12: TCA cycle

Step 1: Formation of Citrate

    The TCA cycle begins with the condensation of acetyl-CoA (a 2-carbon molecule) and oxaloacetate (a 4-carbon dicarboxylic acid), forming a 6-carbon molecule called citrate. This reaction is catalyzed by the enzyme citrate synthase, a key regulatory enzyme that operates through an ordered sequential mechanism, meaning oxaloacetate must bind first to induce a conformational change that allows acetyl-CoA to bind. This is an energetically favorable step and effectively "pulls" acetyl-CoA into the cycle.  From a protein biotech perspective, citrate synthase is often studied for conformational changes and allosteric regulation, making it a model for protein folding and induced fit mechanisms.

• Acetyl-CoA (2C) combines with oxaloacetate (4C) to form citrate (6C).
  • This reaction is catalyzed by the enzyme citrate synthase.

Step 2: Isomerization of Citrate to Isocitrate

    Citrate undergoes isomerization to form isocitrate through the action of aconitase, an iron-sulfur protein. This two-step reaction involves the reversible dehydration of citrate to cis-aconitate, followed by rehydration to isocitrate. Though it seems like a minor rearrangement, this conversion is crucial—it repositions the hydroxyl group on the molecule, making it a better target for subsequent oxidation. Aconitase is unique because its [4Fe–4S] cluster allows it to act as both a catalyst and a sensor of cellular oxidative stress, thus linking metabolism to redox signaling.  In drug discovery, aconitase has been studied as a potential target in neurodegenerative diseases and oxidative damage, where disrupted iron metabolism is implicated.

• Citrate is rearranged to isocitrate,
  • This makes the molecule more reactive and ready for oxidative decarboxylation.
  • This process is catalyzed by aconitase.

Step 3: Oxidative Decarboxylation of Isocitrate

    Isocitrate is oxidized and decarboxylated by isocitrate dehydrogenase (IDH) to form α-ketoglutarate, a 5-carbon molecule. This reaction also produces NADH and releases the first molecule of CO₂. IDH exists in NAD⁺-dependent (mitochondrial) and NADP⁺-dependent (cytosolic and mitochondrial) isoforms, each with distinct roles in metabolism and biosynthesis. Mutations in IDH1 and IDH2 isoforms are implicated in several cancers, particularly gliomas and acute myeloid leukemia, because they result in a neomorphic activity that produces the oncometabolite 2-hydroxyglutarate, which disrupts epigenetic regulation. 

• Isocitrate is oxidized and decarboxylated by isocitrate dehydrogenase, producing:
  • α-ketoglutarate (5C)
  • CO₂
  • NADH

Step 4: Oxidative Decarboxylation of α-Ketoglutarate

    The next step is the oxidative decarboxylation of α-ketoglutarate into succinyl-CoA, catalyzed by the α-ketoglutarate dehydrogenase complex (α-KGDH). This multi-enzyme complex functions similarly to the pyruvate dehydrogenase complex and uses thiamine pyrophosphate (TPP), lipoate, FAD, NAD⁺, and CoA as cofactors. This reaction produces another molecule of NADH and the second CO₂ of the cycle. 

• α-Ketoglutarate is converted to succinyl-CoA (4C) by α-ketoglutarate dehydrogenase, producing:
  • Another CO₂
  • Another NADH

Step 5: Conversion of Succinyl-CoA to Succinate

    The energy-rich succinyl-CoA is converted to succinate by succinyl-CoA synthetase (or succinate thiokinase), coupled with the substrate-level phosphorylation of GDP (or ADP) to GTP (or ATP). This is one of the few steps in metabolism that produces a high-energy phosphate bond directly without involving the electron transport chain. 

• Succinyl-CoA is converted to succinate, catalyzed by succinyl-CoA synthetase.
• This step generates GTP (or ATP, depending on the tissue) via substrate-level phosphorylation.

Step 6: Oxidation of Succinate to Fumarate

    Succinate dehydrogenase catalyzes the oxidation of succinate to fumarate, generating FADH₂. Unique among TCA enzymes, succinate dehydrogenase is embedded in the inner mitochondrial membrane and doubles as Complex II of the electron transport chain (ETC). This dual role makes it a bridge between the TCA cycle and oxidative phosphorylation. Because FADH₂ donates electrons directly into the ETC, it yields less ATP than NADH. Succinate dehydrogenase is a hotspot for studying flavoprotein chemistry, membrane-bound enzymes, and hereditary tumors (e.g., mutations in SDH genes cause paragangliomas and pheochromocytomas).

• Succinate is oxidized to fumarate by succinate dehydrogenase, producing:
  • FADH₂
• Note: Succinate dehydrogenase is part of both the TCA cycle and Complex II of the electron transport chain.

Step 7: Hydration of Fumarate to Malate

    Fumarate is then hydrated to form malate via the enzyme fumarase (fumarate hydratase). This is a simple hydration reaction, but it's clinically relevant—mutations in fumarase are linked to hereditary leiomyomatosis and renal cell cancer (HLRCC). As such, fumarase is a tumor suppressor protein, and its deficiency causes a buildup of fumarate, leading to epigenetic and signaling alterations. Biotech applications include screening for fumarase inhibitors or stabilizers as part of metabolic therapy approaches.

• Fumarase hydrates fumarate to form malate.

Step 8: Oxidation of Malate to Regenerate Oxaloacetate

     Finally, malate is oxidized to oxaloacetate by malate dehydrogenase, producing the third NADH of the cycle. This reaction is slightly endergonic, but it is driven forward by the rapid consumption of oxaloacetate in the citrate synthase reaction (step 1), demonstrating a beautiful example of metabolic flux and equilibrium shifting. Malate dehydrogenase is a key enzyme for studying thermodynamics in enzymology, and it’s widely used in lab protocols for coupled assays, where it helps regenerate NAD⁺ for other enzyme reactions.

• Malate dehydrogenase oxidizes malate to regenerate oxaloacetate, producing:
  • Another NADH

Overall Outputs per Acetyl-CoA Molecule

     From one turn of the TCA cycle (per Acetyl-CoA), the following high-energy products are generated:

• 3 NADH
• 1 FADH₂
• 1 GTP (or ATP)
• 2 CO₂

     Each NADH yields approximately 2.5 ATP, and each FADH₂ yields about 1.5 ATP during oxidative phosphorylation. Thus, the TCA cycle indirectly drives significant ATP production. These reduced cofactors are essential in maintaining the cell’s redox state, and their balance is crucial in bioreactor design, mitochondrial disease therapy, and pharmacokinetics modeling.

Targeting TCA Enzymes in Cancer and Disease

    The tricarboxylic acid (TCA) cycle plays a central role not only in energy metabolism but also in supporting the anabolic demands of rapidly proliferating cancer cells. Many tumors exhibit altered TCA cycle activity—a phenomenon often tied to the Warburg effect, where cells rely more heavily on glycolysis even in the presence of oxygen. A particularly striking example involves mutations in isocitrate dehydrogenase (IDH), especially the IDH1 and IDH2 isoforms. These mutations are frequently found in cancers such as gliomas and acute myeloid leukemia (AML). Mutant IDH enzymes gain a new (neomorphic) function—they reduce α-ketoglutarate to an abnormal metabolite called 2-hydroxyglutarate (2-HG). This oncometabolite interferes with DNA and histone demethylation, causing widespread epigenetic dysregulation and contributing to tumor progression. Understanding the structural and kinetic properties of these mutant enzymes enabled the development of targeted inhibitors such as Ivosidenib (AG-120) and Enasidenib (AG-221). These small molecules bind specifically to the mutant IDH1 and IDH2 enzymes, respectively, suppressing 2-HG production and are now FDA-approved therapies for treating IDH-mutant AML. This illustrates how detailed biochemical knowledge of TCA cycle enzymes directly informs precision oncology and rational drug design.

Enzyme Kinetics in TCA Enzyme Studies

    Enzyme kinetics offers powerful tools for understanding and manipulating the function of TCA enzymes. Through kinetic assays, researchers can explore reaction mechanisms, such as whether an enzyme operates through a ping-pong mechanism (as seen in α-ketoglutarate dehydrogenase) or a ternary complex mechanism (where all substrates must bind before the reaction proceeds). These kinetic characterizations inform drug design, especially when looking to inhibit enzymes in a competitive, non-competitive, or uncompetitive manner. For example, citrate synthase can be inhibited competitively by molecules mimicking acetyl-CoA, and the efficacy of such inhibitors can be tested using Michaelis-Menten or Lineweaver-Burk plots. Standard assays for TCA enzymes often involve measuring the production or consumption of NADH or FADH₂, which absorb strongly at 340 nm, making them ideal for colorimetric or spectrophotometric quantification. These methods are foundational in high-throughput drug screening platforms, where hundreds of compounds can be tested for their effect on enzymatic rates, thereby identifying lead candidates for further development.

Metabolic Engineering in Biotechnology

    In industrial biotechnology, the TCA cycle is often manipulated or rewired to optimize the production of valuable chemicals. For instance, overexpressing citrate synthase in certain microbial systems like Aspergillus niger or engineered yeast strains can boost the yield of citric acid, which is used widely as a food preservative, flavoring agent, and even in pharmaceuticals. Similarly, redirecting carbon flux through succinate-producing branches of the TCA cycle allows the generation of succinic acid, a building block for biodegradable plastics (e.g., polybutylene succinate) and other green chemicals. Metabolic engineers often introduce or overexpress genes encoding specific TCA enzymes, adjust cofactor balances (NADH/NAD⁺), and eliminate competing pathways to increase the efficiency of bioproduction. These engineered strains become biological factories, illustrating how deep biochemical understanding translates into practical, large-scale bioproduction platforms.

    The centrality of the TCA cycle in cellular metabolism makes its enzymes and intermediates highly relevant in clinical pharmacology. Several drugs and toxic agents target different aspects of this pathway. For instance, Ivosidenib, as discussed, inhibits mutant IDH1 to block the production of the oncometabolite 2-HG in cancer. Metformin, a first-line treatment for type 2 diabetes, indirectly affects TCA cycle function by inhibiting Complex I of the electron transport chain. This reduces NADH oxidation, thereby altering TCA flux and limiting hepatic gluconeogenesis. In toxicology, substances like fluoroacetate act as suicide inhibitors of aconitase, halting the TCA cycle and causing the accumulation of citrate, leading to cellular toxicity—a mechanism exploited in rodenticides. Similarly, arsenite is a potent inhibitor of α-ketoglutarate dehydrogenase, disrupting NADH production and inducing energy failure. Understanding these interactions is critical in both drug safety evaluation and the treatment of poisonings.

Amino Acid and Fatty Acid Metabolism

     The TCA cycle acts as a metabolic hub, not just for carbohydrate metabolism, but also for the catabolism and biosynthesis of amino acids and fatty acids. Several amino acids are transaminated into TCA cycle intermediates: for example, glutamate is converted to α-ketoglutarate, and aspartate can feed into oxaloacetate. These conversions are vital in nitrogen metabolism, neurotransmitter synthesis, and the urea cycle. In contrast, fatty acids are broken down through β-oxidation to yield acetyl-CoA, which enters the TCA cycle for complete oxidation. However, during prolonged fasting or in diabetes, excess acetyl-CoA from fat breakdown leads to ketogenesis instead of TCA entry. This dual role of the TCA cycle in catabolic degradation (cataplerosis) and anabolic replenishment (anaplerosis) is central in metabolic engineering, where maintaining balanced flux through TCA intermediates is crucial for sustained biosynthesis and cell health.