Krebs Cycle Explained: The Eight Reactions of the Citric Acid Cycle

Entry Point: Acetyl-CoA

The Krebs cycle does not begin with glucose directly. Glucose is first broken down to pyruvate by glycolysis, and pyruvate is then converted to acetyl-CoA by the pyruvate dehydrogenase complex. Acetyl-CoA is also produced by the beta-oxidation of fatty acids and by the catabolism of certain amino acids, making it the convergence point where all major fuel molecules enter the cycle. The two-carbon acetyl group carried by coenzyme A enters the cycle by condensing with the four-carbon molecule oxaloacetate to form the six-carbon molecule citrate.

The pyruvate dehydrogenase complex itself is a massive multi-enzyme assembly, one of the largest known enzyme complexes, with a molecular weight exceeding 9 million daltons in mammals. It requires five coenzymes (thiamine pyrophosphate, lipoamide, coenzyme A, FAD, and NAD+) and is regulated by phosphorylation and dephosphorylation. When the cell's energy charge is high (abundant ATP, NADH, and acetyl-CoA), a dedicated kinase phosphorylates and inactivates the complex. When energy demand increases, a phosphatase removes the phosphate and reactivates it.

The Eight Reactions

Reaction 1: Citrate synthase catalyzes the condensation of acetyl-CoA (2 carbons) with oxaloacetate (4 carbons) to form citrate (6 carbons), releasing CoA. This is the first committed step of the cycle and is highly exergonic, driven forward by the hydrolysis of the thioester bond in acetyl-CoA. Citrate synthase is inhibited by ATP, NADH, and citrate itself, ensuring that the cycle slows down when the cell's energy charge is high.

Reaction 2: Aconitase catalyzes the isomerization of citrate to isocitrate through a dehydration-rehydration mechanism. The intermediate is cis-aconitate, a tricarboxylic acid that remains bound to the enzyme. This reaction repositions the hydroxyl group from a tertiary carbon (where it cannot be oxidized) to a secondary carbon (where it can be oxidized in the next step). Aconitase contains an iron-sulfur cluster (4Fe-4S) that is essential for catalysis and is sensitive to oxidative damage by superoxide radicals, making aconitase a potential sensor of oxidative stress in the cell.

Reaction 3: Isocitrate dehydrogenase catalyzes the oxidative decarboxylation of isocitrate (6 carbons) to alpha-ketoglutarate (5 carbons), producing one CO2 and one NADH. This is the first of four oxidation reactions in the cycle and the first point where carbon is lost as CO2. Isocitrate dehydrogenase is a key regulatory enzyme, strongly activated by ADP and calcium ions (signaling low energy and increased metabolic demand) and inhibited by ATP and NADH (signaling high energy). This regulation makes step 3 the principal rate-determining step of the cycle under most conditions.

Reaction 4: Alpha-ketoglutarate dehydrogenase complex catalyzes the oxidative decarboxylation of alpha-ketoglutarate (5 carbons) to succinyl-CoA (4 carbons), producing the second CO2 and another NADH. This reaction is mechanistically similar to the pyruvate dehydrogenase reaction and uses the same five coenzymes: thiamine pyrophosphate, lipoamide, FAD, NAD+, and CoA. The complex is inhibited by its products, succinyl-CoA and NADH, and activated by calcium ions, which serve as a signal of increased metabolic demand, particularly in muscle tissue during exercise.

After reactions 3 and 4, two carbons have been released as CO2, balancing the two carbons that entered as the acetyl group. However, isotopic labeling experiments show that these CO2 molecules do not contain the same carbon atoms that entered in the current turn; instead, they carry carbons from previous turns of the cycle. This asymmetry arises because aconitase treats citrate as a prochiral molecule, distinguishing between its two chemically identical arms and always removing the carbon atoms derived from the oxaloacetate rather than from the incoming acetyl group during the first turn.

Reaction 5: Succinyl-CoA synthetase (also called succinate thiokinase) cleaves the high-energy thioester bond of succinyl-CoA to produce succinate, CoA, and one GTP (in animal cells) or ATP (in plant and bacterial cells). GTP is readily converted to ATP by nucleoside diphosphate kinase, so this step represents the only substrate-level phosphorylation in the cycle. The energy of the thioester bond, originally derived from the oxidative decarboxylation in step 4, directly drives the phosphorylation of GDP to GTP.

Reaction 6: Succinate dehydrogenase catalyzes the oxidation of succinate to fumarate, producing FADH2. This enzyme is unique among the cycle's enzymes in two important ways. First, it is embedded in the inner mitochondrial membrane as part of Complex II of the electron transport chain, rather than being a soluble matrix enzyme. This dual role means succinate dehydrogenase simultaneously participates in both the Krebs cycle and the electron transport chain. Second, it uses FAD rather than NAD+ as the electron acceptor because the free energy change of this oxidation (removing two hydrogens from adjacent carbons to form a carbon-carbon double bond) is insufficient to reduce NAD+. The FADH2 produced here passes its electrons directly to ubiquinone (coenzyme Q), entering the electron transport chain at a lower energy level than NADH and consequently yielding approximately 1.5 ATP per electron pair rather than the approximately 2.5 ATP per pair from NADH.

Reaction 7: Fumarase (fumarate hydratase) catalyzes the addition of water across the double bond of fumarate to produce L-malate. This is a simple, stereospecific hydration reaction with no oxidation or reduction and no cofactor requirements. The enzyme produces only the L-isomer of malate, not the D-isomer.

Reaction 8: Malate dehydrogenase catalyzes the oxidation of malate to oxaloacetate, producing the third NADH of the cycle and regenerating the starting molecule. This reaction has a large positive standard free energy change (+7.1 kcal/mol), meaning it is thermodynamically unfavorable under standard conditions. However, in the cell, the reaction is pulled forward because citrate synthase (step 1) rapidly removes oxaloacetate by condensing it with acetyl-CoA, keeping the oxaloacetate concentration extremely low (approximately 10 micromolar). This demonstrates how the coupling of sequential reactions can drive individually unfavorable steps forward in a metabolic pathway.

Energy Yield Per Turn

Each turn of the Krebs cycle produces 3 NADH, 1 FADH2, 1 GTP (or ATP), and 2 CO2. Since each glucose molecule generates two acetyl-CoA molecules (through glycolysis and the pyruvate dehydrogenase reaction), two complete turns of the cycle are required per glucose. The total yield per glucose from the cycle alone is therefore 6 NADH, 2 FADH2, and 2 GTP.

The NADH and FADH2 produced by the cycle carry their electrons to the electron transport chain, where oxidative phosphorylation generates the majority of cellular ATP. Each NADH yields approximately 2.5 ATP, and each FADH2 yields approximately 1.5 ATP. Combined with the GTP produced by substrate-level phosphorylation, each turn of the cycle contributes approximately 10 ATP equivalents, and two turns per glucose contribute approximately 20 ATP. When added to the ATP produced by glycolysis and the pyruvate dehydrogenase step, the complete oxidation of one glucose molecule yields approximately 30 to 32 ATP.

The Cycle as a Metabolic Hub

The Krebs cycle is not merely a catabolic pathway. It also serves as a source of precursors for biosynthetic (anabolic) pathways, earning it the description "amphibolic" (both catabolic and anabolic). Citrate can be exported to the cytoplasm, where it is cleaved by ATP-citrate lyase to provide acetyl-CoA for fatty acid and cholesterol synthesis. Alpha-ketoglutarate is the carbon skeleton for the synthesis of glutamate and, through glutamate, several other amino acids including glutamine, proline, and arginine. Oxaloacetate is the precursor for aspartate synthesis and, through aspartate, for purine and pyrimidine nucleotide synthesis. Succinyl-CoA provides the carbon skeleton for heme biosynthesis, essential for hemoglobin, myoglobin, and cytochrome proteins.

When cycle intermediates are removed for biosynthesis, they must be replenished to keep the cycle running. These replenishment reactions are called anaplerotic reactions. The most important is catalyzed by pyruvate carboxylase, which converts pyruvate to oxaloacetate using CO2 and ATP. This enzyme is activated by acetyl-CoA: when acetyl-CoA accumulates (indicating that the cycle is slowing because oxaloacetate is depleted), pyruvate carboxylase is stimulated to produce more oxaloacetate, a simple and elegant feedback mechanism that ensures the cycle always has enough starting material to continue turning.

Regulation of the Cycle

The Krebs cycle is regulated primarily at three enzymatic steps: citrate synthase (reaction 1), isocitrate dehydrogenase (reaction 3), and alpha-ketoglutarate dehydrogenase (reaction 4). The common regulatory theme is that high energy charge (abundant ATP and NADH) inhibits the cycle, while low energy charge (accumulated ADP and NAD+) activates it. Calcium ions, released during muscle contraction and other energy-demanding processes, activate isocitrate dehydrogenase and alpha-ketoglutarate dehydrogenase, ensuring that the cycle speeds up when the cell needs more ATP.

Substrate availability also regulates flux through the cycle. The rate of acetyl-CoA production by the pyruvate dehydrogenase complex and by fatty acid oxidation determines how much fuel enters the cycle. Oxaloacetate availability, maintained by anaplerotic reactions, determines whether the cycle can process the incoming acetyl-CoA. This multi-level regulation ensures that the cycle operates at a rate precisely matched to the cell's energy demands and biosynthetic needs.