Citric Acid Cycle Notes

Citric Acid Cycle (TCA Cycle)

Discovery and Nomenclature

• The tricarboxylic acid in question was identified as citric acid, leading to the name Citric Acid Cycle.
• Carl Wilhelm Scheele isolated citric acid from citrus fruits in 1780.

Functions of the Citric Acid Cycle

• Final common oxidative pathway: Oxidizes acetyl CoA to $CO_2$.
• Source of reduced co-enzymes: Provides substrates for the respiratory chain.
• Link between catabolic and anabolic pathways: Plays an amphibolic role.
• Precursor provision: Supplies precursors for amino acid and nucleotide synthesis.
• Enzyme control: Cycle components directly or indirectly control key enzymes in other pathways.

Reactions of the Cycle: Preparatory Steps

• Acetyl CoA entry: Enters the cycle and is completely oxidized, trapping energy during the process.

Sources of Acetyl CoA (Fig. 19.1):

• Pyruvate: Derived from glycolysis, oxidatively decarboxylated to acetyl CoA by pyruvate dehydrogenase.
  • This reaction links the TCA cycle and glycolysis.
  • Occurs in the mitochondria; pyruvate enters via a carrier from the cytoplasm.
• Fatty acids: Acetyl CoA derived from beta oxidation is formed in the mitochondria.
• Ketogenic amino acids

Enzyme Location

• All citric acid cycle enzymes are located inside the mitochondria.

First Step: Formation of Citric Acid

• Condensation: 4-carbon oxaloacetate condenses with 2-carbon acetyl CoA to form 6-carbon citrate (a tricarboxylic molecule).
• Enzyme: Citrate synthase (step 1, Fig. 19.2).
• Driving force: Hydrolysis of the thioester bond in acetyl CoA.
• Irreversible step: However, the body can reverse this step using ATP-citrate lyase (see Fig. 12.13).

Second Step: Formation of Isocitrate

• Isomerization: Citrate is isomerized to isocitrate by aconitase (step 2, Fig. 19.2).
• Two-step process:
  • Water removal: A water molecule is removed from citrate, forming cis-aconitate (a transient compound with a very short half-life).
  • Water addition: A water molecule is added to aconitate to form isocitrate, shifting the hydroxyl group position.

Third Step: Formation of Alpha Keto Glutarate

• Two-step process: Catalyzed by isocitrate dehydrogenase (step 3, Fig. 19.2).
  • Dehydrogenation: Isocitrate is dehydrogenated to form oxalosuccinate (unstable).
  • Decarboxylation: Oxalosuccinate undergoes spontaneous decarboxylation to form alpha keto glutarate.
• NADH generation: NADH produced is oxidized in the electron transport chain (ETC) to generate ATP.
• Oxidative decarboxylation: Isocitrate (6 carbons) undergoes oxidative decarboxylation to form alpha keto glutarate (5 carbons), releasing one molecule of $CO_2$.
• Isoenzymes: Isocitrate dehydrogenase has isoenzymes; the mitochondrial form uses $NAD^+$, while the cytoplasmic enzyme is $NADP^+$-dependent.

Fourth Step: Formation of Succinyl CoA

• Oxidative decarboxylation: Alpha keto glutarate is oxidatively decarboxylated to form succinyl CoA by alpha keto glutarate dehydrogenase (step 4, Fig. 19.2).
• NADH generation: NADH produced enters the ETC to generate ATPs.
• $CO<em>2$ removal: Another molecule of $CO</em>2$ is removed.
• Irreversible step: The only irreversible step in the entire cycle.
• Enzyme complex: Alpha keto glutarate dehydrogenase is a multienzyme complex with 3 enzyme proteins and 5 co-enzymes.
  • Similarity to pyruvate dehydrogenase: Similar to the pyruvate dehydrogenase reaction (Compare Fig. 19.3 with Fig. 9.22).
  • First two enzyme activities are similar to corresponding components of pyruvate dehydrogenase complex; the 3rd enzyme is the same in both complexes.

Fifth Step: Generation of Succinate

• Substrate-level phosphorylation: A high-energy phosphate is generated from the energy trapped in the thioester bond of succinyl CoA.
• Enzyme: Succinate thiokinase (step 5, Fig. 19.2).
• GTP formation: GDP is phosphorylated to GTP, and succinate is formed.
• GTP conversion: GTP can be converted to ATP by reacting with an ADP molecule: $GTP + ADP \rightarrow GDP + ATP$
• Succinyl CoA metabolism is shown in Figure 12.11.

Sixth Step: Formation of Fumarate

• Dehydrogenation: Succinate is dehydrogenated to fumarate (an unsaturated dicarboxylic acid) by succinate dehydrogenase (step 6, Fig. 19.2).
• Hydrogen acceptor: Hydrogen atoms are accepted by FAD.
• FADH2 entry: The $FADH_2$ enters the ETC to generate ATPs.
• Enzyme type: Succinate dehydrogenase is a flavoprotein.
• Inhibition: Competitively inhibited by malonate (see Fig. 5.19).

Seventh Step: Formation of Malate

• Hydration: Formation of malate from fumarate is catalyzed by fumarase (step 7, Fig. 19.2).
• Reaction: Addition of a water molecule.
• Stereospecificity: Only L-malate is formed.

Eighth Step: Regeneration of Oxaloacetate

• Oxidation: Malate is oxidized to oxaloacetate by malate dehydrogenase (step 8, Fig. 19.2).
• Coenzyme: $NAD^+$.
• NADH generation: NADH is generated, entering the electron transport chain to produce ATPs.
• Cycle continuation: Oxaloacetate can further condense with another acetyl CoA molecule to continue the cycle (Fig. 19.2).

Oxaloacetate as a Junction Point

• Catalytic role: Oxaloacetate acts as a catalyst, causing complete oxidation of acetyl CoA and regenerating itself without change.

Complete Oxidation of Acetyl CoA

$CO_2$ Removal Steps

• Step 3: Oxalosuccinate to alpha ketoglutarate.
• Step 4: Alpha ketoglutarate to succinyl CoA (Fig. 19.5).
• Acetyl CoA oxidation: Acetyl CoA contains 2 carbon atoms, which are removed as $CO_2$ in steps 3 and 4, resulting in complete oxidation during one cycle turn.

ATP Generating Steps in TCA Cycle

• NADH molecules: 3 NADH molecules are generated per cycle, each yielding 2.5 ATPs upon oxidation in the electron transport chain (ETC), totaling $3 \times 2.5 = 7.5$ ATP.
• $FADH_2$ molecule: Generates 1.5 ATP molecules.
• GTP molecule: One GTP molecule (equivalent to one ATP) is formed by substrate-level phosphorylation.
• Total ATP production: Per cycle turn, 10 high-energy phosphates (ATP) are produced.

Additional Points

• Irreversible step: Alpha ketoglutarate dehydrogenase reaction is the only irreversible step in the cycle.
• Spontaneity: Free energy changes favor spontaneous clockwise operation.
• Energy trapping: Only about 33% of liberated energy is trapped as ATP; the rest maintains body temperature.

Final Common Oxidative Pathway

• Central role: The citric acid cycle is the final common oxidative pathway for all foodstuffs.

Box 19.1: Significance of Citric Acid Cycle

1. Complete oxidation of acetyl CoA
2. ATP generation
3. Final common oxidative pathway
4. Integration of major metabolic pathways
5. Fat is burned on the wick of carbohydrates
6. Excess carbohydrates are converted as neutral fat
7. No net synthesis of carbohydrates from fat
8. Carbon skeletons of amino acids finally enter the citric acid cycle
9. Amphibolic pathway
10. Anaplerotic role

Integration of Major Metabolic Pathways

• Carbohydrates: Metabolized via glycolysis to pyruvate, converted to acetyl CoA for TCA cycle entry.
• Fatty acids: Broken down to acetyl CoA via beta-oxidation, entering the cycle.
• Glucogenic amino acids: Enter at various points in the cycle after transamination (Fig. 19.9).
• Ketogenic amino acids: Converted into acetyl CoA.
• Junction points: Integration achieved by key metabolites like pyruvate, acetyl CoA, and oxaloacetate (Figs 19.1 and 19.4), allowing carbon atoms from one source to be used for synthesizing another.

Fat is Burned on the Wick of Carbohydrates

• Analogy: Oxidation of fat (acetyl CoA) requires oxaloacetate, similar to how a flame needs a wick.
• Oxaloacetate's role: Acts as a catalyst, oxidizing acetyl CoA into two $CO_2$ molecules and regenerating itself.
• Carbohydrate dependence: The major source of oxaloacetate is pyruvate (from carbohydrates). Therefore, carbohydrates are essential for fat oxidation.

Excess Carbohydrates are Converted as Neutral Fat

• Pathway: Glucose to pyruvate to acetyl CoA to fatty acid.
• Irreversibility: Fat cannot be converted back to glucose because the pyruvate dehydrogenase reaction (pyruvate to acetyl CoA) is irreversible (Fig. 19.7).

No Net Synthesis of Carbohydrates from Fat

• Acetyl CoA fate: Acetyl CoA entering the cycle is completely oxidized to $CO_2$ by the time it reaches succinyl CoA (Fig. 19.2).
• Gluconeogenesis limitation: Acetyl CoA cannot be used for gluconeogenesis. Therefore, there is no net synthesis of carbohydrates from fat (Fig. 19.7).

Amino Acids Finally Enter the TCA Cycle

• Ketogenic amino acids: Catabolized to acetyl CoA but not converted to glucose; instead, they enter the TCA cycle or are channeled to ketone body formation (Fig. 19.9).
• Glucogenic amino acids: Converted to intermediates of the TCA cycle.

Amphibolic Pathway

• Nature: TCA cycle is amphibolic (both catabolic and anabolic), unlike purely catabolic (e.g., beta-oxidation) or anabolic (e.g., glycogen synthesis) pathways.
• Influx and efflux: Continuous influx and efflux of 4-carbon units (Fig. 19.8, 19.9).
• "Metabolic traffic circle": Compounds enter and leave the TCA cycle.
  • Anabolic reactions:
    • Oxaloacetate as precursor: Precursor of aspartate.
    • Alpha-ketoglutarate to glutamate conversion.
    • Succinyl CoA use in heme synthesis.
    • Citrate transport to cytoplasm: Mitochondrial citrate is transported to the cytoplasm, where it’s cleaved into acetyl CoA, the starting point for fatty acid synthesis (see Fig. 12.13).

Anaplerotic Role of TCA Cycle

• Precursor source: The citric acid cycle provides precursors for biosynthetic pathways (e.g., heme from succinyl CoA, aspartate from oxaloacetate).
• Anaplerotic reactions: To counterbalance losses and maintain 4-carbon unit concentrations, anaplerotic reactions are essential, "filling up" or "replenishing" the cycle.
• Other amino acids entering the TCA cycle are shown in Figure 19.9.
• Pyruvate carboxylation: Pyruvate can be carboxylated to malate by $NADP^+$-dependent malic enzyme.

REGULATION OF CITRIC ACID CYCLE

Citrate and Citrate Synthase

• Control point: Citrate formation from oxaloacetate and acetyl CoA is an important control step (step 1, Fig. 19.5).
• ATP inhibition: ATP acts as an allosteric inhibitor of citrate synthase.
• Citrate's influence: Citrate allosterically inhibits PFK (a key glycolysis enzyme), stimulates fructose-1,6-bisphosphatase (a key gluconeogenesis enzyme), and activates acetyl CoA carboxylase (a key fatty acid synthesis enzyme).

Availability and Cellular Need of ATP

• Energy charge dependent: When cellular energy charge is low, the cycle operates faster.
• Coupling to respiratory chain: Tightly coupled to the respiratory chain, providing ATP.
• ATP generation: The Krebs cycle is the largest ATP generator among metabolic pathways.
• Anaerobiasis (hypoxia) effect: Inhibits ETC, leading to NADH and $FADH_2$ accumulation, inhibiting the TCA cycle.

Isocitrate Dehydrogenase

• Step 3 regulation: ADP acts as a positive modifier, enhancing substrate binding; NADH is an inhibitor (Fig. 19.5).

Alpha Keto Glutarate Dehydrogenase

• Inhibition: Inhibited by succinyl CoA and NADH.

PDH Complex

• Inhibition: inhibited by Acetyl-CoA and NADH
• Activation: activated by non-acetylated CoA (COASH) and $NAD^+$
• Regulation: by phosphorylation and dephosphorylation.
• Activated by: $Mg^{2+}$ and $Ca^{2+}$

Further Regulation of TCA Cycle

• Regulation points: Regulation, similar to glycolysis, occurs at substrate entry and key cycle reactions.
• Acetyl CoA generation: Acetyl-CoA generation from carbohydrates (PDH complex reaction) is a major control point.
• PDH complex regulation: Inhibited by acetyl CoA and NADH; activated by non-acetylated CoA (COASH) and $NAD^+$.
• PDH regulation by phosphorylation: Pyruvate dehydrogenase activities are regulated by phosphorylation (by PDH kinase) and dephosphorylation (by PDH phosphatase).
  • Phosphorylation inhibits PDH, decreasing pyruvate oxidation.
  • PDH kinase activated by NADH and acetyl CoA; inhibited by pyruvate, ADP, COASH, $Ca^{2+}$ and $Mg^{2+}$.
  • PDH phosphatase is activated by $Mg^{2+}$ and $Ca^{2+}$.
• $NAD^+$/NADH ratio: Cellular ratio of $NAD^+$/NADH significantly impacts carbon flux through the TCA cycle, as three TCA cycle reactions and PDH utilize $NAD^+$.
• Substrate availability: Can regulate TCA flux; reduced oxaloacetate availability limits the citrate synthase reaction.
• Product inhibition: Controls TCA flux (e.g., citrate inhibits citrate synthase, a-KGDH is inhibited by NADH and succinyl CoA).
• Allosteric regulation: Key TCA cycle enzymes are allosterically regulated by $Ca^{2+}$, ATP and ADP.
• Thiamine requirement: TCA cycle operation requires Thiamine due to TPP co-enzyme requirement in PDH and Alpha KGDH reactions.
• Enzyme association: Enzymes of the TCA cycle are closely physically associated, enabling substrate channeling.
• Metabolon: A new term for such enzyme arrangements in a pathway.

Inhibitors of TCA Cycle

Non-physiological Inhibitors

• Aconitase inhibition: Inhibited by fluoroacetate (non-competitive).
• Alpha ketoglutarate dehydrogenase inhibition: Inhibited by Arsenite (non-competitive).
• Succinate dehydrogenase inhibition: Inhibited by malonate (competitive).