Table of Contents
Overview: Where the Citric Acid Cycle Fits
Once pyruvate has been converted to acetyl‑CoA, its remaining energy is extracted in a cyclic pathway called the citric acid cycle (also: Krebs cycle, tricarboxylic acid cycle, TCA cycle). This cycle operates in the mitochondrial matrix of eukaryotes and in the cytosol (or at the inner membrane) of prokaryotes.
Its central functions are:
- Complete oxidation of the acetyl group to CO$_2$
- Transfer of high‑energy electrons to NAD$^+$ and FAD (forming NADH and FADH$_2$)
- Regeneration of the starting molecule oxaloacetate
- Production of a small amount of ATP (or GTP) directly
The cycle does not itself use oxygen, but it depends on the electron transport chain (which uses O$_2$) to reoxidize NADH and FADH$_2$. If the electron transport chain stops, the citric acid cycle also comes to a halt.
Entry into the Cycle: Acetyl‑CoA and Oxaloacetate
Each turn of the cycle begins when a two‑carbon acetyl group (from acetyl‑CoA) is added to a four‑carbon acceptor, oxaloacetate, forming a six‑carbon compound (citrate). Coenzyme A (CoA) is released and can be reused in the pyruvate dehydrogenase reaction or in other pathways.
Key point:
- The carbons that leave as CO$_2$ in the first turn do not come from the newly added acetyl group but from the oxaloacetate pool. Over several turns, the acetyl carbons are also lost as CO$_2$.
One Turn of the Citric Acid Cycle: Step‑by‑Step
Below is the sequence for one acetyl‑CoA entering the cycle. For each step, only the essential transformation and energetic outcome are highlighted.
1. Citrate Formation
Reaction:
Acetyl‑CoA (2 C) + oxaloacetate (4 C) → citrate (6 C) + CoA‑SH
Enzyme: Citrate synthase
- Highly exergonic, effectively irreversible under cellular conditions
- Commits the acetyl group to oxidation in the cycle
- CoA is released and recycled
2. Isomerization of Citrate to Isocitrate
Reaction:
Citrate (6 C) ⇌ isocitrate (6 C)
Enzyme: Aconitase
- Two‑step process: dehydration to cis‑aconitate and rehydration to isocitrate
- Rearranges the hydroxyl group to form a molecule that can be more easily oxidized in the next step
- Overall reaction is near equilibrium and reversible
3. First Oxidative Decarboxylation: Isocitrate to α‑Ketoglutarate
Reaction:
Isocitrate (6 C) + NAD$^+$ → α‑ketoglutarate (5 C) + CO$_2$ + NADH + H$^+$
Enzyme: Isocitrate dehydrogenase
- First CO$_2$ is released in the cycle
- One molecule of NADH is generated
- Step is strongly regulated (rate‑limiting) and essentially irreversible
4. Second Oxidative Decarboxylation: α‑Ketoglutarate to Succinyl‑CoA
Reaction:
α‑Ketoglutarate (5 C) + CoA‑SH + NAD$^+$ → succinyl‑CoA (4 C) + CO$_2$ + NADH + H$^+$
Enzyme: α‑Ketoglutarate dehydrogenase complex
- Structurally and mechanistically similar to pyruvate dehydrogenase
- Second CO$_2$ is released
- Another NADH is produced
- Forms a high‑energy thioester (succinyl‑CoA)
- Another major control point of the cycle
By the end of step 4, the original 6‑carbon skeleton that entered as citrate has lost two carbons as CO$_2$.
5. Substrate‑Level Phosphorylation: Succinyl‑CoA to Succinate
Reaction (in mammals):
Succinyl‑CoA + GDP + P$_i$ ⇌ succinate + CoA‑SH + GTP
Enzyme: Succinyl‑CoA synthetase (also called succinate thiokinase)
- The energy of the thioester bond in succinyl‑CoA is used to form GTP (or ATP in some tissues and organisms)
- GTP can be readily converted to ATP by nucleoside diphosphate kinase
- This is the only substrate‑level phosphorylation step in the citric acid cycle
6. Oxidation of Succinate to Fumarate
Reaction:
Succinate + FAD ⇌ fumarate + FADH$_2$
Enzyme: Succinate dehydrogenase
- Generates FADH$_2$, a reduced electron carrier
- Succinate dehydrogenase is embedded in the inner mitochondrial membrane and is part of the electron transport chain (complex II)
- Reaction is reversible and its direction depends on substrate/product levels
7. Hydration of Fumarate to Malate
Reaction:
Fumarate + H$_2$O ⇌ L‑malate
Enzyme: Fumarase (fumarate hydratase)
- Adds water across the double bond, producing L‑malate
- Reversible hydration reaction
8. Oxidation of Malate to Oxaloacetate
Reaction:
L‑Malate + NAD$^+$ ⇌ oxaloacetate + NADH + H$^+$
Enzyme: Malate dehydrogenase
- Produces NADH
- Thermodynamically unfavorable in isolation (ΔG°’ is positive), but the rapid consumption of oxaloacetate by citrate synthase pulls the reaction forward in the context of the cycle
At this point, oxaloacetate is regenerated and is ready to react with another acetyl‑CoA, continuing the cycle.
Net Yield per Acetyl‑CoA and Per Glucose
For one turn of the cycle (i.e., per acetyl‑CoA):
- 2 CO$_2$
- 3 NADH
- 1 FADH$_2$
- 1 GTP (≈ 1 ATP)
- Oxaloacetate is regenerated (no net gain or loss)
Each molecule of glucose yields two acetyl‑CoA molecules. Therefore, per glucose, the citric acid cycle runs twice, giving:
- 4 CO$_2$
- 6 NADH
- 2 FADH$_2$
- 2 GTP (or ATP)
The NADH and FADH$_2$ formed here donate their electrons to the electron transport chain, leading to the majority of ATP formation in cellular respiration.
Regulation of the Citric Acid Cycle
The citric acid cycle is tightly regulated to match energy supply with demand. Cells adjust flux through the cycle mainly by controlling key irreversible steps.
Main Regulatory Enzymes
- Citrate synthase
- Inhibited by: ATP, NADH, succinyl‑CoA, citrate (products or signals of high energy status)
- Isocitrate dehydrogenase
- Activated by: ADP (signal of low energy), Ca$^{2+}$ (in muscle, indicates activity)
- Inhibited by: ATP, NADH
- α‑Ketoglutarate dehydrogenase
- Inhibited by: NADH, succinyl‑CoA
- Activated by: Ca$^{2+}$ in muscle
High ratios of ATP/ADP or NADH/NAD$^+$ generally slow the cycle, while high ADP and NAD$^+$ accelerate it.
Dependence on the Electron Transport Chain
The cycle’s operation depends on a continuous supply of the oxidized cofactors NAD$^+$ and FAD. If the electron transport chain is blocked or O$_2$ is unavailable:
- NADH and FADH$_2$ accumulate
- NAD$^+$ and FAD become scarce
- Dehydrogenase reactions in the cycle slow or stop
Thus, although the cycle does not directly use oxygen, it is effectively an aerobic pathway.
Anaplerotic and Cataplerotic Roles (Link with Other Pathways)
The citric acid cycle is not only catabolic; it is also a hub that connects many metabolic routes.
Anaplerotic Reactions (Refilling the Cycle)
Intermediates can be withdrawn from the cycle for biosynthesis. To keep the cycle running, cells use anaplerotic (“refilling”) reactions. Key examples:
- Pyruvate carboxylase:
Pyruvate + CO$_2$ + ATP → oxaloacetate + ADP + P$_i$
(Refills oxaloacetate, especially important in liver and other tissues)
Other amino acid degradation pathways can also yield intermediates like α‑ketoglutarate, succinyl‑CoA, fumarate, and oxaloacetate.
Cataplerotic Uses (Draining the Cycle)
Citric acid cycle intermediates serve as precursors for:
- Amino acids: e.g., glutamate from α‑ketoglutarate; aspartate from oxaloacetate
- Heme and other porphyrins: from succinyl‑CoA
- Fatty acid and sterol synthesis: citrate exported from mitochondria provides acetyl units and can influence lipid metabolism
- Gluconeogenesis: via oxaloacetate and malate in tissues capable of making glucose
Because of these bidirectional flows, the cycle functions as a central metabolic hub, integrating energy production with biosynthetic demands.
Summary of Key Features
- Cyclic pathway in the mitochondrial matrix (eukaryotes) that oxidizes acetyl‑CoA to CO$_2$
- Produces reduced electron carriers (3 NADH, 1 FADH$_2$ per acetyl‑CoA) and 1 GTP/ATP
- Regulated primarily at citrate synthase, isocitrate dehydrogenase, and α‑ketoglutarate dehydrogenase
- Depends on the electron transport chain to regenerate NAD$^+$ and FAD
- Serves both in energy release (catabolism) and in providing intermediates for biosynthesis, requiring anaplerotic reactions to maintain its function