Citric Acid Cycle Notes

Citric Acid Cycle (TCA Cycle/Kreb's Cycle)

Learning Objectives

• Explain the role of the Pyruvate Dehydrogenase (PDH) complex in linking glycolysis to the TCA cycle.
• Analyze the steps of the TCA cycle, highlighting key regulatory mechanisms and the energetic yield of the cycle.

Citric Acid Cycle

• The common pathway leading to the complete oxidation of carbohydrates, fatty acids, and amino acids to $CO_2$.
• Some ATP is produced.
• NADH produced will enter the ETC to generate ATP.
• A pathway providing many precursors for biosynthesis of amino acids and nucleotides.
• Acts as both catabolic and anabolic (amphibolic role).

TCA Cycle and Amino Acids

• The TCA cycle is connected to amino acid metabolism.
  • Alanine can be converted to pyruvate.
  • Aspartate can be converted to oxaloacetate.
  • Glutamate and glutamine can be synthesized from α-ketoglutarate.

Fate of Pyruvate

• For energy synthesis, pyruvate enters the mitochondria by active transport with the help of pyruvate translocase, a transport protein.

Pyruvate Dehydrogenase Complex

• Pyruvate is converted to Acetyl-CoA through the pyruvate dehydrogenase complex.

Pyruvate Oxidation

• Overall reaction:
  $Pyruvate + CoA + NAD^+ \rightarrow Acetyl-CoA + CO_2 + NADH + H^+$
• Pyruvate Dehydrogenase converts pyruvate to acetyl-CoA.

Coenzymes Required

• The Pyruvate Dehydrogenase complex requires 5 coenzymes:
  • TPP (Thiamine Pyrophosphate)
  • Lipoic Acid
  • Coenzyme A
  • FAD (Flavin Adenine Dinucleotide)
  • $NAD^+$ (Nicotinamide Adenine Dinucleotide)

Sources and Fate of Acetyl CoA

Precursors

• Fatty acids
• Glucose
• Pyruvate
• Amino acids

Products

• $CO<em>2 + H</em>2O$ + energy (ATP)
• Ketone bodies
• Triglycerides
• Phospholipids
• Eicosanoids
• Cholesterol
• Steroid hormones
• Bile salts
• Acetyl CoA is a central intermediate in lipid metabolism

Steps of the Citric Acid Cycle

1. Condensation: Acetyl-CoA + Oxaloacetate → Citrate (Citrate synthase).
2. Dehydration: Citrate → cis-Aconitate (Aconitase).
3. Hydration: cis-Aconitate → Isocitrate (Aconitase).
4. Oxidative Decarboxylation: Isocitrate → Oxalosuccinate → α-Ketoglutarate (Isocitrate Dehydrogenase, producing NADH).
5. Oxidative Decarboxylation: α-Ketoglutarate → Succinyl-CoA (α-Ketoglutarate Dehydrogenase complex, producing NADH and $CO_2$).
6. Substrate-Level Phosphorylation: Succinyl-CoA → Succinate (Succinyl-CoA synthetase, producing GTP).
7. Dehydrogenation: Succinate → Fumarate (Succinate Dehydrogenase, producing $FADH_2$).
8. Hydration: Fumarate → Malate (Fumarase).
9. Dehydrogenation: Malate → Oxaloacetate (Malate Dehydrogenase, producing NADH).

Key Reactions and Enzymes

• Citrate Synthase: Acetyl CoA + Oxaloacetate → Citrate.
• Isocitrate Dehydrogenase: Isocitrate → Oxalosuccinate → α-Ketoglutarate (produces NADH).
• Alpha Keto glutarate Dehydrogenase: Alpha-ketoglutarate → Succinyl CoA (produces NADH).
• Succinate Thiokinase: Succinyl CoA → Succinate (produces GTP).
• Succinate DH: Succinate → Fumarate (produces $FADH_2$).
• Malate DH: Malate → Oxaloacetate (produces NADH).

Regulation of the Citric Acid Cycle

• ATP acts as an allosteric inhibitor of citrate synthase.
• Citrate allosterically inhibits PFK, the key enzyme of glycolysis.
• Increased levels of $NAD^+, FAD$ stimulate the cycle.
• Isocitrate dehydrogenase is stimulated by ADP and inhibited by NADH.
• Alpha-ketoglutarate dehydrogenase is inhibited by succinyl CoA and NADH.

Non-Physiological Inhibitors

• Aconitase is inhibited by Fluoroacetate.
• Alpha-ketoglutarate dehydrogenase is inhibited by Arsenite.
• Succinate dehydrogenase is inhibited by Malonate.

Energetics

• Pyruvate Oxidation: 2.5 ATPs

• Number of ATPs produced per Acetyl-CoA: 10
• Energetics of Kreb's Cycle: One cycle = 10 ATPs
• So total TWO cycles: 20 ATPs

Energetics of Kreb’s Cycle

• Complete oxidation of ONE molecule of glucose:
• Aerobic glycolysis:
• Anaerobic glycolysis:
• Kreb’s cycle:

Malate-Aspartate Shuttle

• Malate shuttle for transferring NADH equivalents into the mitochondria.

Glycerol Phosphate Shuttle

• Glycerol phosphate shuttle for transferring NADH equivalents into the mitochondria.

Significance

• Complete oxidation of acetyl CoA to $CO_2$.
• ATP generation.
• Final oxidative pathway.
• Integrates major metabolic pathways.
• "Fat is burned in the wick of carbohydrates."
• Excess carbohydrates converted to neutral fats.
• Amphibolic pathway.
• Anaplerotic role of TCA cycle.

Final Common Oxidative Pathway

• Citric acid cycle may be considered as the final common oxidative pathway of all foodstuffs.
• All the major ingredients of foodstuffs are finally oxidized through the TCA cycle.

Excess Carbohydrates and Fat Conversion

• Excess calories are deposited as fat in adipose tissue. The pathway is glucose to pyruvate to acetyl CoA to fatty acid.
• Fat cannot be converted to glucose because the pyruvate dehydrogenase reaction (pyruvate to acetyl CoA) is an absolutely irreversible step.

No Net Synthesis of Carbohydrates from Fat

• Acetyl CoA entering in the cycle is completely oxidized to $CO_2$ by the time the cycle reaches succinyl CoA.
• Acetyl CoA is completely broken down in the cycle. Thus acetyl CoA cannot be used for gluconeogenesis.
• Therefore, there is no net synthesis of carbohydrates from fat.

Amino Acids Entering the TCA Cycle

• Some amino acids, such as leucine, catabolized to acetyl CoA are not converted to glucose because the pyruvate to acetyl CoA reaction is irreversible.
• The acetyl CoA molecules either enter the TCA cycle and are completely oxidized, or are channeled to ketone body formation.
• Hence, they are called ketogenic amino acids.

Amphibolic Pathway

• Oxaloacetate is the precursor of aspartate.
• Alpha-ketoglutarate can be made into glutamate.
• Succinyl CoA is used for synthesis of heme.
• Mitochondrial citrate is transported to the cytoplasm, where it is cleaved into acetyl CoA, which then is the starting point of fatty acid synthesis.

Anaplerotic Role of TCA Cycle

• The citric acid cycle acts as a source of precursors of biosynthetic pathways, e.g., heme is synthesized from succinyl CoA and aspartate from oxaloacetate.

Anaplerotic Reactions

• Anaplerotic reactions are “filling up” reactions or “influx” reactions or “replenishing” reactions that supply 4-carbon units to the TCA cycle.
  • Pyruvate to oxaloacetate by pyruvate carboxylase enzyme. It needs ATP.
  • Glutamate is transaminated to alpha ketoglutarate; and aspartate to oxaloacetate. Other important amino acids entering the TCA cycle.
  • Pyruvate can be carboxylated to malate by $NADP^+$ dependent malic enzyme.