Lecture 14: The TCA Cycle 4BBY1013 Biochemistry

The TCA Cycle

Overview

• Alternative names:
  • Krebs cycle
  • Citric acid cycle
  • Tricarboxylic acid cycle
  • Common terminal pathway
• Definition: Oxidation of acetyl CoA to CO_2 and water.
• Location: Mitochondrial matrix.
• Tissues: All tissues with mitochondria (not red blood cells or white muscle fibres).
• Functions: Energy trapping, biosynthesis of intermediates.

Learning Outcomes

• Describe the reaction catalysed by pyruvate dehydrogenase for the conversion of pyruvate to acetyl-CoA.
• Outline the central role of acetyl-CoA in the metabolism of glucose, fatty acids and amino acids.
• Outline the metabolic events of the TCA cycle that result in the conversion of acetyl-CoA into two molecules of CO_2.
• Emphasise the role of the dehydrogenase enzymes in producing reduced cofactors (NADH and FADH_2).
• Indicate the metabolic importance of oxaloacetate and show how it can be produced from pyruvate by the pyruvate carboxylase reaction.
• Indicate how the TCA cycle can act as a source of biosynthetic precursors, e.g. for amino acids and haem.

Catabolism Stages

• Catabolism of sugars, fats & amino acids occurs in 3 stages:
  1. Glycolysis (Glucose to Pyruvate)
  2. β-oxidation (Fatty Acids to Acetyl-CoA) & Transamination (Amino Acids to Acetyl-CoA)
  3. TCA Cycle (Acetyl-CoA to CO_2)
• The Krebs cycle produces CO_2 and reduced cofactors (2H).
• These reduced cofactors are then used in oxidative phosphorylation to produce H_2O and ATP.

Summary of TCA Cycle Events

• Brief overview of events.
• Account for where CO_2 is lost and where carbon atoms are added into the cycle.
• C2 + C4 -> C6 -> C5 + CO2 -> C4 + CO2 -> C4

TCA Cycle Steps

• It is beneficial to learn the names of the enzymes and substrates.
• The following steps are part of the cycle:
  • Citrate
  • Isocitrate
  • α-Ketoglutarate
  • Succinyl-CoA
  • Succinate
  • Fumarate
  • Malate
  • Oxaloacetate
• Key products:
  • CO_2
  • NADH + H^+
  • FADH_2
  • GTP

Conversion of Pyruvate to Acetyl CoA (Link Reaction)

• Very important reaction!
• IRREVERSIBLE reaction!
• Produces 1 NADH and 1 CO_2
• Reaction: CH3 CO COOH + CoASH + NAD^+ [\longrightarrow{\text{Pyruvate dehydrogenase}}] CH3 CO S CoA + CO_2 + NADH + H^+

Coenzyme A

• Coenzyme A forms thioester bonds with carboxylic acids
• CoA-SH + CH3 C-O-H [\longrightarrow{}] CH3 C -S CoA + H_2O
• You don’t need to worry about the Blob

(1) Condensation Reaction

• First reaction to start the TCA cycle.
• KEY reaction to learn!
• Count the carbon atoms!
• Acetyl-CoA + Oxaloacetate -> Citrate (Citrate synthase).

(2) Isomerisation

• Citrate -> Isocitrate (Aconitase)
• Boring reaction, just moving things around

(3) First Loss of CO_2

• Isocitrate -> α-Ketoglutarate + H^+ (Isocitrate dehydrogenase)
• KEY REACTION!
• First loss of CO_2
• Produce NADH

(4) Second Loss of CO_2

• α-Ketoglutarate -> Succinyl CoA + H^+ (α-Ketoglutarate dehydrogenase)
• KEY REACTION!
• Second loss of CO_2
• Produce NADH

(5) Trapping Thioester Bond Energy as GTP

• Succinyl CoA -> Succinate (Succinyl-CoA synthetase)
• KEY REACTION!
• Produce ATP (as GTP)

(6) Conversion of Succinate to Fumarate

• Succinate -> Fumarate (Succinate dehydrogenase)
• KEY REACTION!
• Produce FADH_2

(7) Conversion of Fumarate to Malate

• Fumarate -> Malate (H_2O, Fumarase)
• Boring reaction, just adding water

(8) Conversion of Malate to Oxaloacetate

• Malate -> Oxaloacetate + H^+ (Malate dehydrogenase)
• KEY REACTION!
• Produce NADH
• Re-formed OAA – now ready to start the cycle again!

Question

• If acetyl CoA is only 2 carbon atoms and we have already lost 2 carbons through the previous two decarboxylation reactions, then why do we continue the cycle?
• We need to re-generate Oxaloacetate and also we get more of our high energy intermediates (NADH and FADH_2)

TCA Cycle Complete

• All steps are completed regenerating where we started.

Generation of ATP

• The re-oxidation of NADH to NAD^+ and FADH_2 to FAD via the Electron Transport Chain results in synthesis of ATP from ADP and Pi (Oxidative Phosphorylation) – covered in greater detail in Dr. Hunt’s lecture

Energy Yields of TCA Cycle

• 3 enzyme reactions produce NADH and H^+
• 1 enzyme reaction produces FADH_2
• 1 enzyme reaction produces GTP
• ATP yields:
  • 3 x 3 ATP
  • 1 x 2 ATP
  • 1 GTP
  • Total 12 ATP (old numbers)
  • 3 x 2.5 ATP
  • 1 x 1.5 ATP
  • 1 x GTP
  • Total 10 ATP (new numbers)

Energy Yields from 1 Molecule of Glucose

• How many ATP can we get from the complete oxidation of 1 molecule of glucose?
  • Assume each NADH = 2.5 ATP
  • Assume each FADH_2 = 1.5 ATP
  • Assume each GTP = 1 ATP
• Glycolysis: 2 ATP, 2NADH (2x2.5 = 5 ATP)→ 7 ATP
• Link reaction: 1 NADH (do it 2x for each pyruvate) → 5ATP
• TCA Cycle: 10 ATP per turn (2 turns) → 20 ATP
• Total = 32 ATP!

Irreversibility of Key Stages

• 3 enzyme steps are highly exergonic & irreversible:
  • citrate synthase
  • isocitrate dehydrogenase
  • α-ketoglutarate dehydrogenase
• Why are these three the only irreversible reactions?

Biosynthetic Role of TCA cycle

• Intermediates can be used for biosynthesis:
  • Citrate → Acetyl-CoA
  • α-Ketoglutarate → Glutamate → Other amino acids, purines, pyrimidines
  • Succinyl-CoA → Haem
  • Oxaloacetate → Aspartate → Other amino acids, purines

Anaplerotic 'topping up' of TCA cycle

• Replenishing TCA cycle intermediates:
  • Pyruvate → Oxaloacetate (Pyruvate carboxylase)
  • Pyruvate → Malate (Malic enzyme)

Pyruvate Carboxylase Reaction

• Pyruvate carboxylase reaction also allows regeneration of glucose from pyruvate:
  • Pyruvate --(Pyruvate Kinase)--> Phosphoenol pyruvate --(PEP carboxykinase)--> Oxaloacetate
• Which irreversible enzyme from glycolysis catalyses the reaction from PEP to pyruvate?
  • Pyruvate Kinase!