17. The Citric Acid Cycle
Reading time: ~45 minutes | Key topics: Krebs cycle, acetyl-CoA oxidation, pyruvate dehydrogenase complex, anaplerotic reactions, regulation of TCA flux
Overview of the TCA Cycle
The citric acid cycle (also called the tricarboxylic acid (TCA) cycle or Krebs cycle, after Hans Krebs who elucidated it in 1937) is the central metabolic hub of aerobic organisms. It takes place in the mitochondrial matrix of eukaryotes and in the cytoplasm of prokaryotes. The cycle oxidizes the acetyl group of acetyl-CoA to two molecules of $\text{CO}_2$, while capturing the released energy as reduced electron carriers ($\text{NADH}$ and $\text{FADH}_2$) and one molecule of GTP.
The net reaction for one complete turn of the cycle is:
The reduced coenzymes produced — 3 NADH and 1 $\text{FADH}_2$ — carry high-energy electrons to the electron transport chain, where their oxidation drives ATP synthesis via oxidative phosphorylation. The single GTP produced (equivalent to 1 ATP) arises from substrate-level phosphorylation. In total, the complete oxidation of one acetyl-CoA through the TCA cycle and subsequent oxidative phosphorylation yields approximately 10 ATP equivalents.
Why “Cycle”?
The pathway is cyclic because the four-carbon oxaloacetate (OAA) that accepts the acetyl group at the beginning is regenerated at the end of each turn. The two carbons that enter as the acetyl group are ultimately lost as $\text{CO}_2$, but not in the same turn — isotope-labeling experiments show that the carbons released as $\text{CO}_2$ in any given turn are derived from the oxaloacetate portion, not from the incoming acetyl-CoA.
Pyruvate Dehydrogenase Complex
Before acetyl-CoA can enter the TCA cycle, pyruvate produced by glycolysis must be oxidatively decarboxylated. This is catalyzed by the pyruvate dehydrogenase complex (PDC), a massive multienzyme assembly located in the mitochondrial matrix. The PDC is one of the largest known enzyme complexes, with a molecular mass of approximately 4–10 MDa depending on the organism.
The complex contains three catalytic enzymes and requires five coenzymes:
- E1 — Pyruvate dehydrogenase: Uses thiamine pyrophosphate (TPP) to decarboxylate pyruvate and transfer the resulting hydroxyethyl group to lipoamide.
- E2 — Dihydrolipoyl transacetylase: Transfers the acetyl group from lipoamide to CoA-SH, producing acetyl-CoA. Contains covalently bound lipoamide on a flexible lysine-lipoyl arm.
- E3 — Dihydrolipoyl dehydrogenase: Reoxidizes dihydrolipoamide using FAD as a prosthetic group, ultimately transferring electrons to $\text{NAD}^+$ to produce NADH.
The overall reaction is irreversible under physiological conditions:
Regulation of the PDC is achieved by covalent modification: a dedicated pyruvate dehydrogenase kinase (PDK) phosphorylates E1, inactivating the complex, while pyruvate dehydrogenase phosphatase (PDP) removes the phosphate to reactivate it. PDK is activated by high ratios of [ATP]/[ADP], [NADH]/[NAD$^+$], and [acetyl-CoA]/[CoA], signaling that the energy charge is high. Conversely, pyruvate, ADP, and $\text{Ca}^{2+}$ stimulate PDP, activating the complex when energy demand increases.
The Eight Reactions of the TCA Cycle
The TCA cycle consists of eight sequential enzyme-catalyzed reactions. Three of these reactions are essentially irreversible and serve as regulatory control points. Below is a detailed account of each step.
Step 1: Citrate Synthase
Acetyl-CoA condenses with oxaloacetate to form citrate (6C), releasing CoA-SH. This aldol condensation followed by hydrolysis of the thioester bond makes the reaction highly exergonic.
Step 2: Aconitase
Citrate is isomerized to isocitrate via a dehydration/rehydration mechanism through the intermediate cis-aconitate. Aconitase contains an iron-sulfur ([4Fe-4S]) cluster essential for catalysis. This near-equilibrium reaction repositions the hydroxyl group to enable the subsequent oxidative decarboxylation.
Step 3: Isocitrate Dehydrogenase (Rate-Limiting)
Isocitrate undergoes oxidative decarboxylation to $\alpha$-ketoglutarate, producing the first $\text{CO}_2$ and the first NADH of the cycle. This is the rate-limiting step of the TCA cycle and a major regulatory point.
Step 4: $\alpha$-Ketoglutarate Dehydrogenase Complex
This multienzyme complex is structurally and mechanistically analogous to the pyruvate dehydrogenase complex. It catalyzes the second oxidative decarboxylation, producing succinyl-CoA, $\text{CO}_2$, and NADH. Uses the same five coenzymes: TPP, lipoamide, CoA-SH, FAD, and NAD$^+$.
Step 5: Succinyl-CoA Synthetase
The high-energy thioester bond of succinyl-CoA is cleaved, and the released energy drives substrate-level phosphorylation of GDP to GTP (in animals) or ADP to ATP (in plants and some bacteria). The GTP is readily interconverted with ATP by nucleoside diphosphate kinase.
Step 6: Succinate Dehydrogenase (Complex II)
Succinate is oxidized to fumarate, with FAD (covalently bound to the enzyme) serving as the electron acceptor, producing $\text{FADH}_2$. This is the only TCA cycle enzyme embedded in the inner mitochondrial membrane — it is simultaneously Complex II of the electron transport chain. Electrons from $\text{FADH}_2$ are passed directly to ubiquinone (CoQ).
Step 7: Fumarase
Fumarase catalyzes the stereospecific trans-hydration of the double bond in fumarate, producing L-malate. This is a near-equilibrium reaction.
Step 8: Malate Dehydrogenase
Malate is oxidized to oxaloacetate (OAA), regenerating the cycle's starting substrate and producing the third NADH. Although the $\Delta G^{\circ\prime}$ is highly unfavorable, the reaction is pulled forward in vivo by the very exergonic citrate synthase reaction (Step 1), which rapidly consumes OAA, keeping its steady-state concentration extremely low ($\sim 10^{-6}$ M).
Regulation of the TCA Cycle
The flux through the TCA cycle is tightly regulated to match the cell's energy demands. Regulation occurs primarily at the three irreversible steps, each catalyzed by an enzyme that responds to allosteric effectors reflecting the energy status of the cell. The key principle is that high [NADH]/[NAD$^+$], [ATP]/[ADP], and [succinyl-CoA]/[CoA] ratios signal energy sufficiency and inhibit the cycle, while high [ADP], [NAD$^+$], and $\text{Ca}^{2+}$ signal energy demand and activate it.
| Enzyme | Activators | Inhibitors |
|---|---|---|
| Citrate synthase | ADP, NAD$^+$ | ATP, NADH, succinyl-CoA, citrate |
| Isocitrate dehydrogenase | ADP, Ca$^{2+}$ | ATP, NADH |
| $\alpha$-Ketoglutarate DH | Ca$^{2+}$ | Succinyl-CoA, NADH, ATP |
Calcium ions play a particularly important role in linking TCA cycle flux to muscle contraction and hormonal signaling. During exercise or sympathetic nervous system activation, mitochondrial $[\text{Ca}^{2+}]$ rises, activating isocitrate dehydrogenase and $\alpha$-ketoglutarate dehydrogenase simultaneously to accelerate NADH production and meet increased ATP demand.
Anaplerotic Reactions
Because TCA cycle intermediates are continuously siphoned off for biosynthetic purposes (see Amphibolic Nature below), the pool of cycle intermediates must be replenished. Reactions that replenish these intermediates are called anaplerotic reactions (Greek: “filling up”). The most important anaplerotic reaction in mammals is catalyzed by pyruvate carboxylase:
Pyruvate carboxylase is a biotin-dependent enzyme located in the mitochondrial matrix. It is allosterically activated by acetyl-CoA — an elegant regulatory mechanism: when acetyl-CoA accumulates (because OAA is depleted and citrate synthase cannot run), pyruvate carboxylase is stimulated to produce more OAA, restoring cycle flux.
Other Anaplerotic Reactions
- Glutamate $\rightarrow$ $\alpha$-ketoglutarate: Catalyzed by glutamate dehydrogenase or transamination. Major route for amino acid entry into the cycle.
- Amino acid catabolism $\rightarrow$ succinyl-CoA: Valine, isoleucine, methionine, and threonine are degraded to propionyl-CoA, which is converted to succinyl-CoA via methylmalonyl-CoA (requires vitamin B$_{12}$).
- Aspartate $\rightarrow$ oxaloacetate: Via transamination with $\alpha$-ketoglutarate, catalyzed by aspartate aminotransferase (AST/GOT).
- PEP carboxykinase (reverse): In some tissues, PEP + $\text{CO}_2$ $\rightarrow$ OAA can function anaplerotically.
Amphibolic Nature of the TCA Cycle
The TCA cycle is amphibolic — it serves both catabolic (energy-generating) and anabolic (biosynthetic) functions. Several intermediates are drawn off as precursors for major biosynthetic pathways:
| Intermediate | Biosynthetic Destination |
|---|---|
| Citrate | Exported to cytoplasm; cleaved by ATP-citrate lyase to acetyl-CoA + OAA for fatty acid synthesis and cholesterol biosynthesis |
| $\alpha$-Ketoglutarate | Transamination to glutamate; precursor for amino acid synthesis (glutamine, proline, arginine) and purine nucleotides |
| Succinyl-CoA | Porphyrin and heme biosynthesis (condensation with glycine to form $\delta$-aminolevulinic acid) |
| Oxaloacetate | Gluconeogenesis (converted to PEP by PEP carboxykinase); transamination to aspartate for pyrimidine synthesis |
This dual role means that the TCA cycle is not simply a “furnace” for burning fuel. It is a metabolic roundabout connecting carbohydrate, lipid, and amino acid metabolism. The balance between its catabolic and anabolic functions is maintained by anaplerotic reactions, which replenish intermediates as they are consumed for biosynthesis.
Python: TCA Cycle Energetics
The following Python code generates two plots: (1) a bar chart of the standard free energy change ($\Delta G^{\circ\prime}$) for each TCA cycle step, with regulated enzymes highlighted, and (2) a cumulative plot of NADH, $\text{FADH}_2$, GTP, and $\text{CO}_2$ production through the cycle. A summary table of energetics and ATP equivalents is also printed.
TCA Cycle Energetics & Product Yield
PythonBar chart of free energies and cumulative product generation through the Krebs cycle
Click Run to execute the Python code
Code will be executed with Python 3 on the server
Key Concepts
1. The TCA cycle oxidizes the acetyl group of acetyl-CoA to 2 $\text{CO}_2$, yielding 3 NADH, 1 $\text{FADH}_2$, and 1 GTP per turn — equivalent to ~10 ATP via oxidative phosphorylation.
2. The pyruvate dehydrogenase complex links glycolysis to the TCA cycle and is regulated by phosphorylation/dephosphorylation in response to energy status.
3. Three irreversible steps (citrate synthase, isocitrate dehydrogenase, $\alpha$-ketoglutarate dehydrogenase) are the primary regulatory control points, responding to [NADH]/[NAD$^+$], [ATP]/[ADP], and $\text{Ca}^{2+}$.
4. The thermodynamically unfavorable malate dehydrogenase reaction ($\Delta G^{\circ\prime} = +29.7$ kJ/mol) is driven forward by the highly favorable citrate synthase step, which keeps [OAA] very low.
5. Anaplerotic reactions (especially pyruvate carboxylase) replenish TCA intermediates drained by biosynthetic pathways. Acetyl-CoA allosterically activates pyruvate carboxylase.
6. The TCA cycle is amphibolic: citrate feeds fatty acid synthesis, $\alpha$-KG feeds amino acid synthesis, succinyl-CoA feeds heme synthesis, and OAA feeds gluconeogenesis.
7. Succinate dehydrogenase (Step 6) is unique: it is the only TCA enzyme embedded in the inner mitochondrial membrane and simultaneously serves as Complex II of the electron transport chain.