Unit 4: Metabolism I - Subjective Questions

ATP (Adenosine Triphosphate) is a nucleotide that serves as the universal energy carrier in living cells.

Structure:

• Adenine (nitrogenous base)
• Ribose (5-carbon sugar)
• Three phosphate groups (alpha, beta, gamma) linked in series

Why it is the energy currency:

• The bonds between the phosphate groups are high-energy phosphoanhydride bonds.
• Hydrolysis of the terminal phosphate releases a large amount of free energy:
  
• This energy is coupled to drive endergonic reactions (biosynthesis, transport, movement).
• ATP is continuously regenerated from ADP, making it a renewable intermediate.

Reasons for high energy release:

• Electrostatic repulsion between negatively charged phosphates is relieved.
• Resonance stabilization of products (ADP and ).
• Greater solvation of products.

Thus ATP acts as an intermediate that links energy-releasing (catabolic) and energy-requiring (anabolic) reactions.

Electron carriers are coenzymes that transfer electrons (and hydrogen) between metabolic reactions.

NAD⁺/NADH (Nicotinamide Adenine Dinucleotide):

• Derived from niacin (vitamin B3).
• Accepts 2 electrons and 1 proton (a hydride ion, ):
  
• Mainly involved in catabolic reactions, delivering electrons to the electron transport chain.
• NADH feeds electrons at Complex I, yielding ~2.5 ATP.

FAD/FADH₂ (Flavin Adenine Dinucleotide):

• Derived from riboflavin (vitamin B2).
• Accepts 2 electrons and 2 protons:
  
• Often tightly/covalently bound to enzymes (e.g., succinate dehydrogenase).
• Feeds electrons at Complex II, yielding ~1.5 ATP.

Key differences:

• NAD⁺ is a soluble, freely diffusible carrier; FAD is usually enzyme-bound.
• NADPH (phosphorylated form) is used mainly in anabolic/reductive biosynthesis, whereas NADH is used in energy production.
• FADH₂ yields less ATP than NADH because it enters the chain at a lower energy level.

The preparatory phase (steps 1–5) of glycolysis invests energy to convert glucose into two molecules of glyceraldehyde-3-phosphate.

Steps:

1. Glucose → Glucose-6-phosphate
   • Enzyme: Hexokinase
   • Consumes 1 ATP; irreversible/regulatory step.
2. Glucose-6-phosphate → Fructose-6-phosphate
   • Enzyme: Phosphoglucose isomerase
   • Isomerization (aldose → ketose).
3. Fructose-6-phosphate → Fructose-1,6-bisphosphate
   • Enzyme: Phosphofructokinase-1 (PFK-1)
   • Consumes 1 ATP; committed regulatory step.
4. Fructose-1,6-bisphosphate → DHAP + Glyceraldehyde-3-phosphate
   • Enzyme: Aldolase
5. DHAP ⇌ Glyceraldehyde-3-phosphate
   • Enzyme: Triose phosphate isomerase

Net ATP used: 2 ATP consumed.

Outcome: One glucose molecule yields two G3P molecules that proceed to the payoff phase.

The payoff phase (steps 6–10) harvests energy from the two G3P molecules.

Steps (per G3P, ×2):

6. G3P → 1,3-Bisphosphoglycerate
   • Enzyme: Glyceraldehyde-3-phosphate dehydrogenase
   • Produces NADH; adds inorganic phosphate.
7. 1,3-BPG → 3-Phosphoglycerate
   • Enzyme: Phosphoglycerate kinase
   • Substrate-level phosphorylation → produces ATP.
8. 3-PG → 2-Phosphoglycerate
   • Enzyme: Phosphoglycerate mutase
9. 2-PG → Phosphoenolpyruvate (PEP)
   • Enzyme: Enolase (removes water).
10. PEP → Pyruvate
    • Enzyme: Pyruvate kinase
    • Substrate-level phosphorylation → produces ATP.

Yield (per glucose = 2 G3P):

• ATP produced: 4 (payoff) − 2 (invested) = Net 2 ATP
• NADH produced: 2 NADH
• Pyruvate: 2 molecules

Overall reaction:

Fermentation is the anaerobic process by which cells regenerate NAD⁺ from NADH so that glycolysis can continue in the absence of oxygen. No additional ATP is generated during fermentation itself.

Lactic Acid Fermentation:

• Product: Lactate
• Reaction:
  
• Enzyme: Lactate dehydrogenase
• Organisms/tissues: Muscle cells (during vigorous exercise), lactic acid bacteria.

Alcoholic Fermentation:

• Products: Ethanol +
• Reactions:
  
  
• Organisms: Yeast, some bacteria.

Comparison table:

Common purpose: Both regenerate NAD⁺ to sustain glycolytic ATP production anaerobically.

Gluconeogenesis is the synthesis of glucose from non-carbohydrate precursors such as lactate, pyruvate, glycerol, and glucogenic amino acids. It occurs mainly in the liver (and kidney).

Why glycolysis cannot be simply reversed:

• Three glycolytic reactions are highly exergonic and irreversible (catalyzed by hexokinase, PFK-1, and pyruvate kinase).
• These must be bypassed by different enzymes.

The four bypass reactions:

1. Pyruvate → Oxaloacetate → PEP
   • Enzymes: Pyruvate carboxylase (mitochondria) + PEP carboxykinase (PEPCK)
   • Consumes 1 ATP + 1 GTP.
2. Fructose-1,6-bisphosphate → Fructose-6-phosphate
   • Enzyme: Fructose-1,6-bisphosphatase
3. Glucose-6-phosphate → Glucose
   • Enzyme: Glucose-6-phosphatase (in ER of liver).

Energy cost: Synthesis of one glucose requires 6 high-energy phosphates (4 ATP + 2 GTP) plus 2 NADH.

Overall:

Glycolysis and gluconeogenesis are opposing pathways that share several reversible steps but differ fundamentally.

Reciprocal regulation: The two pathways are regulated so that they do not operate simultaneously at high rates (a futile cycle). For example, fructose-2,6-bisphosphate activates PFK-1 (glycolysis) and inhibits F1,6-BPase (gluconeogenesis).

Glycolysis is regulated at its three irreversible steps.

1. Hexokinase / Glucokinase:

• Hexokinase is inhibited by its product glucose-6-phosphate.
• Glucokinase (liver isoform) has low affinity, not inhibited by G6P — active when glucose is high.

2. Phosphofructokinase-1 (PFK-1) — the main control point:

• Activators: AMP, ADP, fructose-2,6-bisphosphate (F2,6BP).
• Inhibitors: ATP, citrate, low pH.

3. Pyruvate kinase:

• Activator: Fructose-1,6-bisphosphate (feed-forward).
• Inhibitors: ATP, alanine; regulated by phosphorylation (glucagon inactivates it in liver).

Role of Fructose-2,6-bisphosphate:

• Most potent allosteric activator of PFK-1 and inhibitor of fructose-1,6-bisphosphatase.
• Synthesized by PFK-2 and degraded by FBPase-2 (bifunctional enzyme).
• Insulin raises F2,6BP (promotes glycolysis); glucagon lowers it (promotes gluconeogenesis) via cAMP-dependent phosphorylation.

This reciprocal control prevents wasteful futile cycling.

The pentose phosphate pathway (PPP), also called the hexose monophosphate shunt, runs in the cytosol and has two phases. The oxidative phase is irreversible.

Oxidative phase reactions:

1. Glucose-6-phosphate → 6-phosphogluconolactone
   • Enzyme: Glucose-6-phosphate dehydrogenase (G6PD) — rate-limiting.
   • Produces NADPH.
2. 6-phosphogluconolactone → 6-phosphogluconate
   • Enzyme: Lactonase
3. 6-phosphogluconate → Ribulose-5-phosphate + CO₂
   • Enzyme: 6-phosphogluconate dehydrogenase
   • Produces a second NADPH.

Net oxidative phase:

Significance of products:

• NADPH: Provides reducing power for fatty acid, cholesterol and steroid biosynthesis, and maintains reduced glutathione to combat oxidative stress.
• Ribose-5-phosphate: Precursor for nucleotide and nucleic acid synthesis.

Clinical note: G6PD deficiency reduces NADPH, causing hemolytic anemia due to oxidative damage in RBCs.

The non-oxidative phase of the PPP is reversible and allows interconversion of sugars with 3, 4, 5, 6, and 7 carbons. It links the PPP to glycolysis.

Key reactions:

1. Ribulose-5-phosphate is converted to:
   • Ribose-5-phosphate (by phosphopentose isomerase)
   • Xylulose-5-phosphate (by phosphopentose epimerase)
2. Transketolase (requires thiamine pyrophosphate, TPP):
   • Transfers 2-carbon units.
     
3. Transaldolase:
   • Transfers 3-carbon units.
     
4. Transketolase again:
   

Significance:

• Converts excess pentoses into glycolytic intermediates (F6P, G3P).
• Allows the cell to balance its needs for NADPH vs. ribose-5-phosphate.
• Provides flexibility depending on metabolic demand.

Glycogenolysis is the breakdown of glycogen to release glucose (as glucose-1-phosphate/glucose).

Steps:

1. Glycogen phosphorylase:
   • Cleaves α-1,4 glycosidic bonds by phosphorolysis, releasing glucose-1-phosphate.
     
   • Stops 4 residues from a branch point (limit dextrin).
2. Debranching enzyme (bifunctional):
   • Transferase activity: moves 3 glucose units to a nearby chain.
   • α-1,6-glucosidase activity: hydrolyzes the branch-point residue, releasing free glucose.
3. Phosphoglucomutase:
   • Converts glucose-1-phosphate → glucose-6-phosphate.
4. Glucose-6-phosphatase (liver only):
   • Converts G6P → free glucose for release into blood.

Tissue differences:

• Liver: Releases free glucose to maintain blood glucose.
• Muscle: Lacks glucose-6-phosphatase; G6P enters glycolysis for energy.

Regulation: Phosphorylase is activated by glucagon/epinephrine (via cAMP-dependent phosphorylation) and AMP; inhibited by insulin, ATP, and G6P.

Glycogenesis is the synthesis of glycogen from glucose, occurring mainly in liver and muscle.

Steps:

1. Glucose → Glucose-6-phosphate
   • Enzyme: Hexokinase/Glucokinase.
2. Glucose-6-phosphate → Glucose-1-phosphate
   • Enzyme: Phosphoglucomutase.
3. Glucose-1-phosphate → UDP-glucose
   • Enzyme: UDP-glucose pyrophosphorylase
     
   • Hydrolysis of makes the step irreversible (activated glucose donor).
4. Chain elongation:
   • Enzyme: Glycogen synthase — the regulatory enzyme.
   • Adds glucose units via α-1,4 glycosidic bonds.
   • Requires a primer (glycogenin) to start a new chain.
5. Branching:
   • Enzyme: Branching enzyme (amylo-α-1,4→1,6-transglycosylase)
   • Transfers ~6–7 glucose residues to form α-1,6 branches, increasing solubility and the number of ends for rapid synthesis/degradation.

Regulation: Glycogen synthase is activated by insulin (dephosphorylated active form) and inhibited by glucagon/epinephrine (phosphorylated). This is reciprocal to glycogen phosphorylase.

Substrate-level phosphorylation (SLP) is the direct formation of ATP (or GTP) by transferring a phosphate group from a high-energy substrate to ADP, catalyzed by a specific enzyme. It does not require the electron transport chain or oxygen.

Characteristics:

• Occurs in the cytosol (glycolysis) and mitochondria (TCA cycle).
• Involves a phosphorylated intermediate with higher phosphate-transfer potential than ATP.

Examples from glycolysis:

1. 1,3-Bisphosphoglycerate → 3-Phosphoglycerate
   • Enzyme: Phosphoglycerate kinase
     
2. Phosphoenolpyruvate → Pyruvate
   • Enzyme: Pyruvate kinase
     

Significance: SLP is the only means of ATP production during anaerobic conditions, making it essential for tissues like RBCs and exercising muscle.

Pyruvate, the end product of glycolysis, has several fates depending on oxygen availability and cell type.

Under Aerobic Conditions:

• Pyruvate enters the mitochondria.
• Converted to Acetyl-CoA by the pyruvate dehydrogenase complex (PDH):
  
• Acetyl-CoA enters the TCA cycle for complete oxidation, yielding large amounts of ATP.

Under Anaerobic Conditions:

• In muscle/bacteria: Pyruvate → Lactate (lactate dehydrogenase), regenerating NAD⁺.
• In yeast: Pyruvate → Ethanol + CO₂, also regenerating NAD⁺.

Other fates:

• Gluconeogenesis: Pyruvate → Oxaloacetate → Glucose.
• Amino acid synthesis: Pyruvate → Alanine by transamination.

Summary: Aerobic conditions favor complete oxidation for maximal energy; anaerobic conditions favor fermentation to sustain glycolysis by regenerating NAD⁺.

The Cori cycle (lactic acid cycle) is a metabolic pathway that recycles lactate produced by anaerobic glycolysis in muscle back into glucose in the liver.

Steps:

1. In active muscle: Glucose undergoes anaerobic glycolysis producing lactate and 2 ATP.
   
2. Lactate transport: Lactate diffuses into the blood and is carried to the liver.
3. In the liver: Lactate is oxidized to pyruvate and converted to glucose via gluconeogenesis (requires 6 ATP).
   
4. Glucose return: Newly formed glucose returns to muscle via blood.

Physiological importance:

• Prevents lactate accumulation and lactic acidosis.
• Shifts the metabolic energy burden from muscle to liver.
• Allows continued muscle activity under low oxygen.
• Energetically costly: Net 4 ATP are consumed per cycle (the liver spends more ATP than the muscle gains), but it maintains glucose supply.

Glycogen synthesis (glycogenesis) and breakdown (glycogenolysis) are controlled reciprocally to avoid futile cycling. Regulation occurs by allosteric effectors and covalent modification (phosphorylation) driven by hormones.

Key enzymes:

• Glycogen synthase (synthesis)
• Glycogen phosphorylase (breakdown)

Hormonal control:

• Glucagon / Epinephrine (fasting, stress):
  • Activate adenylate cyclase → cAMP → Protein Kinase A (PKA).
  • PKA phosphorylates both enzymes.
  • Phosphorylase becomes active (breakdown ON).
  • Glycogen synthase becomes inactive (synthesis OFF).
• Insulin (fed state):
  • Activates protein phosphatase-1 (PP1).
  • Dephosphorylates both enzymes.
  • Synthase active (synthesis ON); Phosphorylase inactive (breakdown OFF).

Allosteric effectors:

• Muscle phosphorylase activated by AMP, inhibited by ATP/G6P.
• Synthase activated by G6P.

Net effect: Phosphorylation activates degradation and inhibits synthesis; dephosphorylation does the opposite — ensuring coordinated, non-wasteful glycogen metabolism.

Glycogen storage diseases (GSDs) are inherited disorders caused by deficiency of enzymes involved in glycogen synthesis or breakdown, leading to abnormal quantity or structure of glycogen.

Von Gierke's Disease (Type I):

• Deficient enzyme: Glucose-6-phosphatase
• Affected organ: Liver, kidney
• Features:
  • Severe fasting hypoglycemia (cannot release free glucose).
  • Hepatomegaly (enlarged liver from glycogen accumulation).
  • Lactic acidosis, hyperlipidemia, hyperuricemia.

McArdle's Disease (Type V):

• Deficient enzyme: Muscle glycogen phosphorylase (myophosphorylase)
• Affected organ: Skeletal muscle
• Features:
  • Exercise intolerance, muscle cramps, fatigue.
  • Poor lactate rise during exercise.
  • Possible myoglobinuria after strenuous activity.

Key contrast: Von Gierke's affects liver glucose release (systemic hypoglycemia), whereas McArdle's affects muscle energy supply (exercise problems).

Glucose-6-phosphate (G6P) is a central metabolic branch point, but its fate differs between liver and muscle due to enzyme differences and physiological function.

Liver:

• Contains glucose-6-phosphatase.
• Can convert G6P → free glucose for export into blood.
• Role: Maintains blood glucose homeostasis for the whole body (especially brain and RBCs during fasting).
• Uses glucokinase (high ) — active only when glucose is abundant.

Muscle:

• Lacks glucose-6-phosphatase.
• Cannot release free glucose; G6P is trapped and used internally.
• G6P enters glycolysis for ATP production during contraction.
• Uses hexokinase (low , inhibited by G6P).

Metabolic significance:

• Liver is an altruistic organ — supplies glucose to others.
• Muscle is selfish — retains glucose for its own energy needs.

This division of labor ensures both systemic glucose supply and adequate muscle energy.

Anaerobic breakdown consists of glycolysis followed by fermentation.

Energy accounting for glycolysis:

• ATP invested (preparatory phase): −2 ATP
• ATP produced (payoff phase): +4 ATP
• Net ATP = +2 ATP
• NADH produced: 2 NADH

Fermentation:

• The 2 NADH are reoxidized to NAD⁺ (converting pyruvate to lactate/ethanol).
• No additional ATP is produced; the NADH energy is essentially discarded.

Net anaerobic yield:

Comparison with aerobic respiration:

• Aerobic oxidation yields ~30–32 ATP per glucose.
• Reasons anaerobic yield is low:
  • Glucose is only partially oxidized (to lactate/ethanol), not fully to and .
  • The electron transport chain and oxidative phosphorylation are not used.
  • NADH is spent regenerating NAD⁺ rather than driving ATP synthesis.

Conclusion: Anaerobic metabolism is rapid but inefficient; it is used only when oxygen is limited.

Although structurally similar, NADH and NADPH serve distinct metabolic purposes.

Structural difference:

• Both are derived from niacin.
• NADPH has an extra phosphate group on the 2′-hydroxyl of the adenosine ribose; NADH lacks it.

Functional / role differences:

Cellular significance:

• The extra phosphate acts as a recognition tag, allowing enzymes to distinguish the two pools.
• Cells keep a high NAD⁺/NADH ratio (favoring oxidation) and a high NADPH/NADP⁺ ratio (favoring reduction).

This separation lets catabolic and anabolic processes proceed independently in the same cell.