BB ch 16, part 2

Pyruvate Dehydrogenase Complex (PDC)

• Functions as the metabolic “gatekeeper” between glycolysis and the citric acid cycle (CAC).

• Overall reaction (oxidative decarboxylation of pyruvate):
  $\text{Pyruvate} + \text{CoA–SH} + \text{NAD}^+ \;\longrightarrow\; \text{Acetyl-CoA} + \text{CO}_2 + \text{NADH} + H^+$

• Multienzyme assembly (≈60 subunits, ~10 MDa) that channels intermediates—prevents diffusion and increases catalytic rate.

• Three catalytic component enzymes:
  • E1 = Pyruvate dehydrogenase (TPP-dependent)
  • E2 = Dihydrolipoyl transacetylase (lipoate-dependent)
  • E3 = Dihydrolipoyl dehydrogenase (FAD-dependent)

• Five required coenzymes (“biological tethers”):
  • Thiamine pyrophosphate (TPP)
  • Lipoic acid (lipoamide arm on a Lys of E2)
  • Coenzyme A (CoA-SH; carries the product acetyl group)
  • Flavin adenine dinucleotide (FAD)
  • Nicotinamide adenine dinucleotide (NAD^+)

Reaction Mechanism of the PDC

• Step 1 – Decarboxylation: Pyruvate + TPP → hydroxyethyl-TPP (CO₂ lost).

• Step 2 – Oxidation & transfer: Hydroxyethyl group is transferred to the lipoamide arm of E2 ⇒ formation of acetyl-dihydrolipoamide.

• Step 3 – Acetyl-CoA formation: CoA-SH attacks the thioester on reduced lipoamide, releasing Acetyl-CoA.

• Step 4 – Regeneration of oxidized lipoamide: E3 reoxidizes dihydrolipoamide; electrons pass to FAD → FADH₂.

• Step 5 – NADH production: FADH₂ transfers electrons to NAD^+ → NADH + H^+; FAD is regenerated.

• Net result: high-energy thioester (acetyl-CoA) plus reducing equivalents (NADH) enter further oxidation.

Biological Tethers & Swinging Arms

• Lipoamide (on E2) acts as a long, flexible “swinging arm,” ferrying intermediates among active sites.

• Other classic tethers:
  • Biotin (CO₂ carrier; e.g., pyruvate carboxylase)
  • Pantetheine arm of CoA and ACP (fatty-acyl carriers)

• Concept: covalently tethered cofactors increase local concentration, speed, and regulation.

Citric Acid Cycle – Overview

• Central aerobic pathway for oxidation of acetyl-CoA.

• Generates 3 NADH, 1 FADH₂, 1 GTP (or ATP) and releases 2 CO₂ per turn.

• Overall stoichiometry:
  $\text{Acetyl-CoA} + 3\;\text{NAD}^+ + \text{FAD} + \text{GDP} + P<em>i + 2\;\text{H}</em>2\text{O} \rightarrow 2\;\text{CO}<em>2 + 3\;\text{NADH} + 3H^+ + \text{FADH}</em>2 + \text{GTP} + \text{CoA-SH}$

Detailed Reactions of the Citric Acid Cycle

1. Condensation (Citrate Synthase)

   • Oxaloacetate + Acetyl-CoA + H₂O → Citrate + CoA-SH (rate-limiting; highly exergonic due to hydrolysis of thioester).

2. Dehydration / Rehydration (Aconitase)

   • Citrate ⇌ cis-Aconitate + H₂O ⇌ Isocitrate (moves the –OH from C-3 to C-2 to set up oxidation).

3. Oxidative Decarboxylation (Isocitrate Dehydrogenase)

   • Isocitrate + NAD^+ → α-Ketoglutarate + CO₂ + NADH.

4. Oxidative Decarboxylation (α-Ketoglutarate Dehydrogenase Complex; mechanistically analogous to PDC)

   • α-Ketoglutarate + CoA-SH + NAD^+ → Succinyl-CoA + CO₂ + NADH.

5. Substrate-Level Phosphorylation (Succinyl-CoA Synthetase)

   • Succinyl-CoA + GDP + P_i ⇌ Succinate + GTP + CoA-SH (GTP ↔ ATP via nucleoside diphosphate kinase).

6. Dehydrogenation (Succinate Dehydrogenase – inner-membrane flavoprotein, Complex II of ETS)

   • Succinate + FAD ⇌ Fumarate + FADH₂.

7. Hydration (Fumarase)

   • Fumarate + H₂O ⇌ L-Malate.

8. Dehydrogenation (Malate Dehydrogenase)

   • Malate + NAD^+ ⇌ Oxaloacetate + NADH (endergonic; driven forward by citrate synthase step).

Amphibolic Nature of the CAC

• “Amphibolic” = participates in both catabolism (oxidative energy production) and anabolism (biosynthetic precursor supply).

• Intermediates siphoned for synthesis must be replenished (anaplerosis).

The Succinate → Oxaloacetate Trio (Reactions 6-7-8)

• Common metabolic motif:

  1. Oxidation of a single bond → double bond (succinate → fumarate).

  2. Hydration across double bond (fumarate → malate).

  3. Oxidation of alcohol → ketone (malate → oxaloacetate).

• Re-appears in β-oxidation of fatty acids and many amino-acid pathways.

CAC Variants in Anaerobic Bacteria

• Some anaerobes possess an incomplete cycle that stops at succinate; still generates biosynthetic precursors.

• They typically lack α-ketoglutarate dehydrogenase; α-ketoglutarate is siphoned for glutamate family synthesis.

Biosynthetic Uses of CAC Intermediates

• α-Ketoglutarate → glutamate → purines, Arg, Pro, Gln.

• Succinyl-CoA → porphyrins (heme, chlorophyll).

• Fumarate & Oxaloacetate → Asp, Asn, Met, Lys, Thr, Ile and pyrimidines.

• Citrate exported to cytosol can yield acetyl-CoA for fatty-acid and sterol biosynthesis (via ATP-citrate lyase).

Anaplerotic (“Filling Up”) Reactions

• Key to maintaining oxaloacetate flux when intermediates are withdrawn.

Regulation of the CAC & PDC

• Three main pacemakers (irreversible, large −ΔG°’):
  • Citrate synthase – inhibited by ATP, NADH, succinyl-CoA, citrate; activated by ADP & substrate availability.
  • Isocitrate dehydrogenase – inhibited by ATP & NADH; activated by ADP and Ca²⁺ (muscle contraction signal).
  • α-Ketoglutarate dehydrogenase – inhibited by NADH & succinyl-CoA; activated by Ca²⁺.

• PDC regulatory logic:
  • Product inhibition: ATP, NADH, acetyl-CoA.
  • Activation by substrates: NAD^+, CoA-SH, AMP.

Covalent Regulation of Pyruvate Dehydrogenase

• E1 subunit is phosphorylated (inactive) by PDH kinase; dephosphorylated (active) by PDH phosphatase.

• High energy state (↑ATP, ↑NADH/NAD^+, ↑acetyl-CoA/CoA) → kinase active → PDC off.

• Low energy / muscle work (↑ADP, ↑Ca²⁺) → phosphatase active → PDC on.

The Glyoxylate Cycle

• Allows plants, fungi, and some bacteria to convert acetyl-CoA (from fatty acids or acetate) into net carbohydrate.

• Occurs in glyoxysomes (specialized peroxisomes). Bypasses the two decarboxylation steps of CAC.

• Unique “short-circuit” enzymes:
  • Isocitrate lyase: Isocitrate → succinate + glyoxylate.
  • Malate synthase: Glyoxylate + acetyl-CoA + H₂O → malate + CoA-SH.

• Net reaction (two acetyl-CoA → malate → gluconeogenesis):
  $2\;\text{Acetyl-CoA} + \text{NAD}^+ + 2\;\text{H}_2\text{O} \;\longrightarrow\; \text{Succinate} + 2\;\text{CoA-SH} + \text{NADH} + 2H^+$

• Succinate exits glyoxysome, is converted to oxaloacetate in mitochondria (succinate DH, fumarase, malate DH borrowed), then to glucose via gluconeogenesis.

• Biological role: enables germinating oil seeds to grow in the dark until photosynthesis begins.

Coordination Between Glyoxylate & Citric Acid Cycles

• Partitioning control point: Isocitrate dehydrogenase (IDH) vs. isocitrate lyase route.

• IDH is inactivated by phosphorylation → citrate/isocitrate diverted into glyoxylate cycle (biosynthetic mode).

• Dephosphorylation activates IDH → carbon flows through CAC for energy (ATP).

Electron-Transfer Linkage

• Succinate dehydrogenase (CAC step 6) is also Complex II of the respiratory chain; FADH₂ electrons enter ubiquinone, yielding approximately 1.5 ATP per FADH₂.

Practical / Clinical Connections

• Thiamine (vitamin B₁) deficiency → impaired PDC, α-KGDH → lactate acidosis, neurological defects (Wernicke-Korsakoff).

• Fluoroacetate forms fluorocitrate, a suicide inhibitor of aconitase → blocks CAC (used as rodenticide).

Suggested End-of-Chapter Problems

1 – 15, 19 – 25 (apply pathway logic, regulation, energetics, and variant cycles).