Tricarboxylic Acid (TCA) Cycle

Overview of the TCA Cycle

The Tricarboxylic Acid (TCA) Cycle, also known as the Citric Acid Cycle or Krebs Cycle, is a central metabolic pathway that oxidizes acetyl-CoA to CO2 and generates high-energy electron carriers (NADH, FADH2) for ATP production.

• Enzymes: Each step is catalyzed by a specific enzyme (e.g., citrate synthase, isocitrate dehydrogenase).

• Substrates and Products: Key substrates include acetyl-CoA and oxaloacetate; products include CO2, NADH, FADH2, and GTP/ATP.

• Structures: Students should be able to draw the structures of intermediates such as citrate, isocitrate, α-ketoglutarate, succinyl-CoA, succinate, fumarate, malate, and oxaloacetate.

Example: The condensation of acetyl-CoA and oxaloacetate forms citrate, catalyzed by citrate synthase.

Reactions Feeding Into and Out of the TCA Cycle

• Anaplerotic reactions: Replenish TCA intermediates (e.g., pyruvate carboxylase forms oxaloacetate).

• Cataplerotic reactions: Remove intermediates for biosynthesis (e.g., citrate for fatty acid synthesis).

• Transporters:

  • Phosphoenolpyruvate transporter: Moves PEP across mitochondrial membranes.

  • Malate-aspartate shuttle: Transfers reducing equivalents (NADH) between cytosol and mitochondria.

  • Tricarboxylic acid transporter: Exchanges TCA intermediates across membranes.

• Glyoxylate cycle: A variation in plants and bacteria allowing net conversion of acetyl-CoA to glucose.

Thermodynamics of the TCA Cycle

• Standard vs. Cellular Free Energies: Standard free energy change (') is measured under standard conditions; actual cellular free energy () depends on metabolite concentrations.

• Calculating Free Energy:

• K and Q:

  • K (equilibrium constant): Ratio of products to reactants at equilibrium.

  • Q (reaction quotient): Ratio at any point; if Q < K, reaction proceeds forward; if Q > K, reverse.

Regulation of the TCA Cycle

• Substrate Concentration: Levels of acetyl-CoA, oxaloacetate, and NAD+ affect flux.

• Energy Charge: High ATP/ADP ratio inhibits cycle; high ADP stimulates.

• Allosteric Regulation: Enzymes like isocitrate dehydrogenase are allosterically regulated by ADP (activator) and NADH (inhibitor).

• Phosphorylation: Some enzymes are regulated by reversible phosphorylation.

Electron Transport and Oxidative Phosphorylation

Electron Transport Chain (ETC)

The Electron Transport Chain is a series of protein complexes (I-IV) in the inner mitochondrial membrane that transfer electrons from NADH and FADH2 to oxygen, generating a proton gradient.

• Complex I: NADH:ubiquinone oxidoreductase; transfers electrons from NADH to ubiquinone (Q).

• Complex II: Succinate dehydrogenase; transfers electrons from succinate to Q.

• Complex III: Cytochrome bc1 complex; transfers electrons from QH2 to cytochrome c.

• Complex IV: Cytochrome c oxidase; transfers electrons from cytochrome c to O2, forming H2O.

• Electron Carriers: NADH, FADH2, ubiquinone (Q), cytochrome c.

• Proton Transfer: Electron flow is coupled to proton pumping, creating an electrochemical gradient.

Oxidative Phosphorylation

• Substrate Level vs. Oxidative Phosphorylation: Substrate level phosphorylation directly forms ATP in metabolic reactions (e.g., glycolysis), while oxidative phosphorylation uses the proton gradient to drive ATP synthesis.

• ATP Synthase (F0F1-ATPase): Enzyme complex that synthesizes ATP as protons flow back into the mitochondrial matrix.

• Regulation: Controlled by ADP availability, proton gradient, and respiratory control.

• Free Energy Calculations:

  • Free energy change for proton movement:

  • Where is the membrane potential and is Faraday's constant.

Lipid Metabolism

Mobilization of Fatty Acids

• Source: Triacylglycerols stored in adipose tissue.

• Digestion and Transport: Lipases hydrolyze triacylglycerols; fatty acids are transported in blood bound to albumin.

• Storage and Utilization: Fatty acids are stored as triacylglycerols and mobilized for β-oxidation during fasting.

Fatty Acid Activation and β-Oxidation

• Acyl-CoA Synthetase: Activates fatty acids to acyl-CoA using ATP.

• β-Oxidation: Sequential removal of two-carbon units as acetyl-CoA.

• Enzymes: Acyl-CoA dehydrogenase, enoyl-CoA hydratase, hydroxyacyl-CoA dehydrogenase, thiolase.

• Substrates/Products: Fatty acyl-CoA, FADH2, NADH, acetyl-CoA.

• Carbon Oxidation States: Students should be able to determine oxidation states of carbons in intermediates.

Fatty Acid Synthesis vs. Degradation

Ketone Bodies

• Produced from: Acetyl-CoA during fasting or diabetes.

• Main Products: Acetoacetate, β-hydroxybutyrate, acetone.

Regulation of Lipid Metabolism

• Malonyl-CoA: Inhibits carnitine shuttle, preventing fatty acid entry into mitochondria during synthesis.

• Carnitine Shuttle: Transports fatty acyl-CoA into mitochondria for β-oxidation.

• Acetyl-CoA Carboxylase (ACC): Key regulatory enzyme in fatty acid synthesis; activated by citrate, inhibited by palmitoyl-CoA.

Photosynthesis: Light and Dark Reactions

Capture of Light Energy

• Pigments: Chlorophylls and accessory pigments absorb light energy.

• Light Absorption: Excites electrons, initiating electron transfer chains.

Photosystems and Electron Transfer

• Photosystem II (PSII): Absorbs light, splits water, releases O2, and transfers electrons to plastoquinone.

• Photosystem I (PSI): Absorbs light, transfers electrons to NADP+ to form NADPH.

• Cyclic vs. Non-cyclic Electron Flow:

  • Cyclic: Electrons cycle back to PSI, generating ATP but not NADPH.

  • Non-cyclic: Electrons flow from water to NADP+, producing both ATP and NADPH.

  • Source of Electrons: Water (non-cyclic), PSI (cyclic).

  • Ultimate Electron Acceptor: NADP+ (non-cyclic).

• Proton Gradient: Electron flow generates a proton gradient across the thylakoid membrane, driving ATP synthesis.

Organization and Energetics

• Thylakoid Membranes: Photosystems and electron carriers are organized in the thylakoid membranes of chloroplasts.

• Free Energy Calculations: Light energy is converted to chemical energy, and the proton gradient's free energy can be calculated similarly to mitochondria.

Calvin Cycle (Dark Reactions)

• Three Stages:

  1. CO2 fixation (catalyzed by Rubisco)

  2. Reduction of 3-phosphoglycerate to glyceraldehyde-3-phosphate

  3. Regeneration of ribulose-1,5-bisphosphate

• Rubisco: Key enzyme for CO2 fixation; regulated by pH, Mg2+, and activator proteins.

Nitrogen and Amino Acid Metabolism

Nitrogen Fixation

• Definition: Conversion of atmospheric N2 to ammonia (NH3), catalyzed by nitrogenase.

• Symbiosis: Rhizobium bacteria form nodules on plant roots, fixing nitrogen for the plant.

Amino Acid Biosynthesis

• Biosynthetic Families: Amino acids are grouped by their precursor metabolites (e.g., α-ketoglutarate, oxaloacetate).

• Transamination: Transfer of amino groups between amino acids and α-keto acids.

  • General Reaction:

  • Enzyme: Aminotransferase (requires pyridoxal phosphate, PLP).

• Essential vs. Non-essential Amino Acids: Essential amino acids cannot be synthesized by humans and must be obtained from the diet.

Amino Acid Degradation

• Ketogenic Amino Acids: Degraded to acetyl-CoA or acetoacetate (e.g., leucine, lysine).

• Glucogenic Amino Acids: Degraded to pyruvate or TCA intermediates (e.g., alanine, glutamine).

• Classification Table:

  Amino Acid	Ketogenic	Glucogenic
  Leucine	Yes	No
  Lysine	Yes	No
  Alanine	No	Yes
  Phenylalanine	Yes	Yes
  Glutamine	No	Yes
  Additional info: Many amino acids are both ketogenic and glucogenic.

Nitrogen Transport and the Urea Cycle

• Nitrogen Transport: Nitrogen is transported as glutamine or alanine from tissues to the liver.

• Glucose-Alanine Cycle: Transfers amino groups from muscle to liver for urea synthesis.

• Urea Cycle: Converts toxic ammonia to urea for excretion.

  • Location: Reactions occur in both mitochondrial matrix and cytosol of hepatocytes.

  • Inputs: Ammonia, CO2, aspartate.

  • Outputs: Urea, fumarate.

  • Key Intermediates: Carbamoyl phosphate, citrulline, argininosuccinate, arginine, ornithine.

Nitrogen Assimilation and Amino Acid Oxidation

• Glutamate and Glutamine: Central in nitrogen assimilation and transport.

• Oxidative Degradation: Amino acids are deaminated and their carbon skeletons enter central metabolism.