Krebs Cycle Intermediates

Why This Matters

The Krebs cycle isn't just a loop you memorize for an exam—it's the metabolic hub that connects carbohydrate, fat, and protein metabolism into one integrated system. You're being tested on your ability to understand how each intermediate serves dual purposes: generating energy carriers (NADH, FADH₂, GTP) while simultaneously feeding into biosynthetic pathways like amino acid synthesis, gluconeogenesis, fatty acid production, and heme biosynthesis. The cycle's intermediates are constantly being siphoned off and replenished, making this a dynamic crossroads rather than a closed loop.

When exam questions ask about the Krebs cycle, they rarely want you to simply list the intermediates in order. Instead, you'll need to identify where reducing equivalents are generated, which steps release $CO_2$, and how specific intermediates connect to other metabolic pathways. Don't just memorize the sequence—know what each intermediate does and why its position in the cycle matters for cellular metabolism.

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Entry and Cycle Initiation

The cycle begins when two-carbon acetyl units enter and condense with four-carbon acceptors. This condensation reaction is thermodynamically favorable and essentially irreversible, committing carbons to oxidation.

Citrate

• Six-carbon tricarboxylic acid formed by condensation of acetyl-CoA (2C) with oxaloacetate (4C), catalyzed by citrate synthase
• Allosteric regulator of glycolysis and fatty acid synthesis—high citrate signals energy abundance and inhibits phosphofructokinase-1
• Transported to cytosol via the citrate shuttle, where it's cleaved to provide acetyl-CoA for de novo fatty acid synthesis

Isocitrate

• Isomer of citrate formed via the enzyme aconitase, which first dehydrates citrate to cis-aconitate, then rehydrates it
• Branch point intermediate—the isocitrate dehydrogenase reaction is the first of two oxidative decarboxylations in the cycle
• Regulatory checkpoint where isocitrate dehydrogenase is activated by ADP and inhibited by ATP and NADH

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Oxidative Decarboxylation Steps

These reactions release $CO_2$ and generate NADH, representing the cycle's primary energy-harvesting mechanism. Both decarboxylation complexes require the same five coenzymes: TPP, lipoate, CoA, FAD, and NAD⁺.

α-Ketoglutarate

• Five-carbon α-keto acid produced when isocitrate dehydrogenase removes $CO_2$ and transfers electrons to $NAD^+$, generating the cycle's first NADH
• Amino acid metabolism hub—readily interconverts with glutamate via transamination, linking the cycle to nitrogen metabolism and neurotransmitter synthesis
• Substrate for α-ketoglutarate dehydrogenase complex, which is structurally and mechanistically similar to pyruvate dehydrogenase

Succinyl-CoA

• Four-carbon thioester formed when α-ketoglutarate dehydrogenase releases $CO_2$ and generates the cycle's second NADH
• High-energy thioester bond ($\Delta G°' \approx -33 \text{ kJ/mol}$) is conserved as GTP (or ATP) in the next reaction via substrate-level phosphorylation
• Heme synthesis precursor—condenses with glycine to form δ-aminolevulinic acid, the committed step in porphyrin biosynthesis

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Substrate-Level Phosphorylation and Oxidation

This portion of the cycle captures energy directly as GTP and generates $FADH_2$ through a membrane-bound enzyme complex.

Succinate

• Four-carbon dicarboxylic acid formed when succinyl-CoA synthetase cleaves the thioester bond, coupling it to GTP synthesis (the cycle's only substrate-level phosphorylation)
• Oxidized by succinate dehydrogenase (Complex II of the electron transport chain), generating $FADH_2$ rather than NADH because the reaction's $\Delta G°'$ is insufficient to reduce $NAD^+$
• Accumulates in certain cancers—mutations in succinate dehydrogenase cause succinate buildup, stabilizing HIF-1α and promoting tumor growth

Fumarate

• Four-carbon unsaturated dicarboxylic acid with a trans double bond, produced by FAD-dependent oxidation of succinate
• Urea cycle connection—also produced when argininosuccinate is cleaved in the urea cycle, linking nitrogen disposal to energy metabolism
• Hydrated stereospecifically by fumarase to produce only L-malate (not D-malate), demonstrating enzyme specificity

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Cycle Completion and Regeneration

The final steps regenerate oxaloacetate, ensuring the cycle can accept another acetyl-CoA. These reactions also provide key intermediates for gluconeogenesis.

Malate

• Four-carbon hydroxy acid formed by stereospecific hydration of fumarate's double bond by fumarase
• Malate-aspartate shuttle component—cytosolic malate carries reducing equivalents into mitochondria, where it's oxidized to regenerate NADH for the ETC
• Gluconeogenesis intermediate—can exit the mitochondria and be oxidized to oxaloacetate in the cytosol for glucose synthesis

Oxaloacetate

• Four-carbon keto acid regenerated when malate dehydrogenase oxidizes malate, producing the cycle's third NADH
• Acetyl-CoA acceptor—combines with incoming acetyl groups to form citrate, completing the cycle; concentration is limiting and tightly regulated
• Gluconeogenic precursor—converted to phosphoenolpyruvate by PEP carboxykinase, bypassing the irreversible pyruvate kinase reaction

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Quick Reference Table

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Self-Check Questions

1. Which two Krebs cycle reactions release $CO_2$, and what coenzymes do their enzyme complexes share with pyruvate dehydrogenase?

2. Compare the energy-capturing mechanisms at succinate dehydrogenase versus succinyl-CoA synthetase—why does one produce $FADH_2$ while the other produces GTP?

3. A patient has a mutation in succinate dehydrogenase. Which Krebs cycle intermediate would accumulate, and how might this affect cellular signaling beyond metabolism?

4. If you needed to explain how the Krebs cycle connects to both gluconeogenesis and fatty acid synthesis, which two intermediates would you focus on and why?

5. Contrast the roles of α-ketoglutarate and oxaloacetate in amino acid metabolism—which amino acids does each connect to, and what type of reaction interconverts them?