32. Biochemistry | Citric Acid Cycle

*** PDH Complex ***

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What is the function of the pyruvate dehydrogenase complex (PDH)?

Converts pyruvate (3C) → acetyl-CoA (2C) + CO2 + NADH. Links glycolysis to the TCA cycle.

Which enzyme family does PDH belong to?

The α-keto acid dehydrogenase family (includes PDH, α-KG DH, branched-chain ketoacid DH).

What are the three enzymatic subunits of PDH and their roles?

E1: Pyruvate dehydrogenase (TPP-dependent); E2: Dihydrolipoyl transacetylase (requires lipoic acid, CoA); E3: Dihydrolipoyl dehydrogenase (requires FAD, NAD+).

Which five cofactors are required by PDH?

TPP (Vit B1), Lipoic acid, CoA (Vit B5), FAD (Vit B2), NAD+ (Vit B3). Mnemonic: Tender Loving Care For Nancy.

How is PDH regulated by PDH kinase?

PDH kinase phosphorylates and inactivates PDH. Stimulated by ATP, NADH, acetyl-CoA (signals energy sufficiency).

How is PDH regulated by PDH phosphatase?

PDH phosphatase dephosphorylates and activates PDH. Stimulated by ADP, pyruvate, Ca+ (muscle contraction).

What is the clinical presentation of PDH E1 deficiency?

Congenital lactic acidosis, neurologic dysfunction. X-linked dominant inheritance.

What is the biochemical mechanism of arsenic poisoning on PDH?

Arsenite binds lipoic acid (E2), inactivating PDH and α-KG DH → prevents acetyl-CoA formation → lactic acidosis.

What are the clinical features of arsenic poisoning?

Severe lactic acidosis, neurologic disturbance, often fatal.

Which vitamin deficiency affects PDH activity and causes Wernicke-Korsakoff/Beriberi?

Vitamin B1 (Thiamine) deficiency → loss of TPP cofactor.

What are the clinical features of thiamine deficiency?

Wernicke-Korsakoff: confusion, ataxia, ophthalmoplegia, memory loss. Beriberi: neuropathy, cardiomyopathy.

Which enzyme defects may cause Leigh syndrome?

Deficiency of PDH or pyruvate carboxylase.

What are the clinical features of Leigh syndrome?

Lactic acidemia, encephalopathy, respiratory failure.

*** TCA Cycle Overview ***

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Why is the TCA cycle considered the central hub of metabolism?

Because carbohydrates, fatty acids, and amino acids all converge at acetyl-CoA to enter the cycle.

Where does the TCA cycle occur?

In the mitochondrial matrix. It is an aerobic pathway.

What are the two major functions of the TCA cycle?

1. Energy production (NADH, FADH2, GTP/ATP). 2. Provide biosynthetic precursors (amino acids, fatty acids, heme, glucose).

What is the energy yield per acetyl-CoA in the TCA cycle?

3 NADH (7.5 ATP), 1 FADH2 (1.5 ATP), 1 GTP (1 ATP) = ~10 ATP total.

What is the total ATP yield from one glucose after glycolysis, PDH, TCA, and ETC?

36-38 ATP depending on the shuttle system (malate-aspartate vs glycerol phosphate).

Which three enzymes of the TCA cycle are irreversible?

Citrate synthase, Isocitrate dehydrogenase, α-Ketoglutarate dehydrogenase.

Which enzyme is the rate-limiting step of the TCA cycle?

Isocitrate dehydrogenase.

*** TCA Cycle Steps ***

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What reaction does citrate synthase catalyze?

Acetyl-CoA + OAA → Citrate. Irreversible (red).

What inhibits citrate synthase?

Citrate, NADH, Succinyl-CoA (red).

What allosterically regulates citrate synthase?

Oxaloacetate binding facilitates acetyl-CoA binding (blue).

What reaction does aconitase catalyze?

Isomerization of citrate → isocitrate (via cis-aconitate).

What structural feature does aconitase have?

It is an Fe-S protein (blue).

What toxin inhibits aconitase?

Fluoroacetate (red).

What reaction does isocitrate dehydrogenase catalyze?

Isocitrate → α-ketoglutarate + CO2 + NADH.

Why is isocitrate dehydrogenase important?

It is the rate-limiting enzyme of the TCA cycle (red).

Which step of the TCA cycle is the first oxidative decarboxylation?

Step 3, catalyzed by isocitrate dehydrogenase (red).

What activates isocitrate dehydrogenase?

ADP, Ca+ (blue).

What inhibits isocitrate dehydrogenase?

ATP, NADH (red).

What reaction does α-ketoglutarate dehydrogenase catalyze?

α-KG → Succinyl-CoA + CO2 + NADH.

Which step of the TCA cycle is the second oxidative decarboxylation?

Step 4, catalyzed by α-ketoglutarate dehydrogenase (red).

Which cofactors are required by α-ketoglutarate dehydrogenase?

TPP, Lipoic acid, CoA, FAD, NAD+ (blue).

What activates α-ketoglutarate dehydrogenase?

Ca+ (blue).

What inhibits α-ketoglutarate dehydrogenase?

ATP, GTP, NADH, Succinyl-CoA (red).

What reaction does succinyl-CoA synthetase (thiokinase) catalyze?

Succinyl-CoA → Succinate + GTP.

Why is succinyl-CoA synthetase important?

It performs substrate-level phosphorylation (red).

How is GTP converted to ATP in the TCA cycle?

Via nucleoside diphosphate kinase (blue).

What reaction does succinate dehydrogenase catalyze?

Succinate → Fumarate + FADH2.

What is unique about succinate dehydrogenase?

It is embedded in the inner mitochondrial membrane (red) and functions as Complex II of the ETC (blue).

What reaction does fumarase catalyze?

Fumarate + H2O → Malate (hydration reaction).

Is fumarase reversible or irreversible?

It is freely reversible (blue).

What reaction does malate dehydrogenase catalyze?

Malate → OAA + NADH.

What is special about malate dehydrogenase energetics?

ΔG° is positive but pulled forward by the citrate synthase reaction (blue).

How many NADH molecules are produced by malate dehydrogenase?

It produces the 3rd NADH of the cycle (red).

*** Anaplerotic Reactions and Biosynthetic Exits ***

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What is an anaplerotic reaction?

A reaction that replenishes intermediates of the TCA cycle.

What reaction does pyruvate carboxylase catalyze?

Pyruvate + CO2 + ATP → OAA. Requires biotin, activated by acetyl-CoA.

Which amino acids replenish TCA intermediates and where do they enter?

Alanine/serine → pyruvate; glutamate → α-KG; aspartate → OAA; Val, Ile, Met, Thr → Succinyl-CoA; Phe/Tyr → Fumarate.

What is the biosynthetic role of citrate?

Cytosolic fatty acid and cholesterol synthesis.

What is the biosynthetic role of α-ketoglutarate?

Precursor for amino acids and neurotransmitters (glutamate, GABA).

What is the biosynthetic role of succinyl-CoA?

Heme synthesis.

What is the biosynthetic role of malate?

Gluconeogenesis via conversion to OAA.

What is the biosynthetic role of oxaloacetate?

Amino acid synthesis (aspartate) and gluconeogenesis.

*** Cofactors ***

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Which vitamin provides thiamine pyrophosphate (TPP), a cofactor for PDH and α-KG DH?

Vitamin B1 (Thiamine). Deficiency → Beriberi, Wernicke-Korsakoff.

Which vitamin provides coenzyme A (CoA)?

Vitamin B5 (Pantothenic acid). Required for acetyl-CoA formation.

Which vitamin provides FAD as a cofactor?

Vitamin B2 (Riboflavin). Deficiency → cheilitis, glossitis.

Which vitamin provides NAD+ as a cofactor?

Vitamin B3 (Niacin). Deficiency → Pellagra (dermatitis, diarrhea, dementia).

Which molecule is the cofactor lipoic acid used by?

Used by PDH and α-KG DH. Target of arsenic poisoning.

What mnemonic helps remember the 5 cofactors of PDH and α-KG DH?

Tender Loving Care For Nancy: TPP, Lipoic acid, CoA, FAD, NAD+.

*** Clinical Correlates ***

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What is the biochemical defect in PDH deficiency?

Mutation in E1 → impaired conversion of pyruvate to acetyl-CoA → pyruvate shunted to lactate.

What is the inheritance pattern of PDH deficiency?

X-linked dominant.

What are the clinical features of PDH deficiency?

Congenital lactic acidosis, neurologic dysfunction.

What is the biochemical mechanism of arsenic poisoning?

Arsenite binds lipoic acid → blocks PDH and α-KG DH.

What are the clinical features of arsenic poisoning?

Severe lactic acidosis, neurologic disturbance, death.

What is the biochemical defect in thiamine deficiency?

Lack of TPP cofactor impairs PDH and α-KG DH.

What are the clinical features of Wernicke-Korsakoff syndrome?

Confusion, ataxia, ophthalmoplegia, memory loss.

What are the clinical features of Beriberi?

Neuropathy and cardiomyopathy.

Which enzyme deficiencies can cause Leigh syndrome?

PDH deficiency or pyruvate carboxylase deficiency.

What are the clinical features of Leigh syndrome?

Lactic acidemia, encephalopathy, respiratory failure.

A 4-year-old boy presents with developmental delay and recurrent episodes of lethargy. Laboratory studies reveal elevated lactate levels. Genetic testing confirms an E1 subunit deficiency of the pyruvate dehydrogenase complex. Which of the following best explains the biochemical basis of this child’s condition?

A. Failure to regenerate oxaloacetate for the TCA cycle
B. Accumulation of pyruvate shunted to lactate via lactate dehydrogenase
C. Inhibition of α-ketoglutarate dehydrogenase by NADH
D. Decreased activity of succinate dehydrogenase in the ETC
E. Deficiency of biotin-dependent carboxylase enzymes

B

• B (Correct): In PDH E1 deficiency, pyruvate cannot be converted to acetyl-CoA, so it accumulates and is shunted to lactate, causing congenital lactic acidosis.

• A: Relates to pyruvate carboxylase deficiency, not PDH.

• C: NADH inhibits α-ketoglutarate dehydrogenase, but that’s not the cause here.

• D: Succinate dehydrogenase is part of Complex II — unrelated to PDH deficiency.

• E: Biotin deficiency impairs pyruvate carboxylase, not PDH.

A 52-year-old man is brought to the emergency department with confusion, vomiting, and seizures. He recently began working in a pesticide factory. Labs reveal severe lactic acidosis. Arsenic poisoning is suspected. Which cofactor-enzyme interaction is most directly disrupted in this patient?

A. Thiamine with pyruvate carboxylase
B. Biotin with pyruvate carboxylase
C. Lipoic acid with pyruvate dehydrogenase
D. FAD with succinate dehydrogenase
E. NAD⁺ with isocitrate dehydrogenase

C

• C (Correct): Arsenite binds to lipoic acid, blocking E2 of PDH (and also α-ketoglutarate dehydrogenase, BCAA dehydrogenase), preventing acetyl-CoA formation and leading to lactic acidosis.

• A/B: Wrong cofactors (thiamine and biotin).

• D: FAD is part of succinate dehydrogenase, but not arsenic’s target.

• E: NAD⁺ is needed for isocitrate dehydrogenase, but unaffected by arsenic.

A 20-year-old male with a history of alcohol abuse is admitted with confusion, ataxia, and nystagmus. He is diagnosed with Wernicke-Korsakoff syndrome. The deficiency of which cofactor is responsible for his symptoms, and which enzyme is impaired as a result?

A. Biotin; pyruvate carboxylase
B. Thiamine (Vitamin B1); α-ketoglutarate dehydrogenase
C. Riboflavin (Vitamin B2); malate dehydrogenase
D. Niacin (Vitamin B3); citrate synthase
E. Lipoic acid; fumarase

B

• B (Correct): Thiamine deficiency (Vit B1) impairs enzymes requiring TPP, including PDH and α-ketoglutarate dehydrogenase. Classic triad: confusion, ataxia, ophthalmoplegia/nystagmus.

• A: Biotin deficiency → pyruvate carboxylase deficiency, not seen here.

• C: Riboflavin is a precursor for FAD, needed for succinate dehydrogenase, not malate dehydrogenase.

• D: Niacin deficiency impairs NAD⁺-dependent enzymes, but not citrate synthase.

• E: Lipoic acid is a PDH cofactor, but deficiency isn’t the cause of Wernicke-Korsakoff.

A patient with Leigh syndrome is found to have a pyruvate carboxylase deficiency. Which of the following best explains the metabolic consequence of this defect?

A. Failure to replenish oxaloacetate for the TCA cycle
B. Increased GTP production due to excess succinyl-CoA
C. Impaired FADH2 production at succinate dehydrogenase
D. Inhibition of citrate synthase by succinyl-CoA
E. Increased acetyl-CoA entry into fatty acid synthesis

A

• A (Correct): Pyruvate carboxylase (biotin-dependent) normally replenishes oxaloacetate (anaplerosis). Its deficiency → impaired TCA cycle and gluconeogenesis.

• B: Succinyl-CoA doesn’t increase GTP production in this condition.

• C: Succinate dehydrogenase makes FADH2, but unaffected here.

• D: Succinyl-CoA does inhibit citrate synthase, but that’s not the main defect in PC deficiency.

• E: Excess acetyl-CoA actually accumulates since it cannot enter TCA without oxaloacetate.