Pentose Phosphate Pathway, Pyruvate Dehydrogenase & Krebs Cycle Study Notes

Pentose Phosphate Pathway (PPP)

Rationale & Global Functions

• Alternative fate for $\text{Glucose}$/glycolytic intermediates $\text{Glucose-6-P}$ and $\text{Fructose-6-P}$.
• Chief goals, NOT ATP production:
  • Generation of large quantities of $\text{NADPH}$ (principal reductant for anabolic / detoxifying reactions).
  • Provision of $\text{Ribose-5-P}$ for nucleotide & nucleic‐acid synthesis.
• Major in animals and non-photosynthetic organisms; photosynthetic organisms obtain additional $\text{NADPH}$ via photosynthesis.

Oxidative Phase (irreversible)

• Substrate: $\text{Glucose-6-P}$.
• Step 1 – Dehydrogenation to lactone
  • Enzyme: Glucose-6-P dehydrogenase (G6PD).
  • $NADP^+$ reduced → $NADPH$.
  • Generates $\delta$-glucono-lactone.
• Lactone hydrolyzed → 6-phosphogluconate.
• Step 2 – Dehydrogenation + decarboxylation
  • Enzyme: 6-phosphogluconate dehydrogenase.
  • Second $NADP^+ \rightarrow NADPH$, loss of $CO_2$.
  • Produces Ribulose-5-P.
• Isomerization: Ribulose-5-P ⇌ Ribose-5-P (via isomerase) or epimerization to Xylulose-5-P (via epimerase).
• Net reaction
  $\text{Glucose-6-P} + 2 NADP^+ + H<em>2O \rightarrow \text{Ribose-5-P} + 2 NADPH + CO</em>2 + 2 H^+$

Non-Oxidative Phase (reversible carbon-rearrangement)

• Triggered when $\text{NADPH}$ demand > Ribose demand.
• Key enzymes & cofactor:
  • Transketolase – transfers $C_2$ units; requires TPP (thiamine pyrophosphate).
  • Transaldolase – transfers $C_3$ units; Schiff-base mechanism.
• Sequence (one round):
  1. Xylulose-5-P ($C<em>5$) + Ribose-5-P ($C</em>5$) $\xrightarrow{\text{Transketolase}}$ Glyceraldehyde-3-P ($C<em>3$) + Sedoheptulose-7-P ($C</em>7$).
  2. Sedoheptulose-7-P ($C<em>7$) + Glyceraldehyde-3-P ($C</em>3$) $\xrightarrow{\text{Transaldolase}}$ Erythrose-4-P ($C<em>4$) + Fructose-6-P ($C</em>6$).
  3. Erythrose-4-P ($C<em>4$) + Xylulose-5-P ($C</em>5$) $\xrightarrow{\text{Transketolase}}$ Glyceraldehyde-3-P ($C<em>3$) + Fructose-6-P ($C</em>6$).
• Two complete turns (starting from 6 Ribose-5-P):
  • 6 $C<em>5$ → 5 $C</em>6$ (Fructose-6-P / Glucose-6-P) + 1 $C_3$ (Glyceraldehyde-3-P).
  • Carbon accounting: one original glucose equivalent lost as 6 $CO_2$.

Carbon & Reducing-Power Balance (per 6 entering Glc)

• $6 \text{Glucose-6-P} \Rightarrow 12 \text{NADPH} + 6 CO_2 + 5 \text{Glucose-6-P eq.}$ (via recycling of pentoses).
• One glucose skeleton completely oxidized → supplies electrons.

Physiological Roles of NADPH

• Reductive biosynthesis: fatty acids, cholesterol, steroid hormones, deoxynucleotides.
• Maintenance of reduced glutathione; detoxification of reactive oxygen species (ROS) to $H_2O$.
• Cytochrome P450–dependent drug metabolism.

Clinical Correlation – G6PD Deficiency (Favism)

• X-linked; lowered G6PD → insufficient $NADPH$.
• Inability to reduce oxidized glutathione → accumulation of ROS.
• Erythrocytes especially vulnerable (high $O_2$ load, lack mitochondria).
• Triggers: fava beans (divicine), antimalarials, sulfa drugs → hemolytic anemia.

Pyruvate Dehydrogenase Complex (PDC)

Cellular Compartment & Composition

• Occurs in mitochondrial matrix (eukaryotes).
• Multienzyme aggregate: E1 (pyruvate dehydrogenase + TPP), E2 (dihydrolipoamide transacetylase + Lipoate & CoA), E3 (dihydrolipoamide dehydrogenase + FAD).
• Five cofactors: $TPP,\ lipoate,\ CoA,\ FAD,\ NAD^+$.

Mechanistic Steps (substrate channelling)

1. E1: Decarboxylation of pyruvate → $TPP$-bound hydroxyethyl; release $CO_2$ (irreversible).
2. Transfer to oxidized lipoamide (E2) → acetyldihydrolipoamide (tioester); lipoate reduced.
3. Trans-tioesterification: Acetyl group passed to CoA → Acetyl-CoA leaves.
4. E3: Re-oxidation of lipoamide via FAD → $FADH_2$.
5. $FADH_2$ electrons passed to $NAD^+$ → $NADH + H^+$ (soluble; enters ETC).

Net PDC Reaction

$\text{Pyruvate} + CoA + NAD^+ \rightarrow \text{Acetyl-CoA} + CO_2 + NADH + H^+$

Significance

• Bridges glycolysis and TCA cycle.
• Regulation: allosteric (ATP, NADH, Acetyl-CoA inhibit), covalent (phosphorylation inactivates E1).
• Reaction is non-reversible in vivo.

Citric Acid Cycle (Tricarboxylic Acid Cycle, Krebs Cycle)

Input, Output & Location

• Matrix of mitochondria; succinate dehydrogenase embedded in inner membrane.
• Per turn (per acetyl unit):
  • Inputs: Acetyl-CoA ($C<em>2$) + $3 NAD^+ + FAD + GDP + P</em>i + H_2O$.
  • Outputs: $2 CO<em>2 + 3 NADH + FADH</em>2 + GTP (\approx ATP) + CoA + 3 H^+$.

Eight Sequential Steps

1. Citrate synthase – Condensation: Oxaloacetate ($C<em>4$) + Acetyl-CoA → Citrate ($C</em>6$); hydrolysis of high-energy tioester drives reaction.
2. Aconitase – Isomerization: Citrate ⇌ Isocitrate via cis-aconitate; OH relocated.
3. Isocitrate dehydrogenase – Oxidative decarboxylation: Isocitrate → $\alpha$-ketoglutarate; yield $CO_2$ + $NADH$.
4. $\alpha$-Ketoglutarate dehydrogenase – Second oxidative decarboxylation (PDC-like): $\alpha$-KG → Succinyl-CoA; $CO_2$ + $NADH$.
5. Succinyl-CoA synthetase – Substrate-level phosphorylation: Succinyl-CoA + GDP (or ADP) → Succinate + GTP (or ATP).
6. Succinate dehydrogenase – Dehydrogenation: Succinate → Fumarate; $FAD \rightarrow FADH_2$; enzyme is Complex II of ETC (inner membrane-bound flavoprotein).
7. Fumarase – Hydration: Fumarate + $H_2O$ → L-Malate.
8. Malate dehydrogenase – Dehydrogenation: Malate → Oxaloacetate; $NAD^+ \rightarrow NADH$.

Carbon Fate & Labeling Insight

• Two $CO_2$ released originate from oxaloacetate (not the incoming acetyl) during the first turn; acetyl carbons exit in subsequent cycles.

Energetic Summary (per glucose → 2 Acetyl-CoA)

• $2 \times (3 NADH, 1 FADH<em>2, 1 GTP) = 6 NADH + 2 FADH</em>2 + 2 GTP$.
• Coupled oxidative phosphorylation yields approx.
  • $NADH \times 2.5$ ATP = $15$ ATP.
  • $FADH_2 \times 1.5$ ATP = $3$ ATP.
  • Direct: $2$ ATP (GTP).
  • Total from TCA per glucose ≈ $20$ ATP.

Aggregate ATP Yield from Complete Glucose Oxidation

• Glycolysis: $2 \text{ATP} + 2 \text{NADH} (\approx 5 \text{ATP})$.
• PDC: $2 \text{NADH} (\approx 5 \text{ATP})$.
• TCA: $20 \text{ATP}$ (above).
• Overall ≈ $32$ ATP (range $30!–!32$ due to shuttle & organism variation).

Recurring Reaction Strategy (C\rightarrowO insertion)

• Pattern: Dehydrogenation (introduce C=C) → Hydration (add OH) → Oxidation (OH → carbonyl).
• Applied: Succinate → Fumarate → Malate → Oxaloacetate.

Amphibolic Nature & Metabolic Hub

• Intermediates siphoned for biosynthesis (and replenished by anaplerotic reactions):
  • Citrate → cytosolic acetyl-CoA for fatty-acid/cholesterol synthesis.
  • $\alpha$-Ketoglutarate ↔ Glutamate → amino-acid pool.
  • Succinyl-CoA → Porphyrins / Heme.
  • Malate ↔ Pyruvate via malic enzyme; shuttle systems (malate–aspartate).
• Conversely, catabolic pathways feed intermediates directly into the cycle (e.g., propionate → Succinyl-CoA).

Integration & Cellular Decision-Making

• PPP, Glycolysis, Gluconeogenesis & TCA all reside in cytosol + mitochondria; flux direction driven by ATP, $NAD(P)H$ ratios & biosynthetic needs.
• If rapid ATP demand with limited $O_2$: reliance on glycolysis (fermentation) despite low yield.
• Adequate $O_2$: pyruvate funneled into PDC and TCA for maximal ATP.
• Excess NADPH demand with low nucleotide need: oxidative PPP followed by non-oxidative recycling to regenerate glucose skeletons.

Key Numerical & Stoichiometric Facts to Memorize

• Oxidative PPP: 1 Glc-6-P → 2 $NADPH$ + 1 $CO_2$.
• Full PPP recycle (6 Glc): 12 $NADPH$ + 6 $CO_2$ + 5 Glc-6-P.
• PDC: 1 Pyruvate → 1 Acetyl-CoA + 1 $NADH$ + $CO_2$.
• TCA (per Acetyl): 3 $NADH$ + 1 $FADH<em>2$ + 1 GTP + 2 $CO</em>2$.
• OxPhos P/O ratios: $NADH \approx 2.5$ ATP, $FADH_2 \approx 1.5$ ATP.

Ethical / Clinical / Practical Implications

• Drug-induced hemolysis in G6PD-deficient patients – importance in pharmacogenetics.
• Metabolic adaptations during hypoxia, anemia, or intense exercise.
• Centrality of TCA intermediates in cancer metabolism (anaplerosis, oncometabolites like 2-hydroxyglutarate).