Unit 6: Metabolism

I. Orientation

Metabolism is the integrated network of enzyme-catalyzed reactions that converts nutrients into usable energy, reducing power, biosynthetic precursors, and waste products. Catabolic pathways generally release energy, whereas anabolic pathways consume energy to build cellular components.

• Compartmentation: Glycolysis and the pentose phosphate pathway occur in the cytosol; the TCA cycle occurs mainly in the mitochondrial matrix; the electron transport chain and ATP synthase are located in the inner mitochondrial membrane.
• Energy currency: ATP couples energy-releasing reactions to energy-requiring processes; NADH and FADH₂ carry high-energy electrons to the respiratory chain; NADPH mainly supports reductive biosynthesis.
• Redox convention: Oxidation is loss of electrons or hydrogen, while reduction is gain of electrons or hydrogen. Electron carriers cycle between oxidized and reduced forms.
• Pathway control: Flux is regulated by substrate availability, products, energy charge, covalent modification, and hormones. Irreversible reactions commonly serve as control points.
• Compartment and transport: Metabolites must cross membranes through specific carriers; for example, cytosolic NADH transfers its reducing equivalents into mitochondria through shuttle systems.

II. Bioenergetics — Energy flow in biochemical systems

Bioenergetics applies thermodynamic principles to determine whether metabolic reactions can proceed and how cells capture released energy.

A. Bioenergetics

Bioenergetics describes the relationship between free-energy change, equilibrium, and cellular work.

• Free-energy equation: The reaction free-energy change is:

ΔG = ΔG°′ + RT ln Q

ΔG is actual free-energy change, ΔG°′ is standard transformed free energy at biochemical pH, R is the gas constant, T is absolute temperature, and Q is the reaction quotient.

• Spontaneity: A reaction with ΔG < 0 is thermodynamically favorable, but favorable reactions may still be slow without enzymes. A positive ΔG requires coupling to a favorable process.
• ATP coupling: ATP hydrolysis is favorable:

ATP + H₂O → ADP + Pi

Its released energy drives biosynthesis, transport, and mechanical work.

• Redox energy: Electron transfer from NADH to oxygen is highly favorable. Cells conserve part of this energy by pumping protons rather than releasing all energy as heat.
• Energy charge: The ATP/ADP ratio indicates cellular energy status. High ATP suppresses catabolic pathways, whereas high ADP or AMP stimulates ATP-producing pathways.

III. Glycolysis — Cytosolic glucose breakdown

Glycolysis converts one glucose molecule into two pyruvate molecules through ten reactions in the cytosol, producing ATP and NADH without directly requiring oxygen.

A. Glycolysis and its regulation

Glycolysis has an investment phase, a payoff phase, and three major irreversible control steps.

• Net reaction:

Glucose + 2 ADP + 2 Pi + 2 NAD⁺
→ 2 Pyruvate + 2 ATP + 2 NADH + 2 H₂O + 2 H⁺

• Energy investment: Hexokinase or glucokinase converts glucose to glucose-6-phosphate using ATP. Phosphofructokinase-1 (PFK-1) then converts fructose-6-phosphate to fructose-1,6-bisphosphate.
• Energy payoff: Each of two glyceraldehyde-3-phosphate molecules yields one NADH and two ATP, giving a net gain of 2 ATP and 2 NADH per glucose.
• Substrate-level phosphorylation: ATP is formed directly at phosphoglycerate kinase and pyruvate kinase, unlike oxidative phosphorylation.
• Major regulation: Hexokinase is inhibited by glucose-6-phosphate; PFK-1 is inhibited by ATP and citrate and activated by AMP and fructose-2,6-bisphosphate; pyruvate kinase is activated by fructose-1,6-bisphosphate and inhibited by ATP.
• Aerobic versus anaerobic fate: Aerobically, pyruvate becomes acetyl-CoA. Under anaerobic conditions, lactate dehydrogenase regenerates NAD⁺:

Pyruvate + NADH + H⁺ → Lactate + NAD⁺

IV. TCA cycle — Oxidation of acetyl-CoA

The tricarboxylic acid cycle, also called the citric acid cycle or Krebs cycle, oxidizes the acetyl group of acetyl-CoA and supplies reducing equivalents for ATP production.

A. TCA cycle and its regulation

One turn of the cycle combines acetyl-CoA with oxaloacetate and regenerates oxaloacetate after releasing two carbon atoms as CO₂.

• Overall yield per acetyl-CoA:

Acetyl-CoA + 3 NAD⁺ + FAD + GDP + Pi + 2 H₂O
→ 2 CO₂ + 3 NADH + FADH₂ + GTP + CoA-SH + 3 H⁺

• Sequence: Citrate synthase forms citrate; aconitase forms isocitrate; isocitrate dehydrogenase produces α-ketoglutarate; α-ketoglutarate dehydrogenase produces succinyl-CoA; succinyl-CoA synthetase forms GTP; succinate dehydrogenase forms FADH₂; fumarase forms malate; malate dehydrogenase regenerates oxaloacetate.
• Oxidative decarboxylation: Isocitrate dehydrogenase and α-ketoglutarate dehydrogenase release CO₂ and reduce NAD⁺ to NADH.
• Regulation: Citrate synthase is inhibited by ATP, NADH, succinyl-CoA, and citrate. Isocitrate dehydrogenase is activated by ADP and Ca²⁺ and inhibited by ATP and NADH. α-Ketoglutarate dehydrogenase is inhibited by NADH and succinyl-CoA and activated by Ca²⁺.
• Amphibolic role: TCA intermediates support biosynthesis; citrate supplies acetyl units for fatty-acid synthesis, α-ketoglutarate supports amino-acid metabolism, and succinyl-CoA contributes to heme synthesis. Anaplerotic reactions replenish intermediates, especially pyruvate carboxylase forming oxaloacetate.

V. Pentose phosphate pathway — NADPH and pentose production

The pentose phosphate pathway is a cytosolic alternative route for glucose-6-phosphate oxidation that produces NADPH and ribose-5-phosphate rather than ATP.

A. Pentose phosphate pathway and its significance

The pathway has oxidative and nonoxidative phases and is especially active in liver, adipose tissue, adrenal cortex, and red blood cells.

• Oxidative phase: Glucose-6-phosphate dehydrogenase produces NADPH and 6-phosphogluconolactone; subsequent reactions form ribulose-5-phosphate and release CO₂. Two NADPH are generated per glucose-6-phosphate.
• Nonoxidative phase: Transketolase and transaldolase rearrange pentoses into fructose-6-phosphate and glyceraldehyde-3-phosphate, which can re-enter glycolysis.
• NADPH significance: NADPH reduces glutathione in erythrocytes and supports antioxidant defense:

GSSG + NADPH + H⁺ → 2 GSH + NADP⁺

GSSG is oxidized glutathione and GSH is reduced glutathione.

• Biosynthetic function: NADPH supplies reducing power for fatty-acid, cholesterol, and steroid synthesis. Ribose-5-phosphate supports nucleotide synthesis.
• Regulation and deficiency: Glucose-6-phosphate dehydrogenase is activated by NADP⁺ and inhibited by NADPH. Deficiency limits red-cell protection against oxidants and may cause hemolysis after oxidative stress.

VI. Electron transport chain and oxidative phosphorylation — Mitochondrial ATP production

The electron transport chain transfers electrons from NADH and FADH₂ to oxygen, while oxidative phosphorylation uses the resulting proton gradient to synthesize ATP.

A. Electron transport chain and oxidative phosphorylation

The respiratory chain consists of four electron-transfer complexes, mobile carriers, and ATP synthase in the inner mitochondrial membrane.

• Electron route: NADH donates electrons to Complex I; FADH₂ donates through Complex II. Electrons then pass to coenzyme Q, Complex III, cytochrome c, and Complex IV, where oxygen becomes water.
• Proton pumping: Complexes I, III, and IV pump H⁺ from the matrix into the intermembrane space. Complex II does not pump protons.
• Chemiosmosis: The proton-motive force combines membrane potential and pH gradient. Proton flow through the F₀ channel of ATP synthase drives rotation and conformational changes in F₁, producing ATP from ADP and Pi.
• Approximate yield: One mitochondrial NADH produces about 2.5 ATP, while one FADH₂ produces about 1.5 ATP; exact yield depends on transport costs and cellular conditions.
• Inhibitors and uncouplers: Rotenone inhibits Complex I, antimycin inhibits Complex III, cyanide blocks Complex IV, and oligomycin blocks ATP synthase. Uncouplers dissipate the proton gradient as heat without making ATP.
• Reactive oxygen species: Incomplete electron reduction can form superoxide. Superoxide dismutase, catalase, and glutathione-dependent systems limit oxidative damage.

VII. ß-oxidation of fatty acids — Mitochondrial fatty-acid catabolism

ß-oxidation repeatedly removes two-carbon acetyl-CoA units from the carboxyl end of fatty acyl-CoA, generating NADH and FADH₂.

A. ß-oxidation of fatty acids

Long-chain fatty acids are activated in the cytosol or outer mitochondrial membrane and transported into the matrix through the carnitine shuttle.

• Activation cost: Acyl-CoA synthetase forms fatty acyl-CoA using ATP and pyrophosphate. Because ATP is converted to AMP, activation costs two ATP equivalents.
• Carnitine transport: Carnitine acyltransferase I forms acyl-carnitine, translocase moves it across the inner membrane, and carnitine acyltransferase II regenerates matrix acyl-CoA. Malonyl-CoA inhibits the entry step.
• Four-step cycle: Acyl-CoA dehydrogenase forms a trans-Δ²-enoyl-CoA and FADH₂; enoyl-CoA hydratase adds water; β-hydroxyacyl-CoA dehydrogenase forms NADH; thiolase releases acetyl-CoA.
• Worked yield: Palmitate (C16) undergoes seven cycles, producing 8 acetyl-CoA, 7 NADH, and 7 FADH₂. Using 2.5 ATP/NADH and 1.5 ATP/FADH₂ gives about 108 ATP after subtracting 2 ATP equivalents for activation.
• Regulation: Low insulin and high glucagon promote lipolysis. Fatty-acid oxidation increases during fasting, whereas malonyl-CoA and high NADH suppress mitochondrial oxidation.

VIII. Synthesis of fatty acids — Cytosolic reductive biosynthesis

Fatty-acid synthesis builds fatty acids from acetyl-CoA in the cytosol, mainly when carbohydrate and energy supplies are abundant.

A. Synthesis of fatty acids

The pathway uses acetyl-CoA, malonyl-CoA, NADPH, and the multifunctional fatty-acid synthase complex to produce mainly palmitate.

• Cytosolic acetyl-CoA: Mitochondrial acetyl-CoA exits as citrate. ATP-citrate lyase cleaves citrate in the cytosol to regenerate acetyl-CoA.
• Committed step: Acetyl-CoA carboxylase (ACC) uses ATP and biotin to form malonyl-CoA:

Acetyl-CoA + HCO₃⁻ + ATP → Malonyl-CoA + ADP + Pi

• Chain elongation: Fatty-acid synthase loads acetyl-CoA and malonyl-CoA, then performs condensation, reduction, dehydration, and reduction. Each cycle adds two carbons and consumes two NADPH.
• End product: After seven cycles, palmitoyl-ACP is hydrolyzed to palmitate (C16:0). Further elongation and desaturation occur mainly in the endoplasmic reticulum.
• Regulation: ACC is activated by citrate and insulin-dependent dephosphorylation, but inhibited by palmitoyl-CoA, phosphorylation, and glucagon or epinephrine signaling.
• Pathway contrast: ß-oxidation occurs in mitochondria, uses CoA-linked intermediates, generates NADH/FADH₂, and removes two-carbon units; synthesis occurs in the cytosol, uses ACP-linked intermediates, consumes NADPH, and adds two-carbon units.