13.6 Bioenergetics, the Citric Acid Cycle, Oxidative Phosphorylation, Muscle Energetics & Nucleotide Metabolism

Why this area matters

The TOS area labeled "musculoskeletal system" lists:

• "bioenergetics and thermodynamics, role of ATP, biologic oxidation, electron transport chain, oxidative phosphorylation";
• "muscle energetics (glycolysis, Krebs cycle, creatine phosphate)";
• "nucleic acid chemistry, diseases associated with purine and pyrimidine nucleotide metabolism".

These connect to cyanide and carbon monoxide poisoning, aspirin overdose, mitochondrial myopathy, rhabdomyolysis, gout and cancer chemotherapy.

Thermodynamic Principles and High-Energy Phosphate Energetics

Chemical reactions within biological systems obey the laws of thermodynamics. The standard free energy change ($\Delta G^{\circ\prime}$) denotes the net energy liberated or consumed under standard physiological conditions ($25^\circ\text{C}$, $1\text{ atm}$, $\text{pH }7.0$, and $1\text{ M}$ reactant concentrations):

$\Delta G = \Delta G^{\circ\prime} + RT \ln \left( \frac{[\text{Products}]}{[\text{Reactants}]} \right)$

• Exergonic Reactions ($\Delta G < 0$): Proceed spontaneously with the net release of usable free energy.
• Endergonic Reactions ($\Delta G > 0$): Thermodynamically non-spontaneous and require energetic coupling to an exergonic driver.
• Equilibrium ($\Delta G = 0$): Forward and reverse reaction rates are equal, precluding net work.

Hydrolysis of the terminal phosphoanhydride bond of ATP to adenosine diphosphate (ADP) and inorganic phosphate ($P_i$) yields a standard free energy change of approximately $-30.5\text{ kJ/mol}$ ($-7.3\text{ kcal/mol}$):

$\text{ATP} + \text{H}_2\text{O} \longrightarrow \text{ADP} + P_i \quad (\Delta G^{\circ\prime} \approx -30.5\text{ kJ/mol})$

Certain intermediate metabolites carry even higher phosphate transfer potentials than ATP, permitting substrate-level phosphorylation. These include phosphoenolpyruvate (PEP, $-61.9\text{ kJ/mol}$), 1,3-bisphosphoglycerate (1,3-BPG, $-49.4\text{ kJ/mol}$), and creatine phosphate ($-43.1\text{ kJ/mol}$). Phosphorylation of ADP by PEP or 1,3-BPG directly yields ATP without requiring mitochondrial oxygen consumption.

The Citric Acid Cycle (Krebs Cycle): Stoichiometry and Regulatory Checkpoints

The Citric Acid Cycle occurs within the mitochondrial matrix, acting as the final common oxidative pathway for carbohydrates, lipids, and amino acids. Acetyl-CoA ($2\text{C}$) condenses with oxaloacetate ($4\text{C}$) to form citrate ($6\text{C}$), catalyzed by citrate synthase.

High-Yield Krebs Cycle Reactions

Stoichiometric Yield of the Citric Acid Cycle

Each turn of the Citric Acid Cycle per molecule of acetyl-CoA produces:

• $3\text{ NADH}$ ($3 \times 2.5 = 7.5\text{ ATP}$ equivalents)
• $1\text{ }FADH_2$ ($1 \times 1.5 = 1.5\text{ ATP}$ equivalents)
• $1\text{ GTP}$ ($= 1.0\text{ ATP}$ equivalent)
• $2\text{ CO}_2$

Total yield: $10\text{ ATP}$ equivalents per acetyl-CoA ($20\text{ ATP}$ per original molecule of glucose).

Accounting for aerobic glycolysis ($2\text{ ATP} + 2\text{ NADH}$) and the PDH reaction ($2\text{ NADH}$), the complete oxidation of one glucose molecule yields 30 to 32 net ATP:

• $32\text{ ATP}$ via the malate-aspartate shuttle (predominant in liver, heart, and kidneys).
• $30\text{ ATP}$ via the glycerol-3-phosphate shuttle (predominant in skeletal muscle and brain, transferring electrons to $FAD$ as $FADH_2$).

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The Electron Transport Chain and Oxidative Phosphorylation

Oxidative phosphorylation couples the transfer of electrons from NADH and $FADH_2$ to molecular oxygen with the generation of ATP via a transmembrane proton gradient across the inner mitochondrial membrane (Peter Mitchell's chemiosmotic hypothesis).

• Complex I (NADH-Ubiquinone Oxidoreductase): Translocates $4\text{ H}^+$ per electron pair from matrix to intermembrane space.
• Complex II (Succinate Dehydrogenase): Oxidizes $FADH_2$ to transfer electrons to ubiquinone (Coenzyme Q). Translocates $0\text{ H}^+$.
• Complex III (Ubiquinol-Cytochrome c Oxidoreductase / Cytochrome $bc_1$): Q-cycle translocates $4\text{ H}^+$ per pair.
• Complex IV (Cytochrome c Oxidase): Contains heme $a/a_3$ and copper centers ($Cu_A/Cu_B$). Reduces $O_2$ to $2\text{ H}_2\text{O}$, translocating $2\text{ H}^+$.
• Complex V ($F_oF_1$-ATP Synthase): Protons re-enter matrix through the $F_o$ stalk, rotating the $\gamma$-subunit and driving the condensation of ADP and $P_i$ into ATP in the $F_1$ head.

Electron Transport Chain Inhibitors versus Uncouplers

Mitochondrial Beta-Oxidation and the Carnitine Shuttle

During fasting and exercise, triacylglycerols stored in adipose tissue are hydrolyzed by adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) (activated by glucagon/epinephrine via PKA-mediated phosphorylation; inhibited by insulin). Free fatty acids circulate bound to albumin and enter peripheral tissues for $\beta$-oxidation within the mitochondrial matrix.

Activation and the Carnitine Shuttle

In the outer mitochondrial membrane, fatty acids are activated to fatty acyl-CoA by fatty acyl-CoA synthetase (thiokinase), consuming two high-energy phosphate equivalents ($\text{ATP} \to \text{AMP} + PP_i$). Fatty acyl-CoA cannot cross the inner mitochondrial membrane and relies on the Carnitine Shuttle (Mnemonic: "CARnitine = CARnage of fatty acids in mitochondria"):

1. Carnitine Palmitoyltransferase-1 (CPT-1): Located on the outer mitochondrial membrane; transfers the acyl group from CoA to carnitine, forming acylcarnitine.
   • Crucial Allosteric Control: CPT-1 is potently inhibited by malonyl-CoA, preventing newly synthesized fatty acids from immediate futile oxidation.
2. Carnitine-Acylcarnitine Translocase: Shuttles acylcarnitine across the inner membrane while returning free carnitine to the intermembrane space.
3. Carnitine Palmitoyltransferase-2 (CPT-2): Located on the inner mitochondrial membrane; regenerates fatty acyl-CoA and free carnitine within the matrix.

The Four-Step $\beta$-Oxidation Spiral

Each turn of the mitochondrial $\beta$-oxidation spiral shortens the acyl chain by two carbons, releasing:

• $1\text{ FADH}_2$ (via Acyl-CoA Dehydrogenase; feeds into Complex II / ubiquinone)
• $1\text{ NADH}$ (via 3-Hydroxyacyl-CoA Dehydrogenase; feeds into Complex I)
• $1\text{ Acetyl-CoA}$ (via $\beta$-Ketothiolase; feeds into the Krebs cycle)
• A shortened fatty acyl-CoA ($n-2$ carbons)

Medium-Chain Acyl-CoA Dehydrogenase (MCAD) Deficiency

MCAD deficiency is the most common inborn error of $\beta$-oxidation (autosomal recessive). Affected infants cannot oxidize medium-chain (C6–C10) fatty acids. During prolonged fasting or intercurrent viral infection, glucose stores are depleted, but hepatic $\beta$-oxidation fails to supply energy or acetyl-CoA for ketogenesis. Manifestations include:

• Severe Hypoketotic Hypoglycemia: Profound hypoglycemia without detectable urinary or plasma ketone bodies.
• Accumulation of C6–C10 acylcarnitines (octanoylcarnitine) in blood, with dicarboxylic aciduria (from compensatory $\omega$-oxidation in peroxisomes and ER).
• Lethargy, seizures, hepatomegaly, hyperammonemia, and sudden death. Treatment involves avoidance of fasting and high-carbohydrate nutrition.

Muscle energetics

• Myoglobin stores oxygen in muscle. It has a hyperbolic dissociation curve and higher affinity than hemoglobin.
• Rhabdomyolysis (crush injury, extreme exertion, statins, heat stroke) releases myoglobin and CK, which can cause acute kidney injury and hyperkalemia.
• Mitochondrial myopathies cause exercise intolerance and lactic acidosis. McArdle disease (no muscle glycogen phosphorylase) and CPT II deficiency cause exercise-induced myoglobinuria.
• Muscle lacks glucose-6-phosphatase, so its glycogen fuels only the muscle itself.

Nucleic acid chemistry

• Purines (two rings): adenine, guanine. Pyrimidines (one ring): cytosine, thymine (DNA), uracil (RNA).
• A nucleoside is a base plus a sugar; a nucleotide is a nucleoside plus phosphate.
• DNA is a double helix with A pairing with T (2 hydrogen bonds) and G pairing with C (3 hydrogen bonds). GC-rich DNA melts at a higher temperature.
• RNA has ribose and uracil and is usually single-stranded. The 2'-OH makes RNA less stable than DNA.
• Phosphodiester bonds link the 3'-OH of one sugar to the 5'-phosphate of the next, and strands run antiparallel.

Purine and pyrimidine metabolism

De novo purine synthesis:

• The ring is built on PRPP, with atoms from glycine, aspartate, glutamine, CO2 and folate (N10-formyl-THF).
• The rate-limiting enzyme is glutamine-PRPP amidotransferase. The first purine made is IMP, which becomes AMP and GMP.
• Salvage reuses bases through HGPRT (hypoxanthine and guanine) and APRT (adenine).

Pyrimidine synthesis:

• The ring is made first: carbamoyl phosphate synthetase II in the cytosol, then aspartate, giving orotic acid.
• It is then attached to PRPP, forming UMP through UMP synthase.
• Thymidylate synthase (which needs N5,N10-methylene-THF) converts dUMP to dTMP.
• Ribonucleotide reductase forms deoxyribonucleotides.

Purine degradation runs from adenosine (by adenosine deaminase) to inosine, then hypoxanthine, then xanthine, and finally uric acid by xanthine oxidase.

Drugs acting on nucleotide metabolism:

• Methotrexate inhibits dihydrofolate reductase.
• 5-fluorouracil inhibits thymidylate synthase.
• Hydroxyurea inhibits ribonucleotide reductase.
• 6-mercaptopurine and azathioprine are purine analogs. Their toxicity rises with allopurinol, because xanthine oxidase helps degrade them.
• Allopurinol and febuxostat inhibit xanthine oxidase.
• Mycophenolate inhibits IMP dehydrogenase in lymphocytes.
• Leflunomide inhibits dihydroorotate dehydrogenase.
• Trimethoprim and pyrimethamine inhibit microbial dihydrofolate reductase.