Comprehensive Study Guide for the Tricarboxylic Acid (TCA) Cycle

Overview of the Tricarboxylic Acid (TCA) Cycle

• The Tricarboxylic Acid Cycle, also known as the Citric Acid Cycle or Krebs Cycle (CHE 3111), is a cyclic series of 8 enzyme-catalyzed reactions.
• It is a metabolic pathway that occurs within the mitochondrial matrix.
• The mitochondrial structure relevant to this cycle includes:   - Outer membrane   - Inner membrane   - Intermembrane space   - Matrix   - Cristae   - DNA   - Ribosome   - Granules   - ATP synthase particles

Transport of Pyruvate into the Mitochondrial Matrix

• Outer Mitochondrial Membrane (OMM): Pyruvate passes through the OMM, probably via a relatively non-specific, voltage-dependent anion channel (VDAC), or porin. This transport is tied to $H^+$ symport to avoid the collapse of the inner mitochondrial $H^+$ gradient.
• Inner Mitochondrial Membrane (IMM): Pyruvate transport across the IMM is facilitated by two Mitochondrial Pyruvate Carriers (MPCs), identified as MPC1 and MPC2.

Oxidative Decarboxylation of Pyruvate to Acetyl CoA

• Under aerobic conditions, pyruvate undergoes oxidative decarboxylation to become Acetyl CoA within the mitochondria.
• Acetyl CoA formation serves as the essential link between Glycolysis and the Citric Acid Cycle.
• Acetyl CoA, including that derived from glucose, is completely oxidized to $CO_2$ in the TCA cycle.
• The TCA cycle is the final common pathway for the oxidation of carbohydrates, amino acids, and fatty acids.

The Pyruvate Dehydrogenase (PDH) Complex

• The formation of Acetyl CoA occurs in 5 distinct steps catalyzed by the multienzyme complex known as pyruvate dehydrogenase.
• Catalytic Sites: The enzyme complex consists of three catalytic sites:   1. Pyruvate dehydrogenase (E1)   2. Dihydrolipoyl transacetylase (E2)   3. Dihydrolipoyl dehydrogenase (E3)
• Reaction Formula:   - $CH_3-C(=O)-COO^- + \text{CoA} + \text{NAD}^+ \rightarrow CH_3-C(=O)-S-\text{CoA} + CO_2 + \text{NADH} + H^+$   - Pyruvate + Acetyl CoA or CoASH results in Acetyl CoA + $CO_2$ + NADH + $H^+$.

Coenzymes and Vitamins in the Pyruvate Dehydrogenase System

• The enzyme complex requires five different coenzymes or prosthetic groups:   - Thiamine pyrophosphate (TPP)   - Flavin adenine dinucleotide (FAD)   - Coenzyme A (CoA or CoA-SH)   - Nicotinamide adenine dinucleotide (NAD)   - Lipoate ($\text{Lip}(S-S)$ is the oxidized form; $\text{Lip}(SH-SH)$ is the reduced form).
• Four essential vitamins in human nutrition are vital components of this system:   - Thiamine: found in TPP.   - Riboflavin: found in FAD.   - Niacin: found in NAD.   - Pantothenate: found in CoA.

Control and Regulation of Pyruvate Dehydrogenase

• Entry of Acetyl CoA derived from carbohydrates is regulated by this enzyme complex. Because mammals have no other pathways to synthesize Acetyl CoA from pyruvate, strict control is vital.
• End Product Inhibition:   - High levels of Acetyl CoA inhibit the E2 component of the enzyme complex.   - High levels of NADH inhibit the E2 component (as both are end products of the reaction).
• Covalent Modification (Phosphorylation/Dephosphorylation):   - Inactivation: The E1 subunit is inactivated by the phosphorylation of a serine residue, catalyzed by pyruvate dehydrogenase kinase in the presence of ATP.   - Kinase Activation: Pyruvate dehydrogenase kinase is activated by the products NADH and Acetyl CoA.   - Kinase Inhibition: The kinase is inhibited by pyruvate, ADP, $Ca^{2+}$, high $Mg^{2+}$, and $K^+$, leading to the activation of pyruvate dehydrogenase.   - Reactivation: The enzyme is reactivated by dephosphorylation (hydrolysis of the phosphoserine residue) by pyruvate dehydrogenase phosphatase.

Sources of Acetyl CoA

• Acetyl CoA is derived from three primary sources:   1. Oxidative decarboxylation of pyruvate.   2. Breakdown of fatty acids.   3. Breakdown of individual amino acids.

The Eight Enzyme-Catalyzed Steps of the TCA Cycle

1. Condensation: Acetyl-CoA ($CH_3-C(-S-\text{CoA})=O$) and Oxaloacetate ($0=C(COO^-)-CH_2-COO^-$) react with $H_2O$ to form Citrate. This releases CoA-SH and is catalyzed by citrate synthase.
2. Dehydration (Isomerization Step A): Citrate is converted to cis-Aconitate by the enzyme aconitase through the removal of $H_2O$.
3. Hydration (Isomerization Step B): cis-Aconitate is converted to Isocitrate by aconitase through the addition of $H_2O$.
4. Oxidative Decarboxylation: Isocitrate is converted to $\alpha$-Ketoglutarate, releasing $CO_2$ and producing NADH from $\text{NAD}^+$ (catalyzed by isocitrate dehydrogenase).
5. Oxidative Decarboxylation: $\alpha$-Ketoglutarate reacts with CoA-SH and $\text{NAD}^+$ to form Succinyl-CoA, releasing $CO_2$ and NADH.
6. Dehydrogenation: Succinate is oxidized to Fumarate by succinate dehydrogenase, producing $FADH_2$ from FAD.
7. Hydration: Fumarate is converted to Malate by the enzyme fumaras with the addition of $H_2O$.
8. Dehydrogenation: Malate is converted back to Oxaloacetate by malate dehydrogenase, producing NADH from $\text{NAD}^+$.

Regulation and Energy Charge of the TCA Cycle

• Citrate Synthase: Allosterically inhibited by ATP. ATP increases the $K_m$ for Acetyl CoA, meaning as ATP levels rise, the enzyme becomes less saturated with Acetyl CoA and less citrate is formed.
• Isocitrate Dehydrogenase:   - Allosterically stimulated by ADP, which enhances substrate affinity.   - Binding of isocitrate, $\text{NAD}^+$, $Mg^{2+}$, and ADP is mutually cooperative.   - Inhibited by NADH (via direct displacement of $\text{NAD}^+$) and inhibited by ATP.
• $\alpha$-Ketoglutarate Dehydrogenase:   - Inhibited by its own products: Succinyl CoA and NADH.   - Inhibited by a high energy charge.
• Summary of Regulation:   - Pyruvate to Acetyl CoA: Inhibited by ATP, Acetyl CoA, and NADH.   - Citrate formation: Inhibited by ATP.   - Isocitrate to $\alpha$-Ketoglutarate: Inhibited by ATP, Stimulated by ADP.   - $\alpha$-Ketoglutarate to Succinyl CoA: Inhibited by Succinyl CoA and NADH.

Amphibolic Nature and Biological Importance of the TCA Cycle

• The TCA cycle is the "HUB" of metabolic systems.
• It accounts for the major portion of carbohydrate, fatty acid, and amino acid oxidation.
• It generates significant biosynthetic precursors (intermediates).
• Amphibolic definition: The cycle is amphibolic because it operates both anabolically (for synthesis) and catabolically (for breakdown).

Anabolic Pathways and Biosynthesis from TCA Intermediates

1. Glucose Biosynthesis: Malate is transported to the cytosol where glucose biosynthesis occurs.
2. Lipid Biosynthesis: Acetyl CoA cannot cross the mitochondrial membrane. Citrate is transported to the cytosol and converted back into Acetyl CoA and Oxaloacetate:    - $\text{Citrate} + \text{ATP} + \text{CoA} \rightleftharpoons \text{ADP} + P_i + \text{OAA} + \text{Acetyl CoA}$
3. Amino Acid Biosynthesis: Uses TCA intermediates in two main ways:    - Reductive Amination: $\alpha\text{-Ketoglutarate} + \text{NAD(P)H} + NH_4^+ \rightleftharpoons \text{Glu} + \text{NAD(P)}^+ + H_2O$.    - Transamination Reactions:      - $\alpha\text{-Ketoglutarate} + \text{Ala} \rightarrow \text{Pyruvate} + \text{Glu}$ (catalyzed by aminotransferase).      - $\text{Oxaloacetate} + \text{Ala} \rightarrow \text{Pyruvate} + \text{Asp}$ (catalyzed by aminotransferase).
4. Porphyrin Biosynthesis: Succinyl CoA is used in the synthesis of heme.

Integrated Metabolic Siphoning and Intermediates

• Siphoning out intermediates:   - Citrate leads to Fatty acids and sterols.   - $\alpha$-Ketoglutarate leads to Glutamate, which can become Purines, Glutamine, Proline, or Arginine.   - Succinyl-CoA leads to Porphyrins and heme.   - Oxaloacetate leads to Aspartate (Pyrimidines, Asparagine) and Phosphoenolpyruvate (PEP).   - PEP leads to Glucose and various amino acids (Serine, Glycine, Cysteine, Phenylalanine, Tyrosine, Tryptophan).
• Replacing/Replenishing intermediates: Intermediates used for biosynthesis must be replaced. Reactions that replenish cycle intermediates are called anaplerotic reactions.

Anaplerotic Reactions and Replenishment

• All biosynthetic reactions using TCA intermediates require free energy, which is produced by the TCA cycle itself.
• Pyruvate Carboxylase Reaction: The main anaplerotic reaction.   - $\text{Pyruvate} + CO_2 + \text{ATP} + H_2O \rightarrow \text{OAA} + \text{ADP} + P_i + 2H^+$   - This enzyme is activated by high levels of Acetyl CoA.
• Specific Anaplerotic Reactions and Locations:   - Pyruvate carboxylase: $\text{Pyruvate} + HCO_3^- + \text{ATP} \rightarrow \text{oxaloacetate} + \text{ADP} + P_i$. Occurs in the Liver and Kidney.   - PEP carboxykinase: $\text{Phosphoenolpyruvate} + CO_2 + \text{GDP} \rightarrow \text{oxaloacetate} + \text{GTP}$. Occurs in Heart and Skeletal muscle.   - PEP carboxylase: $\text{Phosphoenolpyruvate} + HCO_3^- \rightarrow \text{oxaloacetate} + P_i$. Occurs in higher plants, yeast, and bacteria.   - Malic enzyme: $\text{Pyruvate} + HCO_3^- + \text{NAD(P)H} \rightarrow \text{malate} + \text{NAD(P)}^+$. Widely distributed in eukaryotes and prokaryotes.

Catabolic Pathways Leading to TCA Intermediates

• Various catabolic pathways generate TCA intermediates to fuel the cycle or facilitate replenishment:   1. Fatty Acids (odd chain): Breakdown leads to Succinyl CoA.   2. Amino Acids (Ile, Met, Val): Breakdown leads to Succinyl CoA.   3. Transamination/Deamination: Transamination and deamination of amino acids lead to Oxaloacetate (OAA) and $\alpha$-Ketoglutarate.
• These reactions are reversible; depending on metabolic need, they either siphon intermediates from the cycle for synthesis or replenish the cycle for energy production.