acid cycle is closely linked to various other metabolic pathways, serving as a central hub for energy production and biosynthesis
• Acetyl-CoA, the main input of the cycle, is derived from the oxidation of pyruvate (from glycolysis), fatty acids (via β-oxidation), and certain amino acids
• Intermediates of the cycle, such as α-ketoglutarate and oxaloacetate, serve as precursors for the biosynthesis of amino acids (glutamate, aspartate, and their derivatives)
• Succinyl-CoA is a precursor for the synthesis of heme, a key component of hemoglobin and other heme-containing proteins
• Citrate can be exported from the mitochondria to the cytosol, where it is used for fatty acid synthesis and cholesterol biosynthesis
• The reducing equivalents (NADH) generated in the cycle are also used in gluconeogenesis and other anabolic pathways

Cellular Localization and Compartmentalization

• The citric acid cycle takes place in the mitochondrial matrix, the innermost compartment of mitochondria
• Mitochondria are double-membrane organelles that are the primary sites of cellular respiration and energy production in eukaryotic cells
• The enzymes of the citric acid cycle are soluble proteins found in the mitochondrial matrix, with the exception of succinate dehydrogenase, which is embedded in the inner mitochondrial membrane as part of Complex II of the electron transport chain
• The compartmentalization of the citric acid cycle within the mitochondria allows for efficient coupling of the cycle with the electron transport chain and ATP synthase
• The mitochondrial inner membrane is highly impermeable, requiring specific transport proteins (e.g., the malate-aspartate shuttle and the glycerol-3-phosphate shuttle) to transfer reducing equivalents (NADH) from the cytosol to the mitochondrial matrix

Clinical Significance and Disorders

• Defects in the enzymes of the citric acid cycle can lead to various metabolic disorders, often characterized by impaired energy production and accumulation of toxic intermediates
• Mutations in the genes encoding citric acid cycle enzymes can cause rare inherited disorders, such as:
  • Fumarase deficiency (Fumaric aciduria)
  • Succinate dehydrogenase deficiency (Leigh syndrome)
  • α-Ketoglutarate dehydrogenase complex deficiency
• These disorders often present with neurological symptoms, developmental delays, and organ dysfunction due to impaired energy metabolism
• Acquired deficiencies in citric acid cycle enzymes have been implicated in various neurodegenerative diseases, such as Parkinson's disease and Alzheimer's disease
• Mitochondrial dysfunction and impaired citric acid cycle activity are also associated with cancer, as cancer cells often rely on alternative metabolic pathways (e.g., aerobic glycolysis, or the Warburg effect) for energy production and biosynthesis

Applications and Research Developments

• Understanding the citric acid cycle has led to the development of various therapeutic strategies targeting metabolic disorders and cancer
• Inhibitors of citric acid cycle enzymes, such as isocitrate dehydrogenase (IDH) inhibitors, are being investigated as potential treatments for certain types of cancer (e.g., gliomas and acute myeloid leukemia) that harbor mutations in IDH enzymes
• Metabolic profiling and flux analysis of the citric acid cycle intermediates using stable isotope tracers (e.g., 13C-labeled substrates) have provided valuable insights into the metabolic reprogramming of cancer cells and the identification of potential therapeutic targets
• Research on the regulation of the citric acid cycle has revealed the importance of post-translational modifications (e.g., phosphorylation and acetylation) in modulating enzyme activity and metabolic flux
• The development of mitochondria-targeted antioxidants and compounds that enhance mitochondrial function has shown promise in the treatment of neurodegenerative diseases and age-related metabolic disorders associated with impaired citric acid cycle activity
• Synthetic biology approaches have been used to engineer microorganisms with modified citric acid cycle pathways for the production of valuable compounds, such as biofuels and pharmaceuticals