BB ch 16 part 1

Overview: Citric Acid Cycle (Krebs / TCA)

• Central aerobic pathway for oxidation of acetyl-CoA to $2\,CO_2$.
• Functions as a “metabolic wheel,” analogous to a ferris wheel in time-lapse photography: substrates hop on, are transformed, hop off; the wheel itself (oxaloacetate) is regenerated.
• Integrated with glycolysis, β-oxidation, amino-acid catabolism, and oxidative phosphorylation.
• Key guiding question: “How is pyruvate oxidized under aerobic conditions, and what chemical logic dictates the sequence of reactions?”

Historical Context – Hans Krebs

• Demonstrated that acetate is oxidized by a cyclic sequence of reactions.
• Cycle takes its alternative names from him (Krebs cycle) and from the first isolated intermediate (citric acid).

Global Stoichiometry & Energetics

• Complete oxidation of one glucose to $6\,CO_2$ is a 24-e⁻ process.
• One turn of the TCA cycle (per acetyl-CoA):
  • $3\,NADH$ (≈ $7.5\,\text{ATP}$ after OXPHOS)
  • $1\,FADH<em>2$ (≈ $1.5\,\text{ATP}$) • $1\,GTP$ (substrate-level, readily converted to ATP) • $2\,CO</em>2$ (complete oxidation of acetyl carbons)
• Overall ATP equivalent ≈ $10\,\text{ATP}$ per acetyl-CoA.

Pyruvate Entry – Pyruvate Dehydrogenase Complex (PDH)

• Location: mitochondrial matrix; pyruvate imported via specific carrier.
• PDH is a non-covalent megacomplex (≈ 10 nm):
  • E1 = pyruvate dehydrogenase (decarboxylase)
  • E2 = dihydrolipoyl transacetylase
  • E3 = dihydrolipoyl dehydrogenase
• Overall reaction:
  ${\text{Pyruvate}} + CoA!!\text{-SH} + NAD^+ \rightarrow \text{Acetyl-CoA} + CO_2 + NADH + H^+$
• Five required coenzymes
  1. Thiamine pyrophosphate (TPP) – decarboxylation of $\alpha$-keto acids & carbanion stabilization.
  2. Lipoic acid (covalently tethered to E2 via Lys ε-NH₂) – swings acyl groups & couples acyl and e⁻ transfer.
  3. Coenzyme A (CoA-SH) – reactive thiol forms thioesters (high-energy).
  4. FAD/FADH₂ – accepts 2 e⁻ + 2 H⁺, can mediate 1- or 2-e⁻ transfers.
  5. NAD⁺/NADH – soluble, 2-e⁻ (hydride) carrier, regenerates FAD.
• Mechanistic sequence (Fig. 16-5, 16-6):
  • Decarboxylation of pyruvate → hydroxyethyl-TPP (E1).
  • Transfer to oxidized lipoamide (E2) → acetyl-dihydrolipoamide.
  • Formation of acetyl-CoA; reduced lipoamide stays on E2.
  • Re-oxidation of lipoamide by FAD (E3) → FADH₂.
  • Re-oxidation of FADH₂ by NAD⁺ → NADH.

Chemical Logic of the Cycle

• Direct C–C cleavage of acetate is unfavorable; solution: condense acetate with oxaloacetate to form citrate, then perform a sequence of β-cleavage–like oxidations.
• Oxidations are strategically spaced to harvest energy as NADH/FADH₂ and one substrate-level GTP/ATP.
• Steps 1, 3, and 4 have large negative $\Delta G'$ and are essentially irreversible; they serve as key regulatory points.

Detailed Sequence of TCA Reactions

Step 1 — Condensation (Citrate Synthase)

• $\text{Oxaloacetate} + \text{Acetyl-CoA} + H_2O \rightarrow \text{Citrate} + CoA!!\text{-SH}$
• $\Delta G'^\circ = -31.4\,\text{kJ·mol}^{-1}$ (drives cycle entry).
• Ordered mechanism: oxaloacetate binds first → conformational change → pocket for acetyl-CoA.
• Enzyme is dimeric; flexible domains close to exclude water until C–C bond forms.

Step 2 — Dehydration/Rehydration (Aconitase)

• Citrate ⇌ cis-Aconitate + H₂O ⇌ Isocitrate.
• Net $\Delta G'^\circ \approx +13\,\text{kJ·mol}^{-1};$ pulled forward by next exergonic step.
• Contains [4Fe–4S] iron–sulfur cluster that anchors the substrate and facilitates elimination/addition of water.
• Prochiral specificity: although citrate is symmetric, aconitase acts asymmetrically; only one of the two identical carboxylates is susceptible because of enzyme binding geometry.

Step 3 — Oxidative Decarboxylation (Isocitrate Dehydrogenase)

• Isocitrate + NAD⁺ (or NADP⁺, isozyme-specific) → $\alpha$-Ketoglutarate + CO₂ + NADH.
• Involves Mn²⁺ that stabilizes the intermediate oxalosuccinate and promotes decarboxylation.
• $\Delta G'^\circ = -8.4\,\text{kJ·mol}^{-1}.$

Step 4 — Oxidative Decarboxylation (α-Ketoglutarate Dehydrogenase Complex)

• Analogous architecture to PDH; same five coenzymes.
• $\alpha$-KG + CoA-SH + NAD⁺ → Succinyl-CoA + CO₂ + NADH.
• $\Delta G'^\circ = -30\,\text{kJ·mol}^{-1};$ irreversible control point.

Step 5 — Substrate-Level Phosphorylation (Succinyl-CoA Synthetase)

• Succinyl-CoA + GDP + Pᵢ ⇌ Succinate + GTP + CoA-SH (ATP possible in some tissues).
• Mechanism (Fig. Succinyl-CoA synthetase):
  • Pᵢ attacks thioester → succinyl-phosphate.
  • Phospho-His intermediate transfers Pᵢ to GDP.
• $\Delta G'^\circ \approx -3\,\text{kJ·mol}^{-1}$ (near equilibrium).

Step 6 — Dehydrogenation (Succinate Dehydrogenase)

• Succinate + FAD → Fumarate + FADH₂.
• Integral membrane flavoprotein (Complex II of ETC); transfers e⁻ directly to ubiquinone.
• $\Delta G'^\circ \approx 0$; direction driven by Q reduction.
• Competitive inhibitor: malonate (structural analog of succinate) halts cycle experimentally.

Step 7 — Hydration (Fumarase)

• Fumarate + H₂O → L-Malate.
• Highly stereospecific: trans (fumarate) accepted; not the cis (maleate); product is L-malate only.
• $\Delta G'^\circ = -3.8\,\text{kJ·mol}^{-1}.$

Step 8 — Dehydrogenation (Malate Dehydrogenase)

• L-Malate + NAD⁺ ⇌ Oxaloacetate + NADH + H⁺.
• Strongly endergonic: $\Delta G'^\circ = +29.7\,\text{kJ·mol}^{-1}$; pulled forward in vivo by rapid consumption of oxaloacetate in Step 1.

Cycle Products & Their Fates

• NADH / FADH₂ → electron transport chain → oxidative phosphorylation (major ATP yield).
• GTP (or ATP) – used directly or converted by nucleoside diphosphate kinase.
• CO₂ – diffuses out, ultimately exhaled.

Biosynthetic (Anaplerotic) & Cataplerotic Roles

• Intermediates siphoned for:
  • Citrate → cytosolic source of acetyl-CoA for fatty acid & sterol synthesis.
  • α-KG → glutamate → amino acids, neurotransmitters.
  • Succinyl-CoA → porphyrins, heme.
  • OAA → aspartate, pyrimidines, gluconeogenesis.
• Anaplerotic (“filling”) reactions replenish cycle when intermediates are withdrawn, e.g. pyruvate carboxylase:
  $\text{Pyruvate} + CO<em>2 + ATP \rightarrow OAA + ADP + P</em>i$

Regulation of the TCA Cycle

• Energy charge & redox status dictate flux.
• Major control points (irreversible steps):
  1. Citrate synthase – inhibited by ATP, NADH, succinyl-CoA; substrate availability (OAA).
  2. Isocitrate dehydrogenase – activated by ADP, Ca²⁺; inhibited by ATP, NADH.
  3. α-Ketoglutarate dehydrogenase – activated by Ca²⁺; inhibited by NADH, succinyl-CoA.
• PDH (gateway enzyme) also regulated by phosphorylation (PDH kinase/phosphatase) responsive to ATP, NADH, acetyl-CoA, Ca²⁺, ADP, pyruvate.

Structural / Mechanistic Highlights & Stereochemistry

• Citrate symmetry vs. asymmetric reaction: enzyme binding renders molecule prochiral; only one C₃–C₄ bond is cleaved later – explains isotope-tracer results.
• Aconitase Fe–S cluster positions citrate and accepts OH⁻ during dehydration.
• Fumarase proceeds through a carbanion transition state requiring trans geometry.
• Succinyl-CoA synthetase demonstrates energy conversion: thioester → mixed anhydride → phosphohistidine → NTP.

Inhibitors & Experimental Probes

• Malonate competitively inhibits succinate dehydrogenase; stalls cycle for mechanistic studies.
• Fluoroacetate poisons by forming fluorocitrate, a potent aconitase inhibitor (not explicitly in transcript but commonly linked).

Integration with Oxidative Phosphorylation

• NADH oxidation via Complex I yields ≈ $2.5\,ATP$ per pair of electrons; FADH₂ via Complex II yields ≈ $1.5\,ATP$.
• Hence, combined glycolysis + PDH + TCA + OXPHOS gives ≈ $30!!–32\,ATP$ per glucose (to be detailed in follow-up slides marked "TBC").

Practical / Clinical Connections

• Vitamin deficiencies: Thiamine (TPP) deficiency → impaired PDH & α-KGDH → lactic acidosis, Wernicke-Korsakoff.
• Inherited PDH complex defects → neurologic dysfunction due to reduced ATP.
• Ischemia: limits O₂, backup of NADH, slows TCA.

Ethical & Philosophical Implications

• Illustrates evolutionary optimization: coupling energetically unfavorable cleavages to favorable oxidations; cyclic design minimizes need for multiple pathways.
• Understanding regulation is vital for metabolic disease, cancer metabolism (Warburg effect), and drug development targeting cycle enzymes.