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Overview of the Mitochondria and Citric Acid Cycle

Mitochondrial Structure

• Intermembrane Space: The space between the inner and outer membranes of the mitochondrion.

• Outer Membrane: Surrounds the mitochondrion and contains porins for small molecules.

• Inner Membrane: Contains specific transport proteins and is the site of the electron transport chain and ATP synthesis.

• Cristae: Infoldings of the inner membrane that increase surface area for energy production.

• Matrix: The innermost compartment filled with a gel-like matrix where the citric acid cycle occurs.

• Size of Mitochondria: Typically range from 1-2 μm in length and 0.1-0.5 μm in width.

Introduction to the Citric Acid Cycle

• Also known as:
    - Kreb's Cycle
    - **Tricarboxylic Acid Cycle (TCA)

• The Citric Acid Cycle is a crucial metabolic pathway involved in energy production through the oxidation of acetyl CoA derived from carbohydrates, fats, and proteins.

Entry Point and Initial Reaction

• Acetyl CoA: The compound that enters the TCA cycle.

• A 6-carbon molecule is formed from the combination of Oxaloacetate (4 carbons) and Acetyl CoA (2 carbons).

• During the cycle, oxidation of Acetyl CoA results in the production of 2 CO2 molecules, with the cleavage of a carbon-carbon bond facilitated by stabilizing carbonyl groups in transition states.

Step-by-Step Mechanism of the TCA Cycle

Step 1: Citrate Synthesis

• Reaction: Acetyl CoA and Oxaloacetate combine to produce Citroyl CoA which is then converted to Citrate.

• Chemical Equation:
    $ext{Acetyl-CoA} + ext{Oxaloacetate} + ext{H}_2O <br>ightarrow ext{Citrate} + ext{CoA-SH} + ext{H}^+$

• Standard Free Energy Change: $ext{AG}^ ext{'} = -32.2 ext{ kJ/mol}$

• Enzyme: Catalyzed by Citrate Synthase.
    - Mechanism:
      - Formation of citroyl CoA followed by hydrolysis releases citrate.
      - Type of Reaction: Two-part reaction: condensation and hydrolysis.
    - Energy from the thioester bond is utilized to synthesize a larger molecule, preventing hydrolysis of Acetyl CoA without utilizing its energy.

Step 2: Aconitase Reaction

• Reaction: Citrate is converted to cis-Aconitate, and then to D-Isocitrate via a dehydration/hydration mechanism.

• Chemical Equation:
    $ext{Citrate} <br>ightarrow ext{cis-Aconitate} <br>ightarrow ext{D-Isocitrate}$

• Standard Free Energy Change: $ext{AG}^ ext{'} = +6.3 ext{ kJ/mol}$

• Enzyme: Aconitase does this by catalyzing the dehydration and then hydration of citrate.

Step 3: Isocitrate to α-Ketoglutarate

• Reaction: Isocitrate is oxidized and decarboxylated to form α-ketoglutarate, generating NADH.

• Chemical Equation:
    $ext{Isocitrate} + ext{NAD}^+ <br>ightarrow ext{α-Ketoglutarate} + ext{CO}_2 + ext{NADH}$

• Enzyme: Isocitrate Dehydrogenase.

• This step is crucial for capturing high-energy electrons and produces oxidized CO2.

Step 4: α-Ketoglutarate Dehydrogenase Reaction

• Reaction: α-Ketoglutarate undergoes oxidative decarboxylation to form Succinyl CoA, generating another NADH.

• Mechanistic similarity to pyruvate dehydrogenase complex.

• Flow of Materials:
    - $ext{α-Ketoglutarate} + ext{NAD}^+ <br>ightarrow ext{Succinyl CoA} + ext{CO}_2 + ext{NADH}$

Step 5: Succinyl CoA to Succinate

• Reaction: Succinyl CoA is converted to Succinate, generating GTP (or ATP).

• Energy Transformation: The high-energy thioester bond is utilized for phosphorylation of GDP or ADP to form GTP/ATP.

• Enzyme: Succinyl CoA Synthetase.

• Key Mechanism:
    1. Orthophosphate reacts with Succinyl CoA to form succinyl phosphate.
    2. Histidine captures the phosphoryl group and succinate is released.
    3. Phosphohistidine then interacts with bound GDP to produce GTP, resetting the cycle.

Step 6-8: Regeneration of Oxaloacetate

• Overall Pathway: Succinate is regenerated into Oxaloacetate via:
    - Oxidation (Succinate to Fumarate, generating FADH2),
    - Hydration (Fumarate to Malate),
    - Final Oxidation (Malate to Oxaloacetate, generating NADH).
    - The reaction uses water and produces energy carriers.

• Final Reaction:
    $ext{Malate} + ext{NAD}^+ <br>ightarrow ext{Oxaloacetate} + ext{NADH}$
    - This reaction has a large positive free-energy change of $+29.7 ext{ kJ/mol}$.
    - Coupled with the removal of products to drive the reaction forward into the TCA cycle.

Summary of Key Products in TCA Cycle

• 2 Carbon atoms enter the cycle as Acetyl CoA and leave as 2 CO2.

• 4 pairs of hydrogen atoms are released, generating:
    - 3 NADH and 1 FADH2 in oxidation reactions.

• GTP (or ATP) is produced.

• 2 molecules of water consumed.

• The overall reaction:
    $ext{Acetyl-CoA} + 3 ext{NAD}^+ + ext{FAD} + ext{GDP} + ext{Pi} + 2 ext{H}_2 ext{O} <br>ightarrow 2 ext{CO}_2 + 3 ext{NADH} + ext{FADH}_2 + ext{GTP} + ext{CoA} + 3 ext{H}^+$

Regulation of the TCA Cycle

• Importance: The regulation is complex due to the TCA cycle's role in energy production and as a metabolic intermediate source.

• Key Regulatory Enzymes:
    - Citrate Synthase: Inhibited by Succinyl-CoA and NADH (competing substrates).
    - Isocitrate Dehydrogenase: Activated by ADP and inhibited by NADH and ATP.
    - α-Ketoglutarate Dehydrogenase: Negatively regulated by succinyl CoA and NADH.

• Pyruvate Dehydrogenase Regulation:
    - Involves covalent modification and is influenced by product accumulation (kinase activation) as well as response to insulin or exercise (phosphatase activation).

Anaplerotic Reactions

• Significance: Anaplerotic reactions replenish TCA cycle intermediates that are siphoned off for biosynthesis.

• Mechanisms include pyruvate carboxylase and transamination reactions to create oxaloacetate from amino acids like Aspartate.