Metabolic Pathways: Glycolysis, Fermentation & Anaerobic Respiration

Glycolysis (Embden–Meyerhof Pathway)

• Central catabolic route converting glucose → pyruvate; supports both aerobic & anaerobic ATP production.
• Occurs in cytoplasm of virtually all organisms; does not require O₂.
• Overall reaction: $\text{Glucose} + 2\,\text{NAD}^+ + 2\,\text{ADP} + 2\,\text{P}<em>i \;\longrightarrow\; 2\,\text{Pyruvate} + 2\,\text{NADH} + 2\,\text{H}^+ + 2\,\text{ATP} + 2\,\text{H}</em>2\text{O}$
• Energy yield: net $2\,\text{ATP}$ per glucose by substrate-level phosphorylation (SLP) + 2 NADH (≈ 5–7 ATP under aerobic conditions when re-oxidised in mitochondria/ETC).
• Two phases:
– Preparatory phase (investment):
1. Hexokinase (glucokinase in liver) phosphorylates glucose → glucose-6-P (G6P); consumes 1 ATP; traps sugar in cell.
2. Phosphoglucose isomerase converts G6P ↔ fructose-6-P (F6P).
3. Phosphofructokinase-1 (PFK-1) phosphorylates F6P → fructose-1,6-bisP (F-1,6-BP); consumes 1 ATP; major irreversible, rate-limiting, allosterically regulated step (↑AMP/ADP, ↓ATP, ↓citrate).
4. Aldolase splits F-1,6-BP → dihydroxyacetone-P (DHAP) + glyceraldehyde-3-P (G3P).
5. Triose-phosphate isomerase interconverts DHAP ↔ G3P; ensures both 3-carbon fragments enter payoff phase.
– Pay-off phase (harvest):
6. Glyceraldehyde-3-P dehydrogenase (GAPDH) oxidises G3P + $P_i$ + NAD⁺ → 1,3-bisphosphoglycerate (1,3-BPG) + NADH (high-energy acyl-phosphate bond).
7. Phosphoglycerate kinase (PGK) transfers high-energy phosphate: 1,3-BPG → 3-phosphoglycerate (3-PG) + ATP (first SLP).
8. Phosphoglycerate mutase (PGM) shifts phosphate: 3-PG ↔ 2-PG.
9. Enolase dehydrates 2-PG → phosphoenolpyruvate (PEP) + H₂O (PEP contains very high ΔG°′).

10. Pyruvate kinase (PK) converts PEP → pyruvate + ATP (second SLP); activated by F-1,6-BP (feed-forward), inhibited by ATP & alanine.
    • Thermodynamics: overall exergonic; irreversible steps (1, 3, 10) ensure directionality.
    • Anaerobic continuation: NADH must be re-oxidised → lactate (muscle) or ethanol (yeast) so glycolysis can proceed.
    • Physiological context: rapid ATP during sprinting; tumor Warburg effect; RBCs (no mitochondria) rely exclusively on glycolysis.

Pentose Phosphate Pathway (PPP / Hexose-Monophosphate Shunt)

• Operates in cytosol, parallel to glycolysis; branches from G6P.
• Primary functions:
– Generate reducing power as $\text{NADPH}$ for biosynthesis (fatty acids, cholesterol), oxidative stress defense (glutathione reductase).
– Produce ribose-5-P for nucleotide & cofactor synthesis.
– Interconvert sugars (C3–C7) feeding back into glycolysis (F6P, G3P).
• Two phases:

1. Oxidative (irreversible): G6P dehydrogenase → 6-phosphoglucono-δ-lactone, etc.; yields 2 NADPH + CO₂ per G6P.
2. Non-oxidative (reversible): transketolase ((TDP) cofactor) & transaldolase shuffle carbon skeletons.
   • Clinical link: G6PD deficiency → hemolytic anemia on oxidative stress (fava beans, antimalarials).
   • Flux high in liver, adipose, mammary, adrenal cortex, neutrophils.

Fermentation (General Concepts)

• Anaerobic ATP-generating process; substrate-level phosphorylation only; no external terminal electron acceptor/ETC.
• Key requirement: regenerate $\text{NAD}^+$ from NADH produced in glycolysis to sustain glycolytic flux.
• Energy yield: ≈ 2 ATP per glucose (much lower than aerobic ~30-32 ATP).
• Industrial/food relevance: dairy, brewing, biofuels, solvents.

Lactic Acid Fermentation

• Pyruvate reduced → lactate by lactate dehydrogenase (LDH); NADH oxidised → NAD⁺.
• Reaction: $\text{C}<em>6\text{H}</em>{12}\text{O}<em>6 \;\longrightarrow\; 2\,\text{CH}</em>3\text{CHOHCOOH}$ (homolactic).
• Homolactic microbes: Lactobacillus (yogurt), muscle cells under oxygen debt.
• Heterolactic variant (phosphoketolase pathway) forms lactate + ethanol + CO₂ (e.g., Leuconostoc); reaction: $\text{C}<em>6\text{H}</em>{12}\text{O}<em>6 \;\longrightarrow\; \text{CH}</em>3\text{CHOHCOOH} + \text{C}<em>2\text{H}</em>5\text{OH} + \text{CO}_2$.
• Physiological role: allows continued ATP in ischemic tissue; lactate shuttled to liver (Cori cycle).

Alcoholic (Ethanolic) Fermentation

• Yeasts (Saccharomyces cerevisiae) & some bacteria.
• Two-step: (1) Pyruvate decarboxylase: pyruvate → acetaldehyde + CO₂; (2) Alcohol dehydrogenase: acetaldehyde + NADH → ethanol + NAD⁺.
• Overall: $\text{C}<em>6\text{H}</em>{12}\text{O}<em>6 + 2\,\text{ADP} + 2\,\text{P}</em>i \;\longrightarrow\; 2\,\text{C}<em>2\text{H}</em>5\text{OH} + 2\,\text{CO}_2 + 2\,\text{ATP}$.
• Applications: beer, wine, bread (CO₂ leavens dough).

Mixed Acid Fermentation

• Enterobacteriaceae (e.g., Escherichia coli) diversify end products: lactate, acetate, succinate, formate, ethanol, CO₂, H₂.
• Produces extra ATP via acetate kinase (acetyl-P → acetate + ATP).
• Gas production used in IMViC tests (microbiology diagnostics).
• Environmental adaptation: flexibility in redox balancing; competitive advantage in gut.

2,3-Butanediol & Other Fermentations (contextual)

• Butanediol pathway (Enterobacter, Serratia): pyruvate → acetoin → 2,3-butanediol; less acidic; Voges–Proskauer test positive.

Dissimilatory Sulfate Reduction (Anaerobic Respiration)

• Sulfate-reducing bacteria (SRB) use $\text{SO}_4^{2-}$ as terminal e⁻ acceptor when O₂ absent.
• Pathway steps:

1. Sulfate imported; activated by ATP sulfurylase → adenosine-5′-phosphosulfate (APS) (consumes 1 ATP → AMP + PPᵢ).
2. APS reductase: APS + 2e⁻ → sulfite ($\text{SO}_3^{2-}$) + AMP.
3. Sulfite reductase: $\text{SO}<em>3^{2-} + 6e^- + 6H^+ \rightarrow \text{H}</em>2\text{S} + 3\,\text{H}<em>2\text{O}$. • Electrons supplied by organic substrates (lactate, pyruvate, H₂, etc.) via ferredoxin. • Energy yield modest (≈ 1–2 ATP per substrate) yet vital in marine sediments, oil reservoirs, gut. • Ecological/industrial impact: biocorrosion ((\text{H}2\text{S}) reacts with metals), odor, sulfur cycling.

Nitrate Reduction / Denitrification

• Alternative terminal acceptor $\text{NO}<em>3^-$ used in anaerobic respiration by diverse bacteria (e.g., Pseudomonas, Paracoccus). • Sequential reductions: $\text{NO}</em>3^- \xrightarrow[]{\text{nitrate reductase}} \text{NO}<em>2^- \xrightarrow[]{\text{nitrite reductase}} \text{NO} \xrightarrow[]{\text{NO reductase}} \text{N}</em>2\text{O} \xrightarrow[]{\text{N}<em>2\text{O reductase}} \text{N}</em>2$.
• ETC components: NADH dehydrogenase (Complex I), succinate DH (Complex II), cytochrome bc₁ (III), nitrate reductase at membrane periplasmic face; creates proton motive force → ATP synthase.
• Generic reaction (first step): $\text{NO}<em>3^- + 2e^- + 2H^+ \rightarrow \text{NO}</em>2^- + \text{H}<em>2\text{O}$. • Environmental importance: removes bioavailable nitrogen from soils/water; mitigates/fuels greenhouse gas (\text{N}2\text{O}) emissions.

Methanogenesis (Archaea-specific)

• Strictly anaerobic process reducing CO₂, formate, methanol, methylamines, or acetate → methane ($\text{CH}_4$).
• Key pathway (CO₂ + H₂):

1. CO₂ + Fdred → formyl-Methanofuran (formyl-MF) (formyl-MF dehydrogenase).
2. Formyl-MF → methenyl-MF (cyclohydrolase) – water removed.
3. Methenyl-MF + Fdred → methylene-MF (methenyl-MF reductase).
4. Methylene-MF → methyl-MF (methyl transferase).
5. Methyl-MF + coenzyme M (CoM-SH) → methyl-CoM.
6. Methyl-CoM + coenzyme B (CoB-SH) → methane + CoM–S–S–CoB (methyl-CoM reductase; nickel cofactor F₄₃₀).
   • Energy conservation: sodium/proton gradients coupled to heterodisulfide reductase, F420H₂ dehydrogenase, Ech-complex → ATP synthase yields ~1 ATP per 4 CH₄.
   • Habitats: wetlands (largest natural CH₄ source), rumen, landfills, hydrothermal vents.
   • Climate link: CH₄ = potent greenhouse gas; methanogenesis vs methanotrophy balance.

Comparative Energetics & Biological Significance

• Aerobic respiration: O₂ terminal acceptor; complete glucose oxidation → CO₂; ΔG°′ ≈ −2870 kJ; ~30–32 ATP.
• Anaerobic respiration (nitrate, sulfate, CO₂): lower ΔE₀′ yields; ATP varies (nitrate ≈ 20-22, sulfate < 5, methanogenesis minimal but sufficient for niche survival). • Fermentation: no ETC; 2 ATP; rapid but inefficient; favoured when electron transport limited or speed > yield (e.g., sprinting, fast-growing microbes in sugar-rich niches).
• Metabolic flexibility enables microbes to thrive across redox gradients; shapes biogeochemical cycles (C, N, S).
• Industrial/medical ethics: manipulation for bioenergy (biogas), waste valorisation; need to mitigate pathogen or greenhouse outputs.