The Tricarboxylic Acid (TCA) Cycle

Lecture 2: Biochemistry II: Carbohydrate Digestion and Metabolism

• This lecture focuses on the Tricarboxylic Acid (TCA) cycle, also known as the Krebs cycle or Citric Acid Cycle.
• The lecturer is Najeeb Alwais.

Learning Outcomes

• Define oxidative decarboxylation.
• Understand the components and regulation of the TCA cycle.
• Differentiate between substrate-level phosphorylation and oxidative phosphorylation.

The TCA Cycle

• Glycolysis yields 2 ATP, which is a low amount of energy.
• In eukaryotes, the TCA cycle occurs in the mitochondrial matrix.
• The Krebs Cycle serves both catabolic (degradation) and anabolic (biosynthetic) functions, making it amphipathic.

Role in Metabolism

• The Krebs Cycle plays a central role in metabolism.
• Degradation products from carbohydrates, lipids, and proteins enter the TCA cycle for oxidative metabolism, releasing energy.
• Metabolites from the TCA cycle can be used to synthesize glucose, lipids, and amino acids.

Overview of Catabolic Processes

• Stage I: Macromolecules break down into building blocks with no useful energy.
• Stage II: Products from Stage I are oxidized to acetyl-CoA with limited energy.
• Stage III: Acetyl-CoA is oxidized to CO₂ and H₂O, releasing energy.

Mitochondrial Compartments

• The matrix contains TCA cycle enzymes and handles the oxidative decarboxylation of pyruvate.
• The inner membrane, with its large surface area due to cristae (invaginations), houses electron transport chain proteins, transport proteins, establishes an electrochemical gradient of H+, and contains ATP synthase.
• The outer membrane contains channel proteins that allow molecules up to 5 kDa to enter the intermembrane space.

Acetyl Coenzyme A (Acetyl CoA)

• Acetyl CoA contains phosphate, pantothenic acid (vitamin B5), 3',5'-adenosine diphosphate, and 2-aminoethanethiol, ending in acetate.

Oxidative Decarboxylation of Pyruvate

• This is an irreverisble link from glycolysis to the TCA cycle via pyruvate dehydrogenase complex occurring in the mitochondrion.

Pyruvate Process

• Pyruvate is oxidized, releasing CO₂ in decarboxylation (C3 becomes C2).
• The acetate unit (C2) within pyruvate binds to Coenzyme A via a thioester bond, forming acetyl CoA.
• This step commits carbon atoms to oxidation via the TCA cycle or to lipid synthesis.
• Requires ATP or when two-carbon fragments are needed for lipid biosynthesis

Pyruvate Oxidative Decarboxylation Components

• The pyruvate dehydrogenase complex is a multi-enzyme complex with three enzymes.
• Pyruvate dehydrogenase (E1) uses thiamine pyrophosphate (TPP).
• Dihydrolipoamide acetyltransferase (E2) uses lipoamide.
• Dihydrolipoamide dehydrogenase (E3) uses FAD.
• The complex requires TPP attached to E1, lipoamide attached to E2, FAD attached to E3, and NAD+ and Coenzyme A as free coenzymes.

TCA Cycle Overview

• Acetyl CoA (2C) enters the cycle by joining with oxaloacetate (4C) to form citrate for futher stages
• There are eight steps in the TCA cycle.
• Cycle requires sufficient oxidizing capability, provided by oxygen.

Steps of the TCA Cycle

Step 1: Citrate Synthase

• Oxaloacetate binds to acetyl CoA to form citryl CoA (condensation).
• Citryl CoA is hydrolyzed by water releasing CoA and citrate.
• Citrate accumulation moves to the cytoplasm.
• This in turn inhibits phosphofructokinase (PFK), thus stopping glycolysis.

Step 2: Aconitase

• Citrate undergoes dehydration followed by hydration of cis-Aconitate.
• This forms isocitrate (isomerization).

Step 3: Isocitrate Dehydrogenase

• Isocitrate is oxidized, forming an unstable intermediate (oxalosuccinate).
• Alpha-ketoglutarate is formed via oxidative decarboxylation.

Step 4: α-Ketoglutarate Dehydrogenase

• A similar reaction mechanism to pyruvate dehydrogenase results in oxidative decarboxylation.

Step 5: Succinyl CoA Synthetase

• Energy from the thioester is transferred into phosphoryl-group transfer potential.
• Substrate-level phosphorylation forms GTP at the expense of succinyl CoA.

Step 6: Succinate Dehydrogenase

• Succinate is oxidized, and FAD is reduced.
• A carbon-carbon double bond (C=C) is formed via oxidation.

Step 7: Fumarase

• Water is used to perform the hydration of fumarate.
• This forms malate

Step 8: Malate Dehydrogenase

• Malate oxidation forms oxaloacetate.
• This oxaloacetate is ready to react with another acetyl CoA to repeat the cycle.

The TCA Cycle Oxidizes Acetyl CoA (Summary)

• For each acetyl CoA entering the cycle:
  • Acetyl CoA binds to the acetate portion (2C) with oxaloacetate (4C).
  • 8 steps (catalyzed by enzymes) convert acetate to 2 CO₂ molecules.
  • 3 NAD⁺ and one FAD are reduced to NADH and FADH₂
  • 1 GDP (= 1 ATP) is phosphorylated.
  • The initial acceptor molecule (oxaloacetate) is reformed and recycled.

Overall Reaction

• For each turn of the cycle: Acetyl CoA + 3NAD+ + FAD + GDP + Pi + 2H₂O results in releasing 2CO₂ + 3NADH + FADH₂ + GTP + 3H+ + CoA.

Energy Output per Acetyl CoA

• 3 NADH, 1 FADH₂, and 1 GTP are produced.
• Each glucose yields 2 pyruvate, hence 2 Acetyl CoA.
  • 2 × 3 NADH = 6 NADH
  • 2 × FADH₂ = 2 FADH₂
  • 2 × GTP = 2 GTP

Before the Cycle

• 2 NADH are produced from converting 2 pyruvate to 2 Acetyl CoA.

Regulation of the TCA Cycle

• Primarily regulated by ATP and NADH concentrations.
• Pyruvate dehydrogenase (entry to cycle):
  • Inhibited by acetyl CoA, ATP, and NADH.
  • Activated by NAD+, CoA-SH, ADP, and pyruvate.
• Isocitrate dehydrogenase (control point).
  • Inhibited by ATP and NADH.
  • Activated by ADP.
• α-Ketoglutarate dehydrogenase:
  • Inhibited by succinyl CoA, ATP, and NADH.
• Citrate synthase in bacteria:
  • Inhibited by ATP.

Pathways To and From the TCA Cycle

• Metabolites from the TCA cycle can go to several destinations including Amino acids, urea, pyrimidine nucleotides, carbohydrates and lipids.

Complete Oxidation of Glucose

• 2 ATP from glycolysis.
• 2 NADH from glycolysis (some energy lost in transport into mitochondria).
• 2 NADH from pyruvate dehydrogenase.
• 6 NADH from the TCA cycle.
• 2 FADH₂ from the TCA cycle.
• 2 GTP (ATP) from the TCA cycle.
• Calculations:
  • 2 + (2 × 1.5) + (2 × 2.5) + (6 × 2.5) + (2 × 1.5) + 2 = 30 ATP / glucose
  • 2 + (2 × 2.5) + (2 × 2.5) + (6 × 2.5) + (2 × 1.5) + 2 = 32 ATP / glucose
• 2 NADH from glycolysis.

Additional Information

• In eukaryotes, the ETC and ATP synthase are in the mitochondrial inner membrane.
• In TCA cycle reactions, NAD+ gains 2 electrons and is reduced.
• In the ETC, NADH loses electrons to other electron carriers and is oxidized.
• ATP synthase is an enzyme that synthesizes ATP using a proton gradient.
• ETC = series of redox reactions.

ATP and ADP

• Energy is required to add the third phosphate.
• Energy is released when a phosphate is removed.
• ADP-ATP conversions are a major means of transferring energy.

ATP-ADP Cycle

• Includes phophoanhydride bonds
• Cycle transforms energy from sunlight or food to cellular, or chemical synthesis.

ATP Production

• Substrate-level phosphorylation: energy released directly from a substrate (e.g., phosphoenolpyruvate + ADP forming pyruvate + ATP).
• Oxidative phosphorylation: phosphate comes from a solution and requires energy input from a proton gradient

Key terms

• Oxidative decarboxylation
• Substrate level phosphorylation
• Inner membrane
• Cristae
• Amphipathic
• Redox reaction