Unit 6: Metabolism - Subjective Questions

Bioenergetics is the study of energy changes and energy transformations in living systems.\n\n- Gibbs free energy (): It predicts whether a reaction can occur spontaneously at constant temperature and pressure.\n - If , the reaction is exergonic and releases energy.\n - If , the reaction is endergonic and requires energy.\n - If , the reaction is at equilibrium.\n- Enthalpy (): It represents the heat content or total energy change of a system.\n- Entropy (): It measures the degree of randomness or disorder.\n\nThe relationship among these quantities is . Cells drive unfavorable reactions by coupling them with favorable reactions, especially ATP hydrolysis. ATP serves as an energy currency because its hydrolysis releases usable free energy for biosynthesis, transport, and mechanical work.

ATP, or adenosine triphosphate, consists of adenine, ribose, and three phosphate groups. The terminal phosphoanhydride bonds have a high group-transfer potential.\n\n- ATP hydrolysis occurs as .\n- The reaction has a large negative standard free-energy change.\n- ATP can transfer phosphate groups to substrates, thereby activating them.\n- It couples energy-releasing reactions with energy-requiring processes.\n\nATP supports:\n- Biosynthesis: formation of proteins, nucleic acids, and lipids.\n- Active transport: movement of ions and molecules against concentration gradients.\n- Muscle contraction: interaction of actin and myosin.\n- Phosphorylation: regulation of enzymes and signaling proteins.\n\nATP is continuously regenerated from ADP and inorganic phosphate through substrate-level phosphorylation and oxidative phosphorylation.

Glycolysis is a ten-step pathway that occurs in the cytosol and converts one glucose molecule into two pyruvate molecules. It has two phases.\n\nEnergy-investment phase:\n- Glucose is phosphorylated to glucose-6-phosphate by hexokinase or glucokinase.\n- It is converted to fructose-6-phosphate and then fructose-1,6-bisphosphate.\n- The six-carbon compound splits into two three-carbon molecules.\n\nEnergy-payoff phase:\n- Glyceraldehyde-3-phosphate is oxidized, producing NADH.\n- Substrate-level phosphorylation produces ATP.\n- Phosphoenolpyruvate is converted to pyruvate by pyruvate kinase.\n\nThe overall reaction is:\n\n\nThe net yield per glucose is 2 ATP, 2 NADH, and 2 pyruvate molecules.

Glycolysis is regulated mainly at three irreversible reactions.\n\n- Hexokinase or glucokinase:\n - Hexokinase has a low and is inhibited by glucose-6-phosphate.\n - Glucokinase in the liver has a high , is active when blood glucose is high, and is induced by insulin.\n- Phosphofructokinase-1 (PFK-1): This is the major rate-limiting enzyme.\n - It is inhibited by ATP and citrate.\n - It is activated by AMP, ADP, and fructose-2,6-bisphosphate.\n- Pyruvate kinase:\n - It is activated by fructose-1,6-bisphosphate through feed-forward activation.\n - It is inhibited by ATP and alanine.\n - In the liver, phosphorylation by glucagon-dependent protein kinase reduces its activity.\n\nInsulin generally promotes glycolysis, whereas glucagon suppresses hepatic glycolysis during fasting.

The fate of pyruvate depends on oxygen availability and the metabolic requirements of the cell.\n\n- Aerobic conditions: Pyruvate enters mitochondria and is converted to acetyl-CoA by the pyruvate dehydrogenase complex. Acetyl-CoA enters the TCA cycle.\n- Anaerobic conditions in animal cells: Pyruvate is reduced to lactate by lactate dehydrogenase.\n \n- Alcoholic fermentation in microorganisms: Pyruvate is converted to acetaldehyde and then ethanol, regenerating NAD.\n\nFermentation does not produce additional ATP beyond glycolysis. Its main significance is the regeneration of NAD, which allows glyceraldehyde-3-phosphate dehydrogenase to continue functioning and permits glycolysis to produce a small amount of ATP in the absence of oxygen.

The pyruvate dehydrogenase complex converts pyruvate into acetyl-CoA, linking glycolysis with the TCA cycle. The overall reaction is:\n\n\nThe complex contains three enzymes:\n- E1: Pyruvate dehydrogenase, requiring thiamine pyrophosphate.\n- E2: Dihydrolipoyl transacetylase, requiring lipoamide and CoA.\n- E3: Dihydrolipoyl dehydrogenase, requiring FAD and NAD.\n\nRegulation occurs through covalent modification and product inhibition:\n- Pyruvate dehydrogenase kinase phosphorylates and inactivates E1.\n- Pyruvate dehydrogenase phosphatase dephosphorylates and activates E1.\n- ATP, NADH, and acetyl-CoA inhibit the complex.\n- Pyruvate, ADP, and calcium promote activity, especially in muscle.

The tricarboxylic acid cycle occurs mainly in the mitochondrial matrix. Acetyl-CoA combines with oxaloacetate to form citrate. The sequence is:\n\n1. Citrate is formed from acetyl-CoA and oxaloacetate.\n2. Citrate is converted to isocitrate.\n3. Isocitrate undergoes oxidative decarboxylation to form -ketoglutarate.\n4. -Ketoglutarate is converted to succinyl-CoA.\n5. Succinyl-CoA is converted to succinate with substrate-level phosphorylation.\n6. Succinate is oxidized to fumarate.\n7. Fumarate is hydrated to malate.\n8. Malate is oxidized to oxaloacetate.\n\nFor each acetyl-CoA, the cycle produces:\n- 3 NADH\n- 1 FADH\n- 1 GTP or ATP\n- 2 CO\n\nThe reduced coenzymes transfer electrons to the electron transport chain, while oxaloacetate is regenerated to continue the cycle.

The TCA cycle is regulated according to the cell's energy requirements and availability of substrates.\n\nImportant regulatory enzymes:\n- Citrate synthase: Inhibited by ATP, NADH, succinyl-CoA, and citrate.\n- Isocitrate dehydrogenase: Activated by ADP and calcium; inhibited by ATP and NADH.\n- -Ketoglutarate dehydrogenase: Inhibited by NADH and succinyl-CoA; activated by calcium in muscle.\n\nThe cycle is amphibolic because it participates in both catabolism and anabolism.\n- Catabolic role: oxidizes acetyl-CoA and produces NADH, FADH, and GTP.\n- Biosynthetic role: provides citrate for fatty acid synthesis, -ketoglutarate for amino acid synthesis, succinyl-CoA for porphyrins, and oxaloacetate for aspartate and gluconeogenesis.\n\nWhen intermediates are withdrawn, anaplerotic reactions replenish them. For example, pyruvate carboxylase converts pyruvate to oxaloacetate.

The pentose phosphate pathway, also called the hexose monophosphate shunt, occurs in the cytosol. It has an oxidative phase and a nonoxidative phase.\n\nOxidative phase:\n- Glucose-6-phosphate is oxidized by glucose-6-phosphate dehydrogenase.\n- Ribulose-5-phosphate, NADPH, and carbon dioxide are produced.\n- The pathway produces two molecules of NADPH for each glucose-6-phosphate processed.\n\nNonoxidative phase:\n- Ribulose-5-phosphate is converted into ribose-5-phosphate and other pentose phosphates.\n- Transketolase and transaldolase rearrange carbon skeletons.\n- Products can be converted into fructose-6-phosphate and glyceraldehyde-3-phosphate, which enter glycolysis.\n\nThe pathway is particularly active in liver, adipose tissue, adrenal cortex, lactating mammary glands, testes, ovaries, and red blood cells.

The pentose phosphate pathway has two major functions.\n\n- NADPH production: NADPH provides reducing power for fatty acid and cholesterol synthesis. It also maintains glutathione in its reduced form, protecting cells from oxidative damage.\n- Ribose-5-phosphate production: Ribose-5-phosphate is required for synthesis of nucleotides and nucleic acids.\n\nIn red blood cells, NADPH is essential for maintaining reduced glutathione because these cells lack mitochondria.\n\nGlucose-6-phosphate dehydrogenase deficiency:\n- Reduces NADPH production.\n- Limits regeneration of reduced glutathione.\n- Makes red blood cells vulnerable to oxidative stress.\n- May cause hemolytic anemia after exposure to infections, certain drugs, or fava beans.\n\nThe disorder is often associated with Heinz bodies and bite cells in affected erythrocytes.

The electron transport chain is located in the inner mitochondrial membrane. It transfers electrons from NADH and FADH to oxygen and uses the released energy to pump protons.\n\n- Complex I: NADH dehydrogenase transfers electrons from NADH to ubiquinone and pumps protons.\n- Complex II: Succinate dehydrogenase transfers electrons from FADH to ubiquinone but does not pump protons.\n- Coenzyme Q: Transfers electrons from complexes I and II to complex III.\n- Complex III: Transfers electrons to cytochrome and pumps protons.\n- Cytochrome : Carries electrons to complex IV.\n- Complex IV: Transfers electrons to oxygen, forming water, and pumps protons.\n\nThe resulting proton gradient across the inner membrane creates a proton-motive force. This electrochemical gradient drives ATP synthesis by ATP synthase.

Oxidative phosphorylation is the synthesis of ATP using energy released during the oxidation of NADH and FADH. It occurs through the chemiosmotic mechanism.\n\n- Electron transport through complexes I, III, and IV pumps protons from the mitochondrial matrix into the intermembrane space.\n- This produces both a proton concentration gradient and a membrane potential.\n- Together, these form the proton-motive force.\n- Protons flow back into the matrix through the portion of ATP synthase.\n- Proton movement causes rotation and conformational changes in the portion, allowing ADP and inorganic phosphate to form ATP.\n\nThe approximate ATP yield is 2.5 ATP per NADH and 1.5 ATP per FADH, although the exact yield depends on shuttle systems and cellular conditions. Oxygen is the final electron acceptor and is reduced to water.

Inhibitors block electron transfer or ATP synthesis at specific sites.\n\n- Rotenone inhibits Complex I.\n- Antimycin A inhibits Complex III.\n- Cyanide, carbon monoxide, and azide inhibit Complex IV.\n- Oligomycin blocks the proton channel of ATP synthase.\n\nUncouplers disrupt the proton gradient without directly stopping electron transport. They allow protons to return to the matrix without producing ATP.\n\n- 2,4-Dinitrophenol is a chemical uncoupler.\n- Thermogenin, or uncoupling protein 1, is present in brown adipose tissue and produces heat.\n\nInhibition decreases both oxygen consumption and ATP production. Uncoupling increases oxygen consumption but decreases ATP synthesis, with the energy released as heat.

-oxidation is the mitochondrial pathway in which fatty acids are degraded into acetyl-CoA units.\n\nSteps in each cycle:\n1. Oxidation: Acyl-CoA dehydrogenase forms a double bond and produces FADH.\n2. Hydration: Enoyl-CoA hydratase adds water.\n3. Oxidation: -Hydroxyacyl-CoA dehydrogenase produces NADH.\n4. Thiolysis: Thiolase releases acetyl-CoA.\n\nPalmitoyl-CoA contains 16 carbon atoms. It undergoes 7 cycles and produces:\n- 8 acetyl-CoA\n- 7 NADH\n- 7 FADH\n\nUsing modern ATP values, the approximate net yield from palmitate is 106 ATP, after subtracting the 2 ATP equivalents required for activation of the fatty acid. The acetyl-CoA molecules are oxidized further in the TCA cycle.

Long-chain fatty acyl-CoA molecules cannot cross the inner mitochondrial membrane directly. They use the carnitine shuttle.\n\n- CPT-I: Located on the outer mitochondrial membrane; transfers the acyl group from CoA to carnitine.\n- Translocase: Transports acyl-carnitine into the matrix while moving free carnitine outward.\n- CPT-II: Transfers the acyl group back to CoA inside the matrix.\n\nThe rate-limiting step is controlled by CPT-I. Malonyl-CoA inhibits CPT-I, preventing simultaneous fatty acid synthesis and degradation.\n\n-oxidation is also regulated by:\n- The availability of fatty acids and CoA.\n- The NADH-to-NAD and FADH-to-FAD ratios.\n- The energy state of the cell.\n\nHigh ATP and NADH inhibit fatty acid oxidation, whereas increased energy demand promotes it.

Odd-chain fatty acids undergo repeated cycles of -oxidation, producing acetyl-CoA until a three-carbon propionyl-CoA molecule remains. Propionyl-CoA is converted to succinyl-CoA through three reactions:\n\n1. Propionyl-CoA carboxylase converts propionyl-CoA to methylmalonyl-CoA. This reaction requires biotin and ATP.\n2. Methylmalonyl-CoA epimerase converts the stereoisomer.\n3. Methylmalonyl-CoA mutase converts it to succinyl-CoA. This enzyme requires vitamin B.\n\nSuccinyl-CoA enters the TCA cycle and can contribute to gluconeogenesis. Therefore, odd-chain fatty acids are partly glucogenic, unlike even-chain fatty acids, whose acetyl-CoA cannot produce a net synthesis of glucose. Vitamin B deficiency can impair this pathway and cause methylmalonic acid accumulation.

Fatty acid synthesis occurs mainly in the cytosol, especially in liver and adipose tissue. Acetyl-CoA is transported from mitochondria as citrate.\n\n- Citrate is cleaved in the cytosol to release acetyl-CoA.\n- Acetyl-CoA carboxylase converts acetyl-CoA to malonyl-CoA using ATP and biotin. This is the committed and rate-limiting step.\n- Fatty acid synthase uses one acetyl-CoA primer and repeated malonyl-CoA units.\n- Each cycle includes condensation, reduction, dehydration, and reduction. NADPH supplies reducing equivalents.\n- After seven cycles, palmitoyl-ACP is hydrolyzed to palmitate.\n\nThe overall process requires acetyl-CoA, ATP, and NADPH. The NADPH is supplied mainly by the pentose phosphate pathway and the malic enzyme reaction.

Fatty acid synthesis and oxidation are reciprocally regulated to prevent futile cycling.\n\nRegulation of synthesis:\n- Acetyl-CoA carboxylase is activated by citrate and inhibited by palmitoyl-CoA.\n- Insulin promotes dephosphorylation and activation of acetyl-CoA carboxylase.\n- Glucagon and epinephrine promote phosphorylation and inhibition.\n- Malonyl-CoA inhibits CPT-I, thereby suppressing fatty acid entry into mitochondria.\n\nRegulation of oxidation:\n- CPT-I is inhibited by malonyl-CoA.\n- Glucagon and epinephrine stimulate lipolysis and fatty acid oxidation.\n- Insulin suppresses lipolysis and promotes storage.\n\nThus, the fed state favors fatty acid synthesis, whereas fasting and exercise favor fatty acid mobilization and -oxidation.

| Pathway | Main location | Major function | Important products |\n|---|---|---|---|\n| Glycolysis | Cytosol | Glucose breakdown | Pyruvate, ATP, NADH |\n| TCA cycle | Mitochondrial matrix | Oxidation of acetyl-CoA | NADH, FADH, GTP, CO |\n| Pentose phosphate pathway | Cytosol | NADPH and pentose production | NADPH, ribose-5-phosphate |\n| -oxidation | Mitochondrial matrix | Fatty acid degradation | Acetyl-CoA, NADH, FADH |\n| Fatty acid synthesis | Cytosol | Fatty acid production | Palmitate, using NADPH |\n\nGlycolysis and -oxidation are mainly catabolic, whereas fatty acid synthesis is anabolic. The TCA cycle is amphibolic, and the pentose phosphate pathway supplies both reducing power and biosynthetic intermediates.

During fasting, insulin levels decrease while glucagon and epinephrine increase. These hormonal changes shift metabolism from storage to mobilization.\n\n- Glycogenolysis: Liver glycogen is broken down to maintain blood glucose during the early fasting period.\n- Gluconeogenesis: The liver produces glucose from lactate, glycerol, and glucogenic amino acids when glycogen stores decline.\n- Lipolysis: Adipose tissue releases fatty acids and glycerol.\n- Fatty acid oxidation: The liver and muscles oxidize fatty acids to produce ATP.\n- Ketogenesis: Excess acetyl-CoA in the liver is converted into acetoacetate, -hydroxybutyrate, and acetone.\n\nKetone bodies serve as an alternative fuel for muscle and, during prolonged fasting, the brain. This reduces the need for amino acid breakdown and helps preserve muscle protein.