TCA Cycle Notes

Glucose

  • Dr. Sneha M. Pinto (s.pinto@surrey.ac.uk)
  • Tricarboxylic Acid (TCA) Cycle
  • Key Molecules:
    • Glucose, Glucose 6-P, Pyruvate
    • Fatty acid synthase (FAS), Glutamine, Malonyl-CoA
    • ATP citrate lyase, Acetyl-CoA
    • Oxaloacetate, Citrate, Cis-aconitate, Itaconate, Malate, Fumarate, Isocitrate, a-Ketoglutarate, Succinyl-CoA, Succinate
  • Key Enzymes:
    • Pyruvate dehydrogenase, Citrate synthase, Aconitase, IRG1/Cis-aconitate decarboxylase, Malate dehydrogenase, Isocitrate dehydrogenase, a-ketoglutarate dehydrogenase, Succinyl-CoA synthetase, Succinate dehydrogenase/CII, Fumarate hydratase
  • Coenzymes/Factors:
    • NAD+, NADH, FAD+, FADH
  • Processes:
    • OXPHOS (Oxidative Phosphorylation), ADP, Pi, ATP

Learning Objectives

  • Describe cellular respiration.
  • Explain the production of Acetyl-CoA.
  • Provide an overview of the TCA/Citric Acid Cycle.
  • Describe intermediates and enzymes of the Citric Acid Cycle.
  • Describe the amphibolic role of the TCA cycle.
  • Discuss key regulatory points within the TCA cycle.

Cellular Respiration

  • Catabolic process where cells consume O<em>2O<em>2 and produce CO</em>2CO</em>2 to break down organic molecules.
  • Carbohydrates, lipids, and amino acids are catabolized in three stages.
  • Provides more energy (ATP) from glucose than glycolysis.

Glycolysis Overview

  • Reaction: Glucose + 2 NAD+ + 2 ADP + 2 Pi → 2 Pyruvate + 2 NADH + 2 H+ + 2 ATP
  • ΔG′°=−146 kJ/mol\Delta G'° = -146 \text{ kJ/mol}
  • Overall Glucose Oxidation: 6 O<em>2O<em>2 → 6 CO</em>2CO</em>2 + 6 H2OH_2O
  • ΔG′°=−2840 kJ/mol\Delta G'° = -2840 \text{ kJ/mol}
  • Only a small amount of energy available in glucose is captured by glycolysis.

Fatty Acid Oxidation Overview

  • Reaction: Palmitoyl-CoA + 7CoA + 7FAD + 7NAD+ + 7H2OH_2O → 8 acetyl-CoA + 7FADH2 + 7NADH + 7H+
  • Acetyl-CoA enters the citric acid/TCA cycle.
  • 8 Acetyl-CoA + 16O<em>2O<em>2 + 80Pi + 80ADP → 8CoA + 80ATP + 16CO</em>2CO</em>2 + 16H2OH_2O
  • Overall from a 16C fatty acid:
  • Palmitoyl-CoA + 23O<em>2O<em>2 + 108Pi + 108ADP → CoA + 108ATP + 16CO</em>2CO</em>2 + 23H2OH_2O

Energy Scale for Food

  • Calories = Energy
  • Carbohydrates: 4 Calories per gram
  • Protein: 4 Calories per gram
  • Fats: 9 Calories per gram

Stages of Cellular Respiration

  1. Acetyl-CoA production
  2. Acetyl-CoA oxidation
  3. Electron transfer and oxidative phosphorylation

Mitochondrion Structure

  • Outer membrane:
    • Freely permeable to small molecules and ions due to porin channels.
  • Inner membrane:
    • Impermeable to most small molecules and ions, including H+.
    • Contains respiratory electron carriers (Complexes I-IV), ADP-ATP translocase, ATP synthase (FoF1), and other membrane transporters.
  • Matrix:
    • Contains pyruvate dehydrogenase complex, citric acid cycle enzymes, fatty acid β-oxidation enzymes, amino acid oxidation enzymes, DNA, ribosomes, ATP, ADP, Pi, Mg2+Mg^{2+}, Ca2+Ca^{2+}, K+K+, and many soluble metabolic intermediates.

Stage 1: Acetyl-CoA Production

  • Organic fuels such as glucose are oxidized to two-carbon fragments in the form of acetyl-CoA.
  • Site of synthesis: mitochondrial matrix
  • Generates ATP/NADH

Acetyl-CoA: Central Molecule in Cellular Metabolism

  • Produced via:
    • Glycogenolysis: Glycogen → Glucose
    • Glycolysis: Glucose → Pyruvate
    • β-Oxidation: Free fatty acids → Acetyl-CoA
    • Lipolysis: Triglyceride → Fatty acids
    • Proteolysis: Protein → Amino acids
  • Acetyl-CoA is used in:
    • TCA cycle
    • Ketone bodies
    • Sterols and fatty acids synthesis
    • Protein acetylation

Conversion of Pyruvate to Acetyl-CoA

  • Oxidative decarboxylation of pyruvate
  • Reaction: Pyruvate + CoA-SH + NAD+ → Acetyl-CoA + CO2CO_2 + NADH
  • Catalyzed by the pyruvate dehydrogenase complex (E1 + E2 + E3), requiring TPP, lipoate, and FAD.
  • ΔG′°=−33.4 kJ/mol\Delta G'° = -33.4 \text{ kJ/mol}

Structure of Coenzyme-A

  • Coenzyme A (CoA) has a reactive thiol group to which the acetyl group is covalently linked, forming a thioester.

Stage 2: Acetyl-CoA Oxidation

  • Also known as the Tricarboxylic acid Cycle/Krebs Cycle.
  • Discovered by Hans Krebs in 1937 (Nobel Prize in Physiology/Medicine, 1953).
  • Central hub of cellular metabolism.
  • Meets cellular energy requirements by oxidizing the products of carbohydrate and fat metabolism.
  • Generates NADH, FADH2, and GTP.

Citric Acid Cycle (TCA Cycle)

  • Overview of Reactions:
    • Acetyl-CoA + Oxaloacetate → Citrate (Citrate Synthase)
      • Claisen condensation: methyl group of acetyl-CoA converted to methylene in citrate
    • Citrate → cis-Aconitate → Isocitrate (Aconitase)
      • Dehydration/rehydration: -OH group of citrate repositioned in isocitrate to set up decarboxylation in the next step.
    • Isocitrate → α-Ketoglutarate (Isocitrate Dehydrogenase) + NADH, CO2CO_2
      • Dehydrogenation: introduction of a double bond initiates methylene oxidation sequence
      • Oxidative decarboxylation: -OH group oxidized to carbonyl, which facilitates decarboxylation by stabilizing the carbanion formed on the adjacent carbon.
    • α-Ketoglutarate → Succinyl-CoA (α-Ketoglutarate Dehydrogenase) + NADH, CO2CO_2
      • Oxidative decarboxylation: pyruvate-dehydrogenase-like mechanism.
    • Succinyl-CoA → Succinate (Succinyl-CoA Synthetase) + GTP
      • Substrate-level phosphorylation: energy of thioester conserved in phosphoanhydride bond of GTP
    • Succinate → Fumarate (Succinate Dehydrogenase) + FADH2
      • Dehydrogenation: oxidation of -OH completes oxidation sequence; generates carbonyl positioned to facilitate Claisen condensation in the next step
    • Fumarate → Malate (Fumarase)
      • Hydration: addition of water across a double bond introduces -OH group for the next oxidation step
    • Malate → Oxaloacetate (Malate Dehydrogenase) + NADH

Step 1: Condensation of Acetyl-CoA and Oxaloacetate

  • Reaction: Acetyl-CoA + Oxaloacetate → Citrate + CoA-SH
  • Enzyme: Citrate Synthase
  • ΔG′°=−32.2 kJ/mol\Delta G'° = -32.2 \text{ kJ/mol}

Step 2: Isomerization by dehydration/rehydration

  • Citrate → cis-Aconitate → Isocitrate
  • Enzyme: Aconitase
  • Two-step process, involving the removal and then the addition of a water molecule.
  • ΔG′°=13.3 kJ/mol\Delta G'° = 13.3 \text{ kJ/mol}

Iron-Sulfur center in Aconitase

  • Shows the iron-sulfur cluster and the binding of citrate.

Step 3: Oxidative decarboxylation by Isocitrate dehydrogenase

  • NAD-dependent enzyme occurs in the mitochondrial matrix and serves in the citric acid cycle.
  • NADP-dependent enzymes are present in both the mitochondrial matrix and the cytosol, generating NADPH necessary for fatty acid and sterol synthesis.

Step 4: Oxidative decarboxylation by α-Ketoglutarate dehydrogenase

  • Catalyzes the conversion of α-ketoglutarate to succinyl-CoA.
  • Produces NADH directly, providing electrons for the respiratory chain.
  • Highly regulated enzyme - determines the metabolic flux through the TCA cycle.

Step 5: Substrate-level phosphorylation by Succinyl-CoA synthetase

  • Reversible conversion of succinyl-CoA to succinate, coupled with the phosphorylation of GDP to GTP.
  • GTP formed by succinyl-CoA synthetase donates its terminal phosphoryl group to ADP to form ATP, in a reversible reaction catalyzed by nucleoside diphosphate kinase.

Step 6: Oxidation of an Alkane to Alkene by Succinate dehydrogenase

  • Reaction: Succinate + ubiquinone (Q) → Fumarate + ubiquinol (QH2QH_2).
  • SDH is embedded in the inner membrane of the mitochondria, allowing FADH2 to directly transfer its electrons into the electron transport chain.
  • Electron flow through these carriers to the final electron acceptor, O2O_2, is coupled to the synthesis of ~1.5 ATP molecules.

Step 7: Hydration of a double bond by Fumarase

  • Reversible hydration/dehydration of fumarate to malate
  • Fumarate → L-Malate
  • ΔG′°=−3.8 kJ/mol\Delta G'° = -3.8 \text{ kJ/mol}

Step 8: Oxidation of Malate to Oxaloacetate by malate dehydrogenase

  • L-Malate → Oxaloacetate
  • ΔG′°=29.7 kJ/mol\Delta G'° = 29.7 \text{ kJ/mol}
  • Regenerates oxaloacetate for the next cycle.

One turn of the Citric Acid Cycle

  • Input: Acetyl-CoA
  • Outputs: 2 CO2CO_2, 3 NADH, 1 FADH2, 1 GTP (ATP)
  • Regenerates: Oxaloacetate

Net result of the Citric Acid Cycle

  • Acetyl-CoA + 3NAD+ + FAD + GDP + Pi + 2 H<em>2OH<em>2O → 2CO</em>2CO</em>2 + 3NADH + FADH2 + GTP + CoA + 3H+

Direct and indirect ATP yield

  • Glycolysis of one molecule of glucose yields 2 NADH and 2ATP.
  • Production of 2 molecules of acetyl-CoA from pyruvate yields 2 NADH.
  • Citric acid cycle of 2 molecules of acetyl-CoA yields 6 NADH, 2 FADH2, and 2ATP

TCA cycle intermediates are amphibolic

  • NOT just oxidation of acetate
  • Hub of metabolism
    • Other 4- and 5-carbon end products enter and serve as fuel
    • Intermediates are also used as precursors in other biosynthetic pathways
  • Anaplerotic reactions replenish depleted cycle intermediates (Greek words "ana" (up) and "plerō" (to fill)

Regulation of the Citric Acid Cycle

  • Regulated at highly thermodynamically favorable (irreversible) steps.
  • General regulatory mechanisms:
    • Activated by substrate availability.
    • Inhibited by product accumulation.
  • Both glycolysis and TCA cycle pathways are inhibited by high levels of ATP, NADH, and citrate.

Summary

  • Cellular respiration
  • Intermediates & enzymes of the Citric Acid Cycle
  • Citric Acid Cycle: (indirect) Energy yield
  • Citric Acid Cycle: Hub of intermediary metabolism
  • Citric Acid Cycle: Regulation

Stage 3: Oxidative phosphorylation

  • Reduced coenzymes are oxidized, and electrons are transferred to O2O_2.
  • Generates lots of ATP.