08 - TCA Cycle

TARGET DECK: MED::I::Signaling Pathways in Health and Disease::Metabolic Biochemistry::08 - TCA Cycle

Aerobic Pyruvate Oxidation

Overview

Acetyl-CoA is a key molecule that connects the final stages of carbohydrate, lipid, and amino acid metabolism.

Stage 1 — Acetyl-CoA Production

• Amino acids, fatty acids, and glucose all converge on Acetyl-CoA
• Glucose → Glycolysis → Pyruvate → Pyruvate dehydrogenase complex → ${\text{CO}}_2$ + Acetyl-CoA

Stage 2 — Acetyl-CoA Oxidation

• Acetyl-CoA enters the TCA cycle for complete oxidation

Stage 3 — Electron Transfer and Oxidative Phosphorylation

• Reduced electron carriers ($\text{NADH}$, ${\text{FADH}}_2$) feed the Respiratory (electron transfer) chain
• $2{\text{H}}^{+}+\frac{1}{2}{\text{O}}_2\to {\text{H}}_2\text{O}$

What are the three stages of aerobic pyruvate oxidation?

Stage 1: Acetyl-CoA production (glycolysis + PDH); Stage 2: Acetyl-CoA oxidation (TCA cycle); Stage 3: Electron transfer and oxidative phosphorylation (respiratory chain).

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Pyruvate Dehydrogenase

The Pyruvate Dehydrogenase Complex (PDH)

Reaction

$\text{Pyruvate}+{\text{NAD}}^{+}+\text{CoASH}\to \text{Acetyl-CoA}+{\text{CO}}_2+\text{NADH}+{\text{H}}^{+}$
This is oxidative decarboxylation.

Irreversibility

This reaction is ABSOLUTELY IRREVERSIBLE. This is the reason why lipids cannot be converted to carbohydrates.

PDH is formed by 3 enzymes (E1, E2, E3) and 3 prosthetic groups (TPP, Lipoic acid, FAD).

$\text{CoA-SH}+{\text{NAD}}^{+}\stackrel{{\text{PDH complex (E}}_1{\text{+E}}_2{\text{+E}}_3\text{)}}{\to }{\text{CH}}_3\text{-CO-S-CoA}+{\text{CO}}_2+\text{NADH}$

What is the overall reaction catalyzed by the Pyruvate Dehydrogenase Complex?

Pyruvate + NAD⁺ + CoASH → Acetyl-CoA + CO₂ + NADH + H⁺ (oxidative decarboxylation).

Anki cloze

The PDH reaction is absolutely {1:irreversible}, which is why {2:lipids} cannot be converted to {3:carbohydrates}.

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Cofactors of PDH

Coenzyme A (Pantothenic acid — Vitamin B5)

Info

Pantothenic acid is a water-soluble vitamin (Vit B5). Coenzyme A contains:

• β-Mercaptoethylamine (with reactive thiol group –SH)
• Pantothenic acid
• Ribose 3’-phosphate
• 3’-Phosphoadenosine diphosphate
• Adenine

The reactive thiol group of CoA-SH forms the high-energy thioester bond in Acetyl-CoA:

CC(=O)SCCNC(=O)CCNC(=O)[C@@H](O)C(C)(C)COP(=O)(O)OP(=O)(O)OC[C@H]1O[C@@H](n2cnc3c(N)ncnc23)[C@H](O)[C@@H]1OP(=O)(O)O

What vitamin is pantothenic acid, and what is its role in PDH?

Pantothenic acid is Vitamin B5. It is the core of Coenzyme A (CoA-SH), which carries the acetyl group as Acetyl-CoA.

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Thiamine Pyrophosphate (TPP — Vitamin B1)

Clinical Pearl

Thiamine is Vitamin B1. Thiamine deficiency induces a severe myocarditis (Beri-beri).

TPP is the prosthetic group of E1 (pyruvate dehydrogenase). It stabilizes a carbanion intermediate via its thiazolium ring.

Enzyme	Pathway	Bond Cleaved	Bond Formed
Pyruvate decarboxylase	Ethanol fermentation	${\text{R}}_1\text{-C-C}$ (α-keto)	${\text{R}}_1\text{-C-H}$
Pyruvate dehydrogenase	Synthesis of Acetyl-CoA	α-keto acid	Thioester (-S-CoA)
α-Ketoglutarate dehydrogenase	Citric acid cycle	α-keto acid	Thioester (-S-CoA)
Transketolase	Pentose phosphate pathway / Carbon-assimilation	${\text{R}}_2{\text{-C-C-R}}_1$	${\text{R}}_2{\text{-C-C-R}}_3$

What disease results from thiamine (Vitamin B1) deficiency, and which PDH cofactor does it affect?

Thiamine deficiency causes Beri-beri (severe myocarditis). It impairs TPP, the prosthetic group of E1 in PDH.

Anki cloze

TPP (Thiamine PyroPhosphate) is the prosthetic group of {1:E1} in the PDH complex, and is derived from Vitamin {2:B1}.

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Lipoic Acid

Info

Lipoic acid is not a Vitamin. It exists in an oxidized (disulfide, S-S) and an acetylated form (reduced, with –SH groups). It is covalently attached to a Lys residue of E2 (dihydrolipoyl transacetylase), forming a flexible lipoyllysine arm that enables substrate channeling.

Is lipoic acid a vitamin?

No. Lipoic acid is not a vitamin. It is a prosthetic group of E2, covalently linked to a lysine residue.

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FAD (Riboflavin — Vitamin B2)

Info

Riboflavin is Vitamin B2. FAD (Flavin adenine dinucleotide) and FMN (Flavin mononucleotide) are both derived from riboflavin. FAD is the prosthetic group of E3 (dihydrolipoyl dehydrogenase).

• $\text{FAD}\to {\text{FADH}}^{\bullet }\text{(semiquinone)}\to {\text{FADH}}_2\text{(fully reduced)}$
• The isoalloxazine ring carries the electrons.

Anki cloze

FAD is derived from Vitamin {1:B2 (Riboflavin)} and is the prosthetic group of {2:E3 (dihydrolipoyl dehydrogenase)} in the PDH complex.

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NAD⁺ (Nicotinamide — Vitamin PP or B3)

Info

Nicotinamide is Vitamin PP or B3. NAD⁺ (oxidized) accepts 2 electrons and 1 H⁺ to become NADH (reduced).
${\text{NAD}}^{+}+2e^{-}+2{\text{H}}^{+}\to \text{NADH}+{\text{H}}^{+}$
In NADP⁺, the 2’-hydroxyl of the adenosine ribose is esterified with phosphate.

What vitamin is NAD⁺ derived from, and what is its role in PDH?

NAD⁺ is derived from Nicotinamide (Vitamin PP or B3). It is the final electron acceptor in the PDH complex, oxidizing FADH₂ on E3 to regenerate FAD and producing NADH.

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Intermediate Reactions of PDH

The five sequential reactions:

${\text{CH}}_3\text{COCOOH}+\text{E1-TPP}\to {\text{CO}}_2+{\text{E1-TPP-[CH}}_3\text{CHO]}$

${\text{E1-TPP-[CH}}_3\text{CHO]}+\text{E2-Lip(S-S)}\to \text{E1-TPP}+\text{E2-Lip}\left(\begin{array}{c}-{\text{SCO-CH}}_3\\ -\text{SH}\end{array}\right)$

$\text{E2-Lip}\left(\begin{array}{c}-{\text{SCO-CH}}_3\\ -\text{SH}\end{array}\right)+\text{CoASH}\to {\text{CH}}_3\text{COSCoA}+{\text{E2-Lip(SH)}}_2$

${\text{E2-Lip(SH)}}_2+\text{E3-FAD}\to \text{E2-Lip(S-S)}+{\text{E3-FADH}}_2$

${\text{E3-FADH}}_2+{\text{NAD}}^{+}\to \text{E3-FAD}+\text{NADH}+{\text{H}}^{+}$

Mnemonic — PDH Reaction Steps

“DC ORT” — Decarboxylation (E1+TPP), Coupling to lipoic acid/oxidation (E1→E2), Oxidative transfer to CoA (E2+CoA), Reduction of FAD (E2→E3), Transfer to NAD⁺ (E3)

Describe the 5 sequential reactions of the PDH complex.

1. E1+TPP: decarboxylation of pyruvate → CO₂ + hydroxyethyl-TPP intermediate.
2. E2 lipoate (S-S): oxidation + transfer of acetyl group → acetyl-lipoamide.
3. E2+CoASH: transacetylation → Acetyl-CoA + dihydrolipoamide.
4. E3+FAD: reoxidation of lipoamide → FADH₂. 5. E3+NAD⁺: reoxidation of FADH₂ → NADH + H⁺.

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E1 Reaction: Decarboxylation

• The α-keto acid (pyruvate) is decarboxylated by E1-TPP
• Resonance stabilizes the hydroxyethyl-TPP carbanion intermediate
• The aldehyde $\text{R-CHO}$ (${\text{CH}}_3\text{CHO}$) is not released free; it is immediately oxidized:
  ${\text{CH}}_3\text{CHO}+\text{Lip(S-S)}\to {\text{CH}}_3\text{COOH}+{\text{Lip(SH)}}_2$
  but oxidised and reduced compounds join together, exploiting the energy of the oxidative reaction:
  $\to {\text{Lip-S-COCH}}_3\text{ (–SH)}$

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E2 Reaction: Oxidation and Transacetylation

• Lipoic acid is the prosthetic group (swinging arm on Lys of E2)
• CoA (CoA-SH) is also involved
• Products: Acetyl-CoA and dihydrolipoamide (Lip-(SH)₂)
• TPP and FAD are regenerated/involved in the cycle

O=C(CCCC[C@@H]1CCSS1)O

(Lipoic acid — oxidized form)

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E3 Reaction: Shuttling Electrons to NAD⁺

• E3 (dihydrolipoyl dehydrogenase) uses FAD to reoxidize lipoamide
• FADH₂ then reduces NAD⁺:
  ${\text{NAD}}^{+}+{\text{FADH}}_2\to \text{NADH}+{\text{H}}^{+}+\text{FAD}$
• This regenerates oxidized lipoyllysine so the cycle can repeat

Substrate Channeling

Substrate channeling: intermediates pass directly from one enzyme to the next without release. The lipoyllysine arm of E2 physically shuttles the acetyl group between E1 and the CoA acceptor, and then carries electrons to E3.

What is substrate channeling in the PDH complex?

Intermediates pass directly from one enzyme (E1→E2→E3) without being released into solution, facilitated by the flexible lipoyllysine “swinging arm” of E2.

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Regulation of Pyruvate Dehydrogenase

Allosteric and Covalent Regulation

• Inhibited by: NADH, ATP, Acetyl-CoA
• Stimulated by: NAD⁺, AMP, CoA
• Covalent regulation:
  • Inhibited by phosphorylation of E1 by PDH kinase (stimulated by ATP)
  • Activated by dephosphorylation by PDH phosphatase

Insulin Effect

Insulin enhances PDH phosphatase activity, thereby activating the enzyme. This favors formation of Acetyl-CoA as a precursor of lipogenesis.

Warburg Connection

In tumor cells, PDH is inactive because PDH kinase (PDHK) is activated → favors glycolysis with formation of lactate (Warburg effect).

How does insulin affect PDH activity?

Insulin enhances PDH phosphatase activity, dephosphorylating and thereby activating PDH. This favors Acetyl-CoA formation and lipogenesis.

Anki cloze

PDH is inhibited by {1:phosphorylation} of E1 by PDH {2:kinase}, which is itself stimulated by {3:ATP}.

Anki cloze

In tumor cells, PDH is inactive because {1:PDH kinase (PDHK)} is activated, favoring glycolysis and {2:lactate} production — the {3:Warburg} effect.

Mnemonic — PDH Inhibitors

“NAA” = NADH, ATP, Acetyl-CoA inhibit PDH
(Activators: NAD⁺, AMP, CoA — the “depleted” forms)

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TCA Cycle (Krebs / Citric Acid Cycle)

Importance

Key Features of the TCA Cycle

• Central energy-yielding pathway: point of convergence of catabolism of fats, carbohydrates, and proteins
• Located in mitochondria
• Intermediates are recycled (cycle)
• Source of precursors for biosynthesis

Where does the TCA cycle occur and why is it central to metabolism?

The TCA cycle occurs in mitochondria. It is the central energy-yielding pathway where catabolism of fats, carbohydrates, and proteins converges; intermediates are recycled and serve as biosynthetic precursors.

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Reactions of the TCA Cycle

Step 1 — Citrate Synthase: Condensation

$\text{Acetyl-CoA}+\text{Oxaloacetate}+{\text{H}}_2\text{O}\stackrel{\text{citrate synthase}}{\to }\text{Citrate}+\text{CoA-SH}$

$\Delta G^{\circ \prime }=-32.2\text{ kJ/mol}$

Important

This reaction is irreversible in vivo because [oxaloacetate] is kept low.
The methyl group of Acetyl-CoA is converted to a methylene group in citrate (aldol condensation).

OC(CC(O)=O)(CC(O)=O)C(O)=O

(Citrate)

What reaction does citrate synthase catalyze, and is it reversible?

Citrate synthase condenses Acetyl-CoA + Oxaloacetate → Citrate + CoA-SH. It is irreversible in vivo due to low oxaloacetate concentration. ΔG°′ = −32.2 kJ/mol.

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Step 2 — Aconitase: Dehydration/Rehydration

$\text{Citrate}\stackrel{\text{aconitase}}{\to }\text{cis-Aconitate}\stackrel{\text{aconitase}}{\to }\text{Isocitrate}$

$\Delta G^{\circ \prime }=+13.8\text{ kJ/mol}$

Info

The –OH group of citrate is repositioned in isocitrate (via cis-aconitate intermediate), which sets up decarboxylation in the next step. Dehydration then rehydration.

Anki cloze

Aconitase converts citrate → cis-aconitate → {1:isocitrate}, repositioning the hydroxyl group to set up {2:decarboxylation} in the next step.

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Step 3 — Isocitrate Dehydrogenase (IDH): Oxidative Decarboxylation

$\text{Isocitrate}+{\text{NAD(P)}}^{+}\stackrel{\text{isocitrate dehydrogenase}}{\to }\alpha \text{-Ketoglutarate}+{\text{CO}}_2+\text{NAD(P)H}+{\text{H}}^{+}$

$\Delta G^{\circ \prime }=-20.9\text{ kJ/mol}$

Important

Regulatory enzyme: inhibited by ATP and NADH; activated by ADP and NAD⁺.

• Specific isozymes: NADP⁺-dependent (cytosolic and mitochondrial) or NAD⁺-dependent (mitochondrial only).
• The reaction is reversible.
• Dehydrogenation: –OH group oxidized to carbonyl → facilitates decarboxylation by stabilizing carbanion on adjacent carbon.

What regulates isocitrate dehydrogenase?

Inhibited by ATP and NADH. Activated by ADP and NAD⁺. It is a regulatory enzyme of the TCA cycle.

Anki cloze

Isocitrate dehydrogenase produces {1:α-ketoglutarate} + {2:CO₂} + {3:NAD(P)H}, and is inhibited by {4:ATP} and {5:NADH}.

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Step 4 — α-Ketoglutarate Dehydrogenase (KGDH): Oxidative Decarboxylation

$\alpha \text{-Ketoglutarate}+\text{CoA-SH}+{\text{NAD}}^{+}\stackrel{\text{α-KG dehydrogenase complex}}{\to }\text{Succinyl-CoA}+{\text{CO}}_2+\text{NADH}$

$\Delta G^{\circ \prime }=-88.5\text{ kJ/mol}$

Important

As in PDH, the KGDH complex consists of E1 (with TPP), E2 (with lipoamide), and E3 (with FAD) and follows the same steps. Reaction is irreversible.

How is the α-Ketoglutarate Dehydrogenase complex similar to PDH?

Both consist of E1 (TPP), E2 (lipoamide), and E3 (FAD) and follow the same mechanistic steps (decarboxylation → acyl-lipoamide → acyl-CoA → FADH₂ → NADH). Both reactions are irreversible.

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Step 5 — Succinyl-CoA Synthetase: Substrate-Level Phosphorylation

$\text{Succinyl-CoA}+\text{GDP}+{\text{P}}_i\stackrel{\text{succinyl-CoA synthetase}}{\to }\text{Succinate}+\text{CoA-SH}+\text{GTP}$

$\Delta G^{\circ \prime }=-2.9\text{ kJ/mol}$

Info

Energy of the thioester bond is conserved in the phosphoanhydride bond of GTP (or ATP). Mechanism involves a phosphohistidyl-enzyme intermediate. The water molecule for the overall stoichiometry may be considered as derived from GDP and Pi dehydration during this reaction.

What type of phosphorylation occurs at the succinyl-CoA synthetase step?

Substrate-level phosphorylation: the energy of the thioester bond of succinyl-CoA is conserved as GTP (or ATP), via a phosphohistidyl-enzyme intermediate.

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Step 6 — Succinate Dehydrogenase: Dehydrogenation

$\text{Succinate}\stackrel{\text{succinate dehydrogenase}}{\to }\text{Fumarate}$

$\Delta G^{\circ \prime }=0\text{ kJ/mol}$

Important

• Natural acceptor is CoQ (Coenzyme Q): succinate-CoQ reductase is Complex II of the respiratory chain.
• The only membrane-bound enzyme of the TCA cycle (mitochondrial inner membrane).
• Introduction of a double bond initiates methylene oxidation sequence.

Why is succinate dehydrogenase unique among TCA cycle enzymes?

It is the only membrane-bound TCA enzyme, embedded in the mitochondrial inner membrane. Its natural electron acceptor is CoQ, making it identical to Complex II of the respiratory chain.

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Step 7 — Fumarase: Hydration

$\text{Fumarate}+{\text{H}}_2\text{O}\stackrel{\text{fumarase}}{\to }\text{L-Malate}$

$\Delta G^{\circ \prime }=-3.8\text{ kJ/mol}$

Info

Addition of water across the double bond introduces a –OH group (hydration), producing L-Malate.

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Step 8 — Malate Dehydrogenase: Dehydrogenation

$\text{L-Malate}+{\text{NAD}}^{+}\stackrel{\text{malate dehydrogenase}}{\to }\text{Oxaloacetate}+\text{NADH}+{\text{H}}^{+}$

$\Delta G^{\circ \prime }=+29.7\text{ kJ/mol}$

Important

Low oxaloacetate levels pull the reaction forward (Le Chatelier’s principle). This step regenerates oxaloacetate for citrate synthesis, completing the cycle.
Oxidation of –OH group completes the oxidation sequence; generates a carbonyl positioned to facilitate the aldol condensation in the next turn.

Why does malate dehydrogenase have a positive ΔG°′, yet still proceeds in vivo?

Because the very low concentration of oxaloacetate (product) pulls the reaction forward by Le Chatelier’s principle.

Anki cloze

The TCA cycle regenerates {1:oxaloacetate} in the last step catalyzed by {2:malate dehydrogenase}, which has a ΔG°′ of {3:+29.7 kJ/mol} but proceeds in vivo because {4:oxaloacetate} concentration is kept very low.

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Mechanisms of NADH Transport

Malate-Aspartate Shuttle

NADH produced in the cytosol must be transported into the mitochondrial matrix:

Cytosol → Mitochondria:

• Oxaloacetate + NADH${}_{\text{cyt}}$ → Malate + NAD⁺ (via malate dehydrogenase, cytosol)
• Malate enters mitochondria via glutamate-aspartate transporter
• Malate + NAD⁺ → Oxaloacetate + NADH${}_{\text{mit}}$ (via malate dehydrogenase, mitochondria)

Transamination arm:

• Oxaloacetate + Glutamate → Aspartate + α-Ketoglutarate (aspartate aminotransferase)
• Aspartate exits mitochondria; glutamate enters

Net effect: ${\text{NADH}}_{\text{cyt}}\to {\text{NADH}}_{\text{mit}}$

What is the net effect of the malate-aspartate shuttle?

It transfers cytosolic NADH into the mitochondrial matrix (NADH_cyt → NADH_mit), enabling oxidative phosphorylation.

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TCA Cycle — Summary Diagram (Reactions)

Step	Enzyme	Reaction	Key Feature
1	Citrate synthase	Acetyl-CoA + OAA → Citrate	Aldol condensation; irreversible
2	Aconitase	Citrate → cis-Aconitate → Isocitrate	Dehydration/rehydration
3	Isocitrate dehydrogenase	Isocitrate → α-KG + CO₂	Oxidative decarboxylation; regulatory
4	α-KG dehydrogenase	α-KG → Succinyl-CoA + CO₂	PDH-like; irreversible
5	Succinyl-CoA synthetase	Succinyl-CoA → Succinate + GTP	Substrate-level phosphorylation
6	Succinate dehydrogenase	Succinate → Fumarate	Complex II; membrane-bound
7	Fumarase	Fumarate → L-Malate	Hydration
8	Malate dehydrogenase	L-Malate → OAA	Regenerates OAA; pulled by low [OAA]

Mnemonic — TCA Cycle Intermediates (in order)

“Citrate Is Krebs’ Starting Substrate For Making Oxaloacetate”
= Citrate → Isocitrate → α-Ketoglutarate → Succinyl-CoA → Succinate → Fumarate → Malate → Oxaloacetate

List the 8 intermediates of the TCA cycle in order.

Citrate → Isocitrate → α-Ketoglutarate → Succinyl-CoA → Succinate → Fumarate → L-Malate → Oxaloacetate (→ back to Citrate).

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TCA Cycle — Stoichiometry

${\text{CH}}_3\text{COSCoA}+3{\text{H}}_2\text{O}\to \text{CoASH}+2{\text{CO}}_2+8\text{H}$

$8\text{H}+2{\text{O}}_2\to 4{\text{H}}_2\text{O}$

Total:
${\text{CH}}_3\text{COSCoA}+2{\text{O}}_2\to \text{CoASH}+2{\text{CO}}_2+{\text{H}}_2\text{O}$

Complete Stoichiometry

One molecule of H₂O may be considered as derived from GDP and Pᵢ dehydration during the succinyl-CoA synthetase reaction:
${\text{HOOCCH}}_2{\text{CH}}_2\text{COSCoA}+\text{GDP-H}+\text{P-OH}\to {\text{HOOCCH}}_2{\text{CH}}_2\text{COOH}+\text{CoASH}+\text{GTP}$

Real (complete) stoichiometry:
${\text{CH}}_3\text{COSCoA}+2{\text{O}}_2+\text{GDP-H}+\text{P-OH}\to \text{CoASH}+2{\text{CO}}_2+{\text{H}}_2\text{O}+\text{GTP}$

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ATP Yield from Aerobic Oxidation of Glucose

Reaction	Reduced Coenzymes / ATP Directly Formed	ATP Ultimately Formed
Glucose → Glucose 6-phosphate	−1 ATP	−1
Fructose 6-phosphate → Fructose 1,6-bisphosphate	−1 ATP	−1
2 G3P → 2 1,3-BPG	2 NADH	3–5
2 1,3-BPG → 2 3-PG	2 ATP	2
2 PEP → 2 Pyruvate	2 ATP	2
2 Pyruvate → 2 Acetyl-CoA	2 NADH	5
2 Isocitrate → 2 α-KG	2 NADH	5
2 α-KG → 2 Succinyl-CoA	2 NADH	5
2 Succinyl-CoA → 2 Succinate	2 ATP (or 2 GTP)	2
2 Succinate → 2 Fumarate	2 FADH₂	3
2 Malate → 2 Oxaloacetate	2 NADH	5
Total		30–32

Info

Calculated as 2.5 ATP per NADH and 1.5 ATP per FADH₂. A negative value indicates consumption.

How many ATP are produced per molecule of glucose in complete aerobic oxidation?

30–32 ATP total (calculated at 2.5 ATP/NADH and 1.5 ATP/FADH₂).

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Regulation of the Citric Acid Cycle

General Principle

Regulated at highly thermodynamically favorable and irreversible steps:

• PDH, Citrate synthase, IDH, and KDH

General regulatory mechanism:

• Activated by substrate availability
• Inhibited by product accumulation
• Overall products of the pathway are NADH and ATP
  • Inhibitors: NADH and ATP
  • Activators: NAD⁺ and AMP

Enzyme	Inhibitors	Activators
PDH complex	ATP, Acetyl-CoA, NADH, fatty acids	AMP, CoA, NAD⁺, Ca²⁺
Citrate synthase	Succinyl-CoA, citrate, ATP	—
Isocitrate dehydrogenase	ATP	Ca²⁺, ADP
α-KG dehydrogenase complex	Succinyl-CoA, NADH	Ca²⁺

What are the four regulated enzymes of the TCA cycle?

PDH (pyruvate dehydrogenase), citrate synthase, isocitrate dehydrogenase (IDH), and α-ketoglutarate dehydrogenase (KDH).

Anki cloze

The TCA cycle is generally inhibited by {1:NADH} and {2:ATP}, and activated by {3:NAD⁺} and {4:AMP}.

Mnemonic — TCA Regulated Enzymes

“PDH Can Inhibit Killing” → PDH, Citrate synthase, Isocitrate dehydrogenase, α-Ketoglutarate dehydrogenase

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Anaplerotic Reactions

Info

Anaplerotic reactions replenish TCA cycle intermediates that have been withdrawn for biosynthesis.

Reaction	Enzyme	Tissue/Organism
Pyruvate + HCO₃⁻ + ATP → Oxaloacetate + ADP + Pᵢ	Pyruvate carboxylase	Liver, kidney
PEP + CO₂ + GDP → Oxaloacetate + GTP	PEP carboxykinase	Heart, skeletal muscle
PEP + HCO₃⁻ → Oxaloacetate + Pᵢ	PEP carboxylase	Higher plants, yeast, bacteria
Pyruvate + HCO₃⁻ + NAD(P)H → Malate + NAD(P)⁺	Malic enzyme	Widely distributed

Important

The most important anaplerotic reaction is catalyzed by pyruvate carboxylase, an allosteric enzyme activated by Acetyl-CoA. Acetyl-CoA thereby acts as a signal to enhance its own oxidation in the TCA cycle.

At fasting, Acetyl-CoA from fatty acid oxidation in the liver may also serve as a signal for gluconeogenesis, which requires oxaloacetate.

What is the most important anaplerotic reaction, and what allosterically activates it?

Pyruvate + HCO₃⁻ + ATP → Oxaloacetate, catalyzed by pyruvate carboxylase, which is obligatorily activated by Acetyl-CoA.

Anki cloze

Anaplerotic reactions replenish {1:TCA cycle intermediates}. The most important is {2:pyruvate carboxylase}, activated by {3:Acetyl-CoA}.

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Pyruvate Carboxylase

Structure

Pyruvate carboxylase is exclusively mitochondrial with four subunits:

1. ATP + HCO₃⁻ binding site
2. Biotin-binding site (mobile arm) — present in many carboxylation reactions
3. Pyruvate-binding site
4. Allosteric site for Acetyl-CoA (obligatory activator)

Mechanism

Step 1 — Carboxylation of biotin (ATP-dependent):
$\text{ATP}+{\text{HCO}}_3^{-}+\text{Biotin-Enz}\to \text{Carboxybiotinyl-Enz}+\text{ADP}+{\text{P}}_i$

Step 2 — Transfer of carboxyl group to pyruvate enolate:
$\text{Pyruvate enolate}+\text{Carboxybiotinyl-Enz}\to \text{Oxaloacetate}+\text{Biotin-Enz}$

Info

Biotin is a water-soluble Vitamin (Vit B7). It acts as a mobile arm carrying the carboxyl group between the two active sites.

What vitamin is biotin, and what is its role in pyruvate carboxylase?

Biotin is Vitamin B7. It is the mobile arm in pyruvate carboxylase that carries the activated carboxyl group (from ATP + HCO₃⁻) to the pyruvate substrate, forming oxaloacetate.

Anki cloze

Pyruvate carboxylase is exclusively {1:mitochondrial} and requires {2:Acetyl-CoA} as an obligatory {3:allosteric activator}. Its prosthetic group is {4:biotin} (Vitamin {5:B7}).

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Biosynthetic Functions of the TCA Cycle

TCA Intermediates as Biosynthetic Precursors

Intermediate	Biosynthetic Pathway
Citrate	Gluconeogenesis; Fatty acids biosynthesis
α-Ketoglutarate	Non-essential amino acids
Succinyl-CoA	Porphyrins
Malate	Gluconeogenesis
Oxaloacetate	Gluconeogenesis; Cholesterol biosynthesis; Purine and pyrimidine rings

Info

PEP carboxykinase converts oxaloacetate → phosphoenolpyruvate (PEP) for gluconeogenesis.
PEP carboxylase converts PEP + HCO₃⁻ → oxaloacetate.
Acetyl-CoA → (via TCA intermediates) → PEP and other biosynthetic building blocks.

Which TCA intermediate is a precursor for porphyrin synthesis?

Succinyl-CoA.

Which TCA intermediates feed into gluconeogenesis?

Citrate, malate, and oxaloacetate.

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Glutaminolysis

Glutaminolysis in Neoplastic Cells

In neoplastic cells, pyruvate dehydrogenase is inhibited:
$\text{Glucose}\to \text{Pyruvate}\to \text{Lactate}$

Neoplastic cells largely utilize glutamine:

For energy/catabolism:
$\text{Glutamine}\to \text{Glutamate}\to \alpha \text{-Ketoglutarate}\to \text{Malate}\to \text{Pyruvate}\to \text{Lactate}$

For membrane lipids:
$\text{Glutamine}\to \text{Glutamate}\to \alpha \text{-Ketoglutarate}\to \text{Isocitrate}\to \text{Citrate}\to \text{membrane lipids}$

What is glutaminolysis and why do cancer cells use it?

Glutaminolysis is the use of glutamine as a major carbon source in neoplastic cells (because PDH is inhibited). Glutamine → glutamate → α-ketoglutarate → TCA intermediates → lactate or membrane lipids.

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Warburg Effect

Definition

Normal cells produce lactate only in the absence of oxygen (anaerobic conditions).
Cancer cells produce lactate in both anaerobic and aerobic environments.

As a result, in tumor cells, pyruvate is not directed to the citric acid cycle.

Metabolic Consequences

• The increased rate of glycolysis in cancer cells allows for tumor growth even in areas with poor blood supply, as these cells overexpress glucose uptake systems.
• Although cancer cells require the same amount of ATP as normal cells, they utilize the TCA cycle for the production of lipids, proteins, and nucleic acids.

Feature	Quiescent Cell	Proliferating (Cancer) Cell
Glucose fate	→ CO₂ (OXPHOS)	→ Lactate (glycolysis)
Glutamine use	Minimal	Major carbon source (glutaminolysis)
NADPH/nucleotide use	Low	High (biomass)
Fatty acid synthesis	Low	High (to biomass)

What is the Warburg effect?

Cancer cells produce lactate even in aerobic conditions (aerobic glycolysis). PDH is inhibited (by active PDHK), diverting pyruvate to lactate rather than TCA cycle. The TCA cycle is instead used for biomass (lipids, proteins, nucleic acids).

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Tumors and Isocitrate Dehydrogenase

IDH Isoforms

Isoform	Location	Cofactor
IDH3	Mitochondria	NAD⁺
IDH2	Mitochondria	NADP⁺
IDH1	Cytosol	NADP⁺

IDH Mutation in Gliomas

Important

Glial cell tumors have a mutant NADPH-dependent isocitrate dehydrogenase:

• Lost function: ability to convert isocitrate → α-ketoglutarate
• Gained (neomorphic) function: ability to convert α-ketoglutarate → 2-hydroxyglutarate (D2HG) using NADPH

$\alpha \text{-KG}\stackrel{{\text{IDH mutant, Mg}}^{2+}\text{, NADPH}}{\to }\text{D2HG (oncometabolite)}$

Oncometabolite

D2HG (D-2-hydroxyglutarate) is an oncometabolite that:

• Is a competitive inhibitor of α-KG-dependent dioxygenases
• Inhibits: histone demethylases, prolyl hydroxylases, collagen prolyl-4-hydroxylase, 5-methylcytosine hydroxylases (TET family)
• Results in: altered histone methylation, DNA hypermethylation, defective collagen maturation
• Promotes cancerogenesis (glioma)

Metabolic Context of IDH Mutations

• Glutaminase converts glutamine → glutamate + NH₄⁺
• Astrocyte-specific glutamine synthase: glutamate + NH₄⁺ → glutamine
• In mutant IDH glioma cells: glutamine → glutamate → α-KG → D2HG (instead of → normal TCA)

What is the neomorphic function gained by mutant IDH in gliomas?

Mutant IDH converts α-ketoglutarate → D-2-hydroxyglutarate (D2HG) using NADPH, instead of the normal reaction (isocitrate → α-KG).

How does D2HG (the oncometabolite from mutant IDH) promote cancerogenesis?

D2HG competitively inhibits α-KG-dependent dioxygenases, including histone demethylases, prolyl hydroxylases, and TET family enzymes, leading to DNA hypermethylation, altered histone methylation, and defective collagen maturation.

Anki cloze

In glial cell tumors, mutant IDH produces {1:D-2-hydroxyglutarate (D2HG)}, an oncometabolite that inhibits {2:α-KG-dependent dioxygenases}, resulting in {3:DNA hypermethylation}.

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Hypoxia-Induced Factor (HIF-1α)

Normal Oxygen Conditions — HIF Inactivation

Proline Hydroxylation Inactivates HIF

Under normal O₂, HIF is hydroxylated on a proline residue and is INACTIVE.

$\alpha \text{-KG}+\text{Pro}+{\text{O}}_2\to \text{Succinate}+{\text{CO}}_2+\text{4-Hydroxyproline}$

Reaction catalyzed by prolyl hydroxylase (PHD).
After hydroxylation, pVHL binds hydroxylated HIF-α and targets it for degradation.

Hypoxia — HIF Activation

Hypoxia and Succinate as a Hypoxia Mimic

• Hypoxia prevents proline hydroxylation → HIF is active
• Succinate inhibits prolyl hydroxylase, thus acting as a hypoxia mimic
• A genetic defect of succinate dehydrogenase acts as an oncogene, promoting inherited tumors as paraganglioma

Consequences of HIF Activation

Activation of HIF induces:

• Metabolic changes: activation of glycolysis, inhibition of OXPHOS
• Angiogenesis
• Metastasis

Signaling Pathway

SDH inhibition → succinate accumulates in mitochondria → succinate transported to cytosol → cytosolic succinate inhibits PHD → HIF-α hydroxylation is prevented → pVHL cannot bind HIF-α → elevated HIF activity → angiogenesis, metastasis, metabolic reprogramming → more aggressive tumors

How does succinate act as a "hypoxia mimic"?

Succinate inhibits prolyl hydroxylase (PHD), preventing hydroxylation of HIF-α. Without hydroxylation, pVHL cannot bind HIF-α for degradation, so HIF remains active — mimicking the effect of hypoxia.

What is the link between succinate dehydrogenase deficiency and cancer?

SDH deficiency leads to succinate accumulation → inhibition of PHD → constitutive HIF activation → angiogenesis and metastasis → inherited tumors, notably paraganglioma.

Anki cloze

Under normal O₂, HIF-α is {1:hydroxylated on proline} by {2:PHD (prolyl hydroxylase)}, using {3:α-KG} and {4:O₂} as substrates, producing {5:succinate} + CO₂. This allows {6:pVHL} to bind and degrade HIF-α.

Anki cloze

Succinate inhibits {1:PHD (prolyl hydroxylase)}, thereby acting as a {2:hypoxia mimic} and constitutively activating {3:HIF}.

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TLDR - 08 - TCA Cycle

Pyruvate Oxidation and TCA Cycle — Key Points

Aerobic Pyruvate Oxidation (3 Stages)

• Stage 1: Pyruvate → Acetyl-CoA (via PDH complex)
• Stage 2: Acetyl-CoA oxidized in TCA cycle
• Stage 3: Reduced carriers (NADH, FADH₂) → oxidative phosphorylation → ATP

Pyruvate Dehydrogenase Complex (PDH)

• Reaction: Pyruvate + NAD⁺ + CoASH → Acetyl-CoA + CO₂ + NADH + H⁺
• Absolutely irreversible → lipids cannot be converted to carbohydrates
• 3 enzymes: E1 (pyruvate decarboxylase), E2 (dihydrolipoyl transacetylase), E3 (dihydrolipoyl dehydrogenase)
• 3 prosthetic groups: TPP (Vit B1), Lipoic acid (not a vitamin), FAD (Vit B2)
• 2 soluble cofactors: CoA (Vit B5/pantothenic acid), NAD⁺ (Vit B3/nicotinamide)
• Substrate channeling via the lipoyllysine swinging arm of E2
• Inhibited by: NADH, ATP, Acetyl-CoA | Activated by: NAD⁺, AMP, CoA
• Covalently regulated: PDH kinase (ATP → phosphorylates/inhibits); PDH phosphatase (dephosphorylates/activates)
• Insulin activates PDH phosphatase → promotes lipogenesis
• Tumor cells: PDHK constitutively active → PDH inactive → Warburg effect

TCA Cycle — 8 Steps

1. Citrate synthase: OAA + Acetyl-CoA → Citrate (irreversible; aldol condensation)
2. Aconitase: Citrate → cis-Aconitate → Isocitrate (dehydration/rehydration)
3. Isocitrate dehydrogenase (IDH): Isocitrate → α-KG + CO₂ + NAD(P)H (regulatory; inhibited by ATP/NADH, activated by ADP/NAD⁺)
4. α-KG dehydrogenase: α-KG → Succinyl-CoA + CO₂ + NADH (PDH-like; irreversible)
5. Succinyl-CoA synthetase: Succinyl-CoA → Succinate + GTP (substrate-level phosphorylation)
6. Succinate dehydrogenase: Succinate → Fumarate (only membrane-bound TCA enzyme = Complex II; uses CoQ)
7. Fumarase: Fumarate → L-Malate (hydration)
8. Malate dehydrogenase: L-Malate → OAA + NADH (pulled by low [OAA])

TCA Stoichiometry per Acetyl-CoA: 3 NADH + 1 FADH₂ + 1 GTP + 2 CO₂
Total ATP from glucose: 30–32 (2.5 ATP/NADH; 1.5 ATP/FADH₂)

TCA Regulation — regulated at PDH, citrate synthase, IDH, α-KGDH:

• Inhibited by NADH, ATP; Activated by NAD⁺, AMP; Ca²⁺ activates PDH, IDH, α-KGDH

Anaplerotic Reactions — replenish TCA intermediates:

• Most important: pyruvate carboxylase (mitochondria; Pyruvate + HCO₃⁻ + ATP → OAA), activated by Acetyl-CoA; biotin (Vit B7) is cofactor
• Others: PEP carboxykinase, PEP carboxylase, malic enzyme

Biosynthetic Functions of TCA:

• Citrate → fatty acid synthesis; α-KG/OAA → amino acids; Succinyl-CoA → porphyrins; OAA → gluconeogenesis; Citrate/malate/OAA → multiple pathways

Warburg Effect:

• Cancer cells produce lactate aerobically (aerobic glycolysis)
• PDH inactive (PDHK constitutively on) → pyruvate → lactate
• TCA cycle used for biomass (lipids, proteins, nucleic acids), not ATP
• Overexpression of glucose transporters; growth in low-vascular areas

Glutaminolysis:

• Neoplastic cells use glutamine as major carbon source (PDH inhibited)
• Glutamine → Glutamate → α-KG → Malate → Pyruvate → Lactate (energy)
• Glutamine → α-KG → Isocitrate → Citrate → membrane lipids

IDH Mutations in Gliomas:

• Mutant IDH: loses isocitrate → α-KG; gains α-KG → D2HG (oncometabolite, using NADPH)
• D2HG inhibits α-KG-dependent dioxygenases → DNA hypermethylation, altered histone methylation, defective collagen maturation → cancerogenesis

HIF-1α and Succinate:

• Normal: PHD + α-KG + O₂ → hydroxylates HIF Pro → pVHL degrades HIF
• Hypoxia or SDH defect: succinate accumulates → inhibits PHD → HIF active → glycolysis ↑, OXPHOS ↓, angiogenesis, metastasis
• Succinate = hypoxia mimic; SDH defect → paraganglioma (oncogene)

Key Vitamins/Cofactors Summary:

Cofactor	Vitamin	Enzyme/Role
TPP	B1 (Thiamine)	E1 of PDH and KGDH; deficiency → Beri-beri
FAD	B2 (Riboflavin)	E3 of PDH and KGDH
NAD⁺	B3 (Niacin/PP)	Final electron acceptor in PDH
CoA	B5 (Pantothenic acid)	Acyl group carrier
Biotin	B7	Pyruvate carboxylase (CO₂ carrier)
Lipoic acid	(Not a vitamin)	E2 of PDH and KGDH