Citric Acid Cycle Notes

TCA Cycle/Kreb's Cycle/Citric Acid Cycle

Overview of Metabolic Stages

• Stage 1: Breakdown of Foods
  • Proteins are broken down into amino acids.
  • Polysaccharides are broken down into simple sugars (e.g., glucose).
  • Fats are broken down into fatty acids and glycerol.
  • This stage occurs in the cytosol.
• Stage 2: Breakdown to Acetyl CoA
  • Simple subunits are broken down into acetyl CoA.
  • Limited amounts of ATP and NADH are produced.
  • Glycolysis converts glucose to pyruvate.
  • Pyruvate is then converted to acetyl CoA, producing $CO_2$ and NADH.
• Stage 3: Complete Oxidation
  • Acetyl CoA is completely oxidized to $H<em>2O$ and $CO</em>2$ in the mitochondrion.
  • Large amounts of ATP are produced via the citric acid cycle and oxidative phosphorylation.
  • The citric acid cycle occurs in the mitochondrial matrix.
  • Oxidative phosphorylation occurs in the inner mitochondrial membrane, utilizing $O<em>2$ to produce $H</em>2O$.

Oxidation of Pyruvate in Mitochondria

• Pyruvate, produced from glycolysis, enters the mitochondria.
• It is converted to acetyl CoA, releasing $CO_2$ and NADH.
• The acetyl CoA then enters the citric acid cycle.
• Oxidative phosphorylation uses the NADH to produce ATP.

Pyruvate Dehydrogenase Complex (PDC)

• Pyruvate must undergo three reactions before entering the citric acid cycle:
  • Decarboxylation (removal of $CO_2$).
  • Oxidation (loss of 2 electrons).
  • Attachment to Coenzyme A.
• These reactions are catalyzed by the pyruvate dehydrogenase complex (PDC).

PDC Enzyme Complex

• PDC comprises multiple copies of three different enzymes: E1, E2, and E3.
• E3 resets the complex, passing electrons to $NAD^+$ to form NADH.

Regulation of PDC

• PDC activity is regulated based on the cell’s energy needs.
• The E1 enzyme (pyruvate dehydrogenase, PDH) is regulated by reversible phosphorylation.
• PDC kinase is stimulated by high concentrations of ATP, NADH, and acetyl CoA.
• When energy charge is high (at rest), PDC kinase is activated, and PDC is phosphorylated, inactivating it.
• Pyruvate and ADP inhibit PDC kinase, switching PDC on when energy charge is low.
• $Ca^{2+}$ stimulates PDC phosphatase, activating PDC during muscle contraction and exercise.

High vs. Low Energy Charge and PDC Regulation

• High Energy Charge:
  • ATP, NADH, and acetyl CoA stimulate PDC kinase.
  • PDC is phosphorylated and inactivated.
• Low Energy Charge:
  • Pyruvate and ADP inhibit PDC kinase.
  • PDC is dephosphorylated and activated.

The Citric Acid Cycle

• Also known as the TCA cycle or Krebs cycle.
• Fully oxidizes the carbons from acetyl CoA, producing 2 molecules of $CO_2$.
• Electrons are passed to $NAD^+$ or FAD.
• Takes place in the mitochondrial matrix.

Steps of the Citric Acid Cycle

• Step 1: Formation of citrate from acetyl CoA and oxaloacetate.
  • Acetyl CoA + Oxaloacetate + $H_2O$ → Citrate + HS-CoA + $H^+$
• Step 2: Isomerization of citrate into isocitrate via cis-aconitate intermediate.
• Step 3: Oxidation and decarboxylation of isocitrate to α-ketoglutarate.
  • Isocitrate + $NAD^+$ → α-ketoglutarate + $CO_2$ + NADH + $H^+$
• Step 4: Oxidative decarboxylation of α-ketoglutarate to succinyl CoA.
  • α-ketoglutarate + $NAD^+$ + HS-CoA → Succinyl-CoA + $CO_2$ + NADH + $H^+$
• Step 5: Cleavage of succinyl CoA to form succinate.
  • Succinyl-CoA + GDP + Pi → Succinate + GTP + HS-CoA
  • Substrate-level phosphorylation. GTP can phosphorylate ADP: GTP + ADP → GDP + ATP.
• Step 6: Oxidation of succinate to fumarate.
  • Succinate + FAD → Fumarate + $FADH_2$
• Step 7: Hydration of fumarate to malate.
  • Fumarate + $H_2O$ → Malate
• Step 8: Oxidation of malate to oxaloacetate.
  • Malate + $NAD^+$ → Oxaloacetate + NADH + $H^+$

Net Reactions of the Citric Acid Cycle

• The NADH and $FADH_2$ formed in the citric acid cycle are oxidized by the electron transport chain.

Fate of Carbons in the Citric Acid Cycle

• The two carbons from acetyl CoA that enter the cycle are converted to $CO_2$ in subsequent turns.

Energy Conservation in the Citric Acid Cycle

• No $O_2$ is directly involved, although this is aerobic respiration.
• Only 1 ATP is formed per round of the cycle.
• Energy is conserved in the form of 3 NADH and 1 $FADH_2$.

Reducing Power of NADH and FADH2

• NADH and $FADH_2$ store energy as reducing power.
• Oxidation of NADH and $FADH_2$ (loss of electrons) is energetically favorable.
• They have a lot of reducing power (the power to reduce other molecules).
• This stored energy is used to make ATP in oxidative phosphorylation.
  • NADH → $NAD^+$ + 2e- + $H^+$ $\Delta Go’ = -62 kJ mol^{-1}$
  • $FADH_2$ → FAD+ 2e- + 2$H^+$ $\Delta Go’ = -42.5 kJ mol^{-1}$

Regulation of the Citric Acid Cycle Enzymes

• Isocitrate dehydrogenase:
  • Inhibited by ATP and NADH.
  • Stimulated by ADP.
• α-ketoglutarate dehydrogenase:
  • Inhibited by ATP, succinyl CoA, and NADH.

Biosynthetic Roles of the Citric Acid Cycle

• Many intermediates of the citric acid cycle are important for the synthesis of other molecules(anabolism) like amino acids and nucleotides.
• The citric acid cycle has a role in biosynthesis (anabolism) as well as energy production (catabolism).
• Regulation of the cycle allows cells to switch between energy production and biosynthesis when energy levels are high.

Clinical Insight: Citric Acid Cycle and Cancer

• Some enzymes of the citric acid cycle are affected in cancer.
• Inhibition of the cycle leads to the Warburg effect: the use of lactic acid fermentation even in aerobic conditions.
• The enzymes for glycolysis are upregulated by transcription factor hypoxia-inducible factor 1 (HIF-1).
• Inhibition of enzymes of the cycle increases levels of HIF-1, switching on glycolysis even in the presence of $O_2$.
• Understanding changes in metabolism in tumor cells could lead to new treatments for cancer.

Citric Acid Cycle Summary

• Pyruvate enters the mitochondrial matrix and is converted to acetyl-CoA by the pyruvate dehydrogenase complex (PDC).
• Acetyl CoA is the substrate for the citric acid cycle.
• Eight different reactions result in oxidation of the 2 carbons to $CO<em>2$, releasing energy as 1 ATP, 3 NADH, and 1 $FADH</em>2$.
• The cycle is regulated according to energy demand.
• The cycle is also a source of intermediates for anabolic pathways.