TCA Cycle Notes

Glucose

Dr. Sneha M. Pinto (s.pinto@surrey.ac.uk)
Tricarboxylic Acid (TCA) Cycle
Key Molecules:
- Glucose, Glucose 6-P, Pyruvate
- Fatty acid synthase (FAS), Glutamine, Malonyl-CoA
- ATP citrate lyase, Acetyl-CoA
- Oxaloacetate, Citrate, Cis-aconitate, Itaconate, Malate, Fumarate, Isocitrate, a-Ketoglutarate, Succinyl-CoA, Succinate
Key Enzymes:
- Pyruvate dehydrogenase, Citrate synthase, Aconitase, IRG1/Cis-aconitate decarboxylase, Malate dehydrogenase, Isocitrate dehydrogenase, a-ketoglutarate dehydrogenase, Succinyl-CoA synthetase, Succinate dehydrogenase/CII, Fumarate hydratase
Coenzymes/Factors:
- NAD+, NADH, FAD+, FADH
Processes:
- OXPHOS (Oxidative Phosphorylation), ADP, Pi, ATP

Learning Objectives

Describe cellular respiration.
Explain the production of Acetyl-CoA.
Provide an overview of the TCA/Citric Acid Cycle.
Describe intermediates and enzymes of the Citric Acid Cycle.
Describe the amphibolic role of the TCA cycle.
Discuss key regulatory points within the TCA cycle.

Cellular Respiration

Catabolic process where cells consume $O2$ and produce $CO2$ to break down organic molecules.
Carbohydrates, lipids, and amino acids are catabolized in three stages.
Provides more energy (ATP) from glucose than glycolysis.

Glycolysis Overview

Reaction: Glucose + 2 NAD+ + 2 ADP + 2 Pi → 2 Pyruvate + 2 NADH + 2 H+ + 2 ATP
$\Delta G'° = -146 \text{ kJ/mol}$
Overall Glucose Oxidation: 6 $O2$ → 6 $CO2$ + 6 $H_2O$
$\Delta G'° = -2840 \text{ kJ/mol}$
Only a small amount of energy available in glucose is captured by glycolysis.

Fatty Acid Oxidation Overview

Reaction: Palmitoyl-CoA + 7CoA + 7FAD + 7NAD+ + 7 $H_2O$ → 8 acetyl-CoA + 7FADH2 + 7NADH + 7H+
Acetyl-CoA enters the citric acid/TCA cycle.
8 Acetyl-CoA + 16 $O2$ + 80Pi + 80ADP → 8CoA + 80ATP + 16 $CO2$ + 16 $H_2O$
Overall from a 16C fatty acid:
Palmitoyl-CoA + 23 $O2$ + 108Pi + 108ADP → CoA + 108ATP + 16 $CO2$ + 23 $H_2O$

Energy Scale for Food

Calories = Energy
Carbohydrates: 4 Calories per gram
Protein: 4 Calories per gram
Fats: 9 Calories per gram

Stages of Cellular Respiration

Acetyl-CoA production
Acetyl-CoA oxidation
Electron transfer and oxidative phosphorylation

Mitochondrion Structure

Outer membrane:
- Freely permeable to small molecules and ions due to porin channels.
Inner membrane:
- Impermeable to most small molecules and ions, including H+.
- Contains respiratory electron carriers (Complexes I-IV), ADP-ATP translocase, ATP synthase (FoF1), and other membrane transporters.
Matrix:
- Contains pyruvate dehydrogenase complex, citric acid cycle enzymes, fatty acid β-oxidation enzymes, amino acid oxidation enzymes, DNA, ribosomes, ATP, ADP, Pi, $Mg^{2+}$ , $Ca^{2+}$ , $K+$ , and many soluble metabolic intermediates.

Stage 1: Acetyl-CoA Production

Organic fuels such as glucose are oxidized to two-carbon fragments in the form of acetyl-CoA.
Site of synthesis: mitochondrial matrix
Generates ATP/NADH

Acetyl-CoA: Central Molecule in Cellular Metabolism

Produced via:
- Glycogenolysis: Glycogen → Glucose
- Glycolysis: Glucose → Pyruvate
- β-Oxidation: Free fatty acids → Acetyl-CoA
- Lipolysis: Triglyceride → Fatty acids
- Proteolysis: Protein → Amino acids
Acetyl-CoA is used in:
- TCA cycle
- Ketone bodies
- Sterols and fatty acids synthesis
- Protein acetylation

Conversion of Pyruvate to Acetyl-CoA

Oxidative decarboxylation of pyruvate
Reaction: Pyruvate + CoA-SH + NAD+ → Acetyl-CoA + $CO_2$ + NADH
Catalyzed by the pyruvate dehydrogenase complex (E1 + E2 + E3), requiring TPP, lipoate, and FAD.
$\Delta G'° = -33.4 \text{ kJ/mol}$

Structure of Coenzyme-A

Coenzyme A (CoA) has a reactive thiol group to which the acetyl group is covalently linked, forming a thioester.

Stage 2: Acetyl-CoA Oxidation

Also known as the Tricarboxylic acid Cycle/Krebs Cycle.
Discovered by Hans Krebs in 1937 (Nobel Prize in Physiology/Medicine, 1953).
Central hub of cellular metabolism.
Meets cellular energy requirements by oxidizing the products of carbohydrate and fat metabolism.
Generates NADH, FADH2, and GTP.

Citric Acid Cycle (TCA Cycle)

Overview of Reactions:
- Acetyl-CoA + Oxaloacetate → Citrate (Citrate Synthase)
  - Claisen condensation: methyl group of acetyl-CoA converted to methylene in citrate
- Citrate → cis-Aconitate → Isocitrate (Aconitase)
  - Dehydration/rehydration: -OH group of citrate repositioned in isocitrate to set up decarboxylation in the next step.
- Isocitrate → α-Ketoglutarate (Isocitrate Dehydrogenase) + NADH, $CO_2$
  - Dehydrogenation: introduction of a double bond initiates methylene oxidation sequence
  - Oxidative decarboxylation: -OH group oxidized to carbonyl, which facilitates decarboxylation by stabilizing the carbanion formed on the adjacent carbon.
- α-Ketoglutarate → Succinyl-CoA (α-Ketoglutarate Dehydrogenase) + NADH, $CO_2$
  - Oxidative decarboxylation: pyruvate-dehydrogenase-like mechanism.
- Succinyl-CoA → Succinate (Succinyl-CoA Synthetase) + GTP
  - Substrate-level phosphorylation: energy of thioester conserved in phosphoanhydride bond of GTP
- Succinate → Fumarate (Succinate Dehydrogenase) + FADH2
  - Dehydrogenation: oxidation of -OH completes oxidation sequence; generates carbonyl positioned to facilitate Claisen condensation in the next step
- Fumarate → Malate (Fumarase)
  - Hydration: addition of water across a double bond introduces -OH group for the next oxidation step
- Malate → Oxaloacetate (Malate Dehydrogenase) + NADH

Step 1: Condensation of Acetyl-CoA and Oxaloacetate

Reaction: Acetyl-CoA + Oxaloacetate → Citrate + CoA-SH
Enzyme: Citrate Synthase
$\Delta G'° = -32.2 \text{ kJ/mol}$

Step 2: Isomerization by dehydration/rehydration

Citrate → cis-Aconitate → Isocitrate
Enzyme: Aconitase
Two-step process, involving the removal and then the addition of a water molecule.
$\Delta G'° = 13.3 \text{ kJ/mol}$

Iron-Sulfur center in Aconitase

Shows the iron-sulfur cluster and the binding of citrate.

Step 3: Oxidative decarboxylation by Isocitrate dehydrogenase

NAD-dependent enzyme occurs in the mitochondrial matrix and serves in the citric acid cycle.
NADP-dependent enzymes are present in both the mitochondrial matrix and the cytosol, generating NADPH necessary for fatty acid and sterol synthesis.

Step 4: Oxidative decarboxylation by α-Ketoglutarate dehydrogenase

Catalyzes the conversion of α-ketoglutarate to succinyl-CoA.
Produces NADH directly, providing electrons for the respiratory chain.
Highly regulated enzyme - determines the metabolic flux through the TCA cycle.

Step 5: Substrate-level phosphorylation by Succinyl-CoA synthetase

Reversible conversion of succinyl-CoA to succinate, coupled with the phosphorylation of GDP to GTP.
GTP formed by succinyl-CoA synthetase donates its terminal phosphoryl group to ADP to form ATP, in a reversible reaction catalyzed by nucleoside diphosphate kinase.

Step 6: Oxidation of an Alkane to Alkene by Succinate dehydrogenase

Reaction: Succinate + ubiquinone (Q) → Fumarate + ubiquinol ( $QH_2$ ).
SDH is embedded in the inner membrane of the mitochondria, allowing FADH2 to directly transfer its electrons into the electron transport chain.
Electron flow through these carriers to the final electron acceptor, $O_2$ , is coupled to the synthesis of ~1.5 ATP molecules.

Step 7: Hydration of a double bond by Fumarase

Reversible hydration/dehydration of fumarate to malate
Fumarate → L-Malate
$\Delta G'° = -3.8 \text{ kJ/mol}$

Step 8: Oxidation of Malate to Oxaloacetate by malate dehydrogenase

L-Malate → Oxaloacetate
$\Delta G'° = 29.7 \text{ kJ/mol}$
Regenerates oxaloacetate for the next cycle.

One turn of the Citric Acid Cycle

Input: Acetyl-CoA
Outputs: 2 $CO_2$ , 3 NADH, 1 FADH2, 1 GTP (ATP)
Regenerates: Oxaloacetate

Net result of the Citric Acid Cycle

Acetyl-CoA + 3NAD+ + FAD + GDP + Pi + 2 $H2O$ → 2 $CO2$ + 3NADH + FADH2 + GTP + CoA + 3H+

Direct and indirect ATP yield

Glycolysis of one molecule of glucose yields 2 NADH and 2ATP.
Production of 2 molecules of acetyl-CoA from pyruvate yields 2 NADH.
Citric acid cycle of 2 molecules of acetyl-CoA yields 6 NADH, 2 FADH2, and 2ATP

TCA cycle intermediates are amphibolic

NOT just oxidation of acetate
Hub of metabolism
- Other 4- and 5-carbon end products enter and serve as fuel
- Intermediates are also used as precursors in other biosynthetic pathways
Anaplerotic reactions replenish depleted cycle intermediates (Greek words "ana" (up) and "plerō" (to fill)

Regulation of the Citric Acid Cycle

Regulated at highly thermodynamically favorable (irreversible) steps.
General regulatory mechanisms:
- Activated by substrate availability.
- Inhibited by product accumulation.
Both glycolysis and TCA cycle pathways are inhibited by high levels of ATP, NADH, and citrate.

Summary

Cellular respiration
Intermediates & enzymes of the Citric Acid Cycle
Citric Acid Cycle: (indirect) Energy yield
Citric Acid Cycle: Hub of intermediary metabolism
Citric Acid Cycle: Regulation

Stage 3: Oxidative phosphorylation

Reduced coenzymes are oxidized, and electrons are transferred to $O_2$ .
Generates lots of ATP.