Glycolysis, Krebs Cycle, and Oxidative Phosphorylation Notes

Glycolysis

Objectives

  • Describe the glycolysis pathway.
  • Describe the regulation of glycolysis.
  • Differentiate between aerobic and anaerobic glycolysis.
  • Explain the role of 2,3-bisphosphoglycerate in red blood cells.

Carbohydrate Metabolism After Meal

After a meal, carbohydrate metabolism involves various processes across different tissues:

  • Small intestine: Absorption of glucose.
  • Pancreas secretes insulin (↑) and glucagon (↓).
  • Liver: Glucose is converted to glycogen, and excess is converted to acetyl CoA which can enter the Krebs cycle or be used for fatty acid synthesis.
  • Muscle: Glucose is converted to glycogen or used in glycolysis to form pyruvate and then acetyl CoA.
  • Adipose tissue: Glucose is converted to glycerol 3-phosphate (Gly 3-P) for triglyceride (TG) synthesis.
  • Brain: Utilizes glucose for energy.
  • Red Blood Cells (RBC): Utilize glucose.

Carbohydrate digestion starts with α\alpha-amylase in the pancreas, breaking down complex carbohydrates into smaller sugars. Disaccharidases like lactase, maltase, and sucrase further break down disaccharides into monosaccharides.
Na+Na^+/ATPase is used in neuron cell membranes.

Release of Chemical Energy

Cellular respiration occurs in three sets of reactions:

  • Glycolysis
  • The citric acid cycle (Krebs cycle)
  • The electron transport chain (oxidative phosphorylation)

Glycolysis

  • Occurs in the cytosol.
  • Can be aerobic or anaerobic (does not require oxygen to proceed).
  • Glucose is converted to pyruvate (aerobic) or lactate (anaerobic).
  • 1st stage: Glucose is phosphorylated at two places, requiring ATP.
  • 2nd stage: The 6-carbon glucose is split into two 3-carbon pyruvate molecules.
  • ATP is synthesized during the second stage.
Phase I of Glycolysis
  • Glucose is converted to glucose 6-phosphate by hexokinase/glucokinase, using ATP.
  • Glucose 6-phosphate is converted to fructose 6-phosphate by phosphoglucose isomerase.
  • Fructose 6-phosphate is converted to fructose 1,6-bisphosphate by phosphofructokinase-1 (PFK-1), using ATP. This is an irreversible reaction.
  • Fructose 1,6-bisphosphate is split into dihydroxyacetone phosphate and glyceraldehyde 3-phosphate by aldolase.
  • Dihydroxyacetone phosphate is converted to glyceraldehyde 3-phosphate by triose phosphate isomerase.
  • Dihydroxyacetone phosphate can be converted to Glycerol 3-P by Glycerol-3P dehydrogenase using NADH.
Phase II of Glycolysis
  • Glyceraldehyde 3-phosphate is converted to 1,3-bisphosphoglycerate by glyceraldehyde-3P dehydrogenase, producing NADH.
  • 1,3-Bisphosphoglycerate is converted to 3-phosphoglycerate by phosphoglycerate kinase, producing ATP.
  • 3-Phosphoglycerate is converted to 2-phosphoglycerate by phosphoglycerate mutase.
  • 2,3-Bisphosphoglycerate can be created from 1,3-Bisphosphoglycerate via 1,3-Bisphosphoglycerate mutase, and degraded back to 3-Phosphoglycerate via 2,3-Bisphosphoglycerate phosphatase.
  • 2-Phosphoglycerate is converted to phosphoenolpyruvate (PEP) by enolase.
  • Phosphoenolpyruvate (PEP) is converted to pyruvate by pyruvate kinase, producing ATP. This is an irreversible reaction.
  • Pyruvate can be converted to lactate by lactate dehydrogenase, using NADH.

Glycerol Phosphate Shuttle

  • Involves the transfer of electrons from NADH in the cytosol to FADH2 in the mitochondria.
  • Dihydroxyacetone phosphate (DHAP) is reduced by NADH to form glycerol-3-phosphate, catalyzed by glycerol-3-phosphate dehydrogenase (G3PDH).
  • Glycerol-3-phosphate is then oxidized back to DHAP by mitochondrial glycerol-3-phosphate dehydrogenase, transferring electrons to FAD to form FADH2.
  • FADH2 then donates electrons to the electron transport chain (ETC).

Malate-Aspartate Shuttle

  • Another mechanism to transfer electrons from cytosolic NADH into the mitochondria.
  • Oxaloacetate (OAA) is reduced by NADH to form malate, catalyzed by malate dehydrogenase (MDH).
  • Malate can then cross the inner mitochondrial membrane.
  • Inside the mitochondria, malate is oxidized back to OAA by mitochondrial MDH, generating NADH. OAA is converted to aspartate (Asp) which can be transported out.
  • Aspartate goes back to the cytosol and through a series of reactions is converted to OAA to begin the process again.

Regulation of Glycolysis

  • Activators:
    • Fructose-2,6-bisphosphate
    • AMP
  • Inhibitors:
    • ATP
    • Citrate
    • Acetyl CoA
    • Alanine
    • Glucagon

Overall Reaction for Glycolysis

Glucose+2ATP+2ADP+2PO<em>43−+2NAD+→2Pyruvate+2NADH+2H</em>2O+4ATPGlucose + 2ATP + 2ADP + 2PO<em>4^{3-} + 2NAD^+ \rightarrow 2 Pyruvate + 2 NADH + 2H</em>2O + 4 ATP

  • Net Energy: 2 ATP
  • Aerobic: Pyruvate enters the TCA cycle, and NADH is used in the electron transport chain (ETC).
  • Anaerobic: No NADH is used; lactate is formed.

Role of 2,3-BPG in RBC

  • 2,3-BPG acts as an allosteric inhibitor of oxygen binding to heme in hemoglobin.
  • It reenters the glycolytic pathway via dephosphorylation to 3-phosphoglycerate.

Anaerobic Glycolysis

  • Occurs in the absence of O2O_2, forming lactate.
  • Tissues dependent on this include RBC, WBC, kidney, eyes, and skeletal muscles.
  • Formation of ATP is faster than aerobic respiration (citric acid cycle and oxidative phosphorylation).
  • ATP yield per mole of glucose is lower.
  • Lactate returns to the liver to be converted to pyruvate and undergo gluconeogenesis (Cori Cycle).
Cori Cycle
  • Lactate produced in the muscle during anaerobic glycolysis is transported to the liver.
  • In the liver, lactate is converted back to glucose via gluconeogenesis.
  • The glucose is then released back into the blood, where it can be taken up by the muscle.

Biosynthetic Functions of Glycolysis

Glycolysis provides precursors for various biosynthetic pathways:

  • Glucose 6-P:
    • Five-carbon sugars (for nucleotide synthesis)
  • Glycerol-P:
    • Triglyceride synthesis
  • 1,3-bis-Phosphoglycerate:
    • Allosteric inhibitor of O2O_2 binding to heme (2,3-bis-Phosphoglycerate)
  • 3-Phosphoglycerate:
    • Serine
  • Pyruvate:
    • Alanine
    • Acetyl CoA (for fatty acid synthesis and TCA cycle)
  • Acetyl CoA:
    • Glutamate and other amino acids

Krebs Cycle

Objectives

  • Explain the conversion of pyruvate to acetyl CoA.
  • Describe the Krebs Cycle and its regulation.
  • Describe the entry and exit of metabolites in the Krebs Cycle.

Krebs Cycle (Overview)

  • Glucose, fatty acids, and amino acids are broken down into pyruvate, acetate, and ketone bodies which are then converted to Acetyl CoA.
  • Acetyl CoA enters the Krebs cycle.
  • Acetyl CoA combines with oxaloacetate to form citrate.
  • Through a series of reactions, oxaloacetate is regenerated, and energy is produced (GTP, NADH, and FADH2), along with the release of carbon dioxide.

Steps of Krebs Cycle

The Krebs Cycle involves the following steps:

  1. Acetyl-CoA combines with oxaloacetate to form citrate (catalyzed by citrate synthase).
  2. Citrate is isomerized to isocitrate (catalyzed by aconitase).
  3. Isocitrate is decarboxylated to α-ketoglutarate (catalyzed by isocitrate dehydrogenase), producing NADH and CO2CO_2.
  4. α-Ketoglutarate is decarboxylated to succinyl-CoA (catalyzed by α-ketoglutarate dehydrogenase), producing NADH and CO2CO_2.
  5. Succinyl-CoA is converted to succinate (catalyzed by succinate thiokinase), producing GTP.
  6. Succinate is oxidized to fumarate (catalyzed by succinate dehydrogenase), producing FADH2.
  7. Fumarate is hydrated to malate (catalyzed by fumarase).
  8. Malate is oxidized to oxaloacetate (catalyzed by malate dehydrogenase), producing NADH.

Function of Krebs Cycle

  • Release of chemical energy (NADH, FADH2, GTP/ATP).
  • Intermediates are precursors of many important compounds:
    • Succinyl CoA → heme synthesis
    • α-Ketoglutarate → neurotransmitter & amino acid synthesis
    • OAA → aspartate
    • Citrate → fatty acid synthesis
    • Malate → gluconeogenesis

Krebs Cycle

  • Acetyl CoA combines with oxaloacetate to form citrate.
  • A series of reactions regenerate oxaloacetate and produce ATP, NADH + H+, FADH2, and carbon dioxide.
  • This cycle can be repeated as long as oxygen and pyruvate are available.

Overall Reaction of Citric Acid Cycle

AcetylCoA+3NAD++FAD+GDP+Pi+2H<em>2O→2CO</em>2+3NADH+FADH2+GTP+3H++CoAAcetyl CoA + 3NAD^+ + FAD + GDP + Pi + 2H<em>2O \rightarrow 2CO</em>2 + 3NADH + FADH_2 + GTP + 3H^+ + CoA

  • Regulatory enzymes: citrate synthase, isocitrate dehydrogenase (allosteric regulation), α-ketoglutarate dehydrogenase
  • General regulatory factors: ADP/ATP and NAD+/NADH

Regulation of TCA Cycle

The TCA cycle is regulated at several points:

  • Citrate synthase is inhibited by ATP, NADH, and citrate.
  • Isocitrate dehydrogenase is activated by ADP and Ca2+Ca^{2+}, and inhibited by ATP and NADH.
  • α-ketoglutarate dehydrogenase is activated by Ca2+Ca^{2+} and inhibited by succinyl-CoA and NADH.

Pyruvate Dehydrogenase

  • Multi-enzyme complex.
  • Pyruvate is converted to Acetyl CoA, linking glycolysis to the TCA cycle.
  • Pyruvate dehydrogenase exists in two forms:
    • Non-phosphorylated form (active)
    • Phosphorylated form (inactive)

Pyruvate Dehydrogenase and Related Pathways

  • Glucose can be converted to pyruvate via glycolysis.
  • Pyruvate can be converted to acetyl-CoA by pyruvate dehydrogenase.
  • Then Acetyl-CoA can enter the Citric Acid Cycle. Succinyl-CoA, Citrate, and α-Ketoglutarate are also citric acid cycle intermediates, creating GABA, Aspartate, and Glutamate.

Pyruvate Dehydrogenase

  • Co-factors: TPP, coenzyme A, NAD+, lipoic acid (vitamins).
  • Acetyl CoA and NADH are negative effectors, while ADP is a positive effector.
  • PDH deficiency (genetic disorder):
    • Lactic acid accumulates, damaging neuron cells, leading to mental retardation (rare).
  • Treatment: supplement with thiamine if E1 subunit is abnormal; lipoic acid if E2 subunit is abnormal and give diet low in carbohydrate.

Regulation of PDH complex

Regulation of the pyruvate dehydrogenase (PDH) complex involves:

  • Phosphorylation/dephosphorylation: PDH kinase phosphorylates and inactivates the PDH complex, while PDH phosphatase dephosphorylates and activates it.
  • Allosteric regulation: ATP, acetyl-CoA, and NADH activate PDH kinase, leading to inactivation of the PDH complex. ADP and pyruvate inhibit PDH kinase, promoting PDH complex activity. Calcium activates PDH phosphatase.

Oxidative Phosphorylation

Objectives

  • Describe the complexes and cofactors in the electron transport chain.
  • Describe chemiosmotic theory and protonmotive force.
  • Describe the aerobic oxidation of cytosolic NADH.
  • Describe the regulation of oxidative phosphorylation.
  • Describe some inhibitors and uncouplers in oxidative phosphorylation.

ETC

  • Electrons are transferred through a series of enzyme complexes on the folds of the inner mitochondrial membrane.
  • Electron energy is transferred to ATP.
  • Oxygen receives the electrons to form water.

Overview of Glycolysis, Krebs Cycle, and ETC

  • Glycolysis starts with glucose and produces pyruvate. ATP and NADH are also produced.
  • Pyruvate is converted into Acetyl CoA. Acetyl CoA enters the Krebs cycle (also known as the citric acid cycle or TCA cycle).
  • The Krebs cycle produces NADH and FADH2, as well as other intermediates. NADH and FADH2 then enter the electron transport chain (ETC).
  • The ETC uses these electron carriers to create a proton gradient, which drives the synthesis of ATP. Oxygen is the final electron acceptor, forming water.

ETC Complexes

  • Complex I: NADH dehydrogenase
  • Complex II: Succinate dehydrogenase
  • Complex III: Cytochrome b-c1 complex
  • Complex IV: Cytochrome oxidase

Electron Transport Chain and ATP Synthesis

The process can be summarized as follows:

  1. NADH donates electrons to Complex I, which pumps protons (H+H^+) from the matrix to the intermembrane space.
  2. Electrons are passed to Coenzyme Q (CoQ).
  3. Succinate donates electrons to Complex II, which passes them to CoQ. No protons are pumped at this step.
  4. CoQH2 passes electrons to Complex III, which pumps protons to the intermembrane space. Electrons are then transferred to cytochrome c (Cyt c).
  5. Cytochrome c passes electrons to Complex IV, which pumps protons to the intermembrane space and reduces oxygen to water.
  6. The electrochemical gradient (proton-motive force) drives protons back into the matrix through ATP synthase (Complex V), which synthesizes ATP from ADP and Pi.

Chemiosmotic Theory of Oxidative Phosphorylation

  • Electron flow through the ETC pumps H+H^+ out from the matrix into the intermembrane space (cytosolic side of the inner membrane).
  • Protons are pumped outside at complexes I, III, and IV.
  • This creates a proton motive force due to pH gradient or membrane potential.
  • H+H^+ gets back into the matrix via FO-F1-ATP synthase, coupling with ATP synthesis and acceptance of electrons by oxygen -> oxidative phosphorylation.

Proton Motive Force

The proton-motive force across the inner mitochondrial membrane is due to:

  • Membrane potential (Δψ): The electrical potential difference, with the matrix side being negative and the cytosolic side being positive.
  • pH gradient (ΔpH): The concentration difference of protons, with the intermembrane space (cytosolic side) being more acidic (higher proton concentration) than the matrix side (alkaline).

Chemiosmosis and ATP Synthesis

Protons accumulate in the outer compartment (intermembrane space), creating an electrochemical gradient. Protons then flow back into the inner compartment (matrix) through ATP synthase, driving the synthesis of ATP from ADP and Pi. Cytochrome oxidase (cytochrome aa3aa_3) is involved in the transfer of electrons to oxygen. It results in ATP generation.

Regulation of Oxidative Phosphorylation

  • If ADP increases -> Oxidative Phosphorylation increases
  • Electron source (substrate)
  • NADH/NAD+ and ATP/ADP
  • Oxygen

Inhibitors of ETC

  • Complex I: Barbiturates, Rotenone, Piericidin A
  • Complex II: Carboxin, TTFA
  • Complex III: Antimycin A, Dimercaprol
  • Complex IV: H2S, cyanide (CN-), CO, sodium azide
  • Inhibitor of Fo-F1 synthase: oligomycin (antibiotic)

Uncoupler of Oxidative Phosphorylation

  • Lipophilic substances permeable to the membrane.
  • Carries proton back into the matrix without going through the ATP synthase pore, so no ATP is formed.
  • Chemical uncouplers:
    • 2,4-Dinitrophenol (slimming drug)
    • Dicumarol (anticoagulant)
    • Aspirin/salicylate poisoning

Brown Fat

  • Adipose tissue rich in mitochondria.
  • In cold conditions, noradrenaline is stimulated, lipase is activated, and fats are oxidized -> FADH2, NADH.
  • ETC is activated, FADH2, NADH are oxidized.
  • Thermogenin in brown fat transports protons from the cytosolic side of the inner mitochondrial membrane into the matrix, generating heat (but no ATP formation).