The TCA cycle

1. The TCA cycle Dr. S. Picksley H17, ext 5935

2. THE PROBLEM Petrol or diesel (hydrocarbons) + oxygen (O2) + spark Energy + C02 + H20 Combustion releases energy very fast & explosively. We need to release energy from food slowly and discretely, This is done by the TCA cycle and oxidative phosphorylation.

3. Learning objectives 1. Understand how complex foodstuffs (carbohydrate, fat & protein) are converted into energy (ATP). 2. The conversion of food to ATP is a two step process, in which catabolites (breakdown products of carbohydrate, fat & protein) are channelled into the TCA cycle and oxidised to produce C02 and energy rich (reduced) molecules of NADH and FADH2 (nucleoside cofactors) and GTP.

4. Learning objectives cont. 3. NADH and FADH2 then transfer their energy to ATP by electron transport / oxidative phosphorylation (the subject of the next lecture!). 4. In effect these two processes represent RESPIRATION - the process by which aerobic cells obtain energy from the oxidation of food by oxygen. 5. The TCA cycle is also a starting point for some biosynthetic pathways (anabolic metabolism, - from simple to complex molecules).

5. Recommended texts ‘Instant notes in biochemistry’ Section L1 The Citric acid cycle. By Hames et al. p291-305. ‘Biochemistry’ by Campbell. p532-571.

6. Overview We derive all our cellular energy from carbohydrates, fat and proteins. Carbohydrates, fat and proteins, are catabolised (broken down) to a common intermediate, acetyl CoA, that enters the TCA cycle. CoA stands for a co-enzyme A (an essential enzyme activator). By a series of enzyme catalysed reactions the two carbon atoms of acetyl CoA is oxidised (loses electrons) to C02 to produce GTP (from GDP) and four pairs of electrons. These electrons are transferred to nucleotide derived coenzymes, NAD+ and FAD, which finally transfer them to O2 (oxygen).

7. Overview cont. Energy is produced and trapped as ATP by oxidative phosphorylation. Energy is also produced during the TCA cycle in the form of GTP (which is formally equivalent to ATP). Energy use in man At rest we will consume half our body weight in ATP per day! Of course we cannot store this amount of ATP. As we consume energy, ATP --> ADP + Pi, we replace it by oxidising food molecules, and regenerating ATP molecules.   The production of ATP is initially carried out by enzymes of the TCA pathway and finally by an electron transport chain.

8. The TCA or Citric acid or Krebs Cycle Its called a cycle because the acetyl CoA reacts with a metabolite (oxaloacetate), that is regenerated by a series of enzyme catalysed reactions. Oxaloacetate + acetyl Coa ------> citrate Hence the alternative name, the Citric Acid Cycle. Citrate has three COO- groups, TriCarboxylic Acid. The cycle takes place in the mitochodria in all mammalian cells.

9. TCA cycle Location: occurs in the mitochondria of eukaryotic cells and in the cytosol of bacteria. Glycolysis Glucose ---> Pyruvate-------> Acetyl CoA Fats, Proteins ------> Acetyl CoA Acetyl CoA is the form in which fuel molecules enter the cycle. This cycle has eight well-characterised stages. We will only consider an overview of the cycle.

10. TCA cycle Acetyl CoA (C2) + oxaloacetate (C4) --> Citric acid (C6) Oxaloacetate (C4) + 2CO2 And then the cycle begins again. 2 carbon atoms enter and 2 leave. Yields per cycle: 1 GTP, 3NADH, 1FADH2, CoA + 2CO2

11. In the text books it will describe the TCA cycle as follows: AcetylCoA Citrate Isocitrate A-ketoglutarate Succinyl CoA Succinate Fumarate Malate oxaloacetate THIS LEVEL OF DETAIL IS NOT REQUIRED. THE AIM IS TO UNDERSTAND THE PRINCIPLES.

13. TCA cycle key points Acetyl CoA + oxaloacetate -----> Citric acid 2 Carbons + 4 Carbons 6 Carbons 2 CO2 Yields: 1 GTP, 3 NADH, 1 FADH2, CoA + 2CO2 Acetyl CoA is oxidised to CO2 This does not involve oxygen

14. Oxidation of acetyl CoA Acetyl CoA is oxidised to CO2 by the donation of electrons (e-) along with the hydrogen (H+) or hydride (H-)ion. H atom = H+ + e- (electron) Hydrogen ion or proton is H+ Hydride ion is H-, H- = H+ + 2e- Oxidation = loss of electrons. Reduction = gain of electrons. Oxidation and reduction occur side by side, as electrons are not created or destroyed

15. Electrons are donated to NAD+ or FAD NAD is nicotinamide adenine di-nucleotide. It is a co-enzyme, i.e. a chemical that is essential for enzyme activity but is easily dissociated from the protein with the loss of activity. NAD+ + 2H+ + 2e- = NADH + H+ (oxidised form) (reduced form) FAD is flavin adenine di-nucleotide. It is a prosthetic group, i.e. a chemical that is essential for activity and that is physically linked to an enzyme. FAD + 2 H+ + 2e- = FADH2 (oxidised form) (reduced form)

17. Oxidised and reduced forms of NAD+ and FAD

18. The TCA reaction Acetyl CoA + 3NAD+ + 1FAD + GDP + Pi + 2H20 CoA + 2CO2 + 3NADH + 3H+ + 1FADH2 + GTP. One complete cycle yields in energy terms: 1 GTP + 3NADH + 1FADH2. (=ATP) GTP is a nucleotide and an energy source just like ATP and is equivalent to ATP. The production of GTP during the TCA cycle is referred to as substrate level phosphorylation, i.e. it is directly produced from GDP + Pi, without an intermediate.

19. NADH & FADH2 are energy-rich carrier molecules NADH and FADH2 are energy-rich molecules, which transfer their energy to ATP molecules by an indirect route of transfering electrons via the electron transport chain to oxygen (oxidative phosphorylation). 1 molecule of NADH ------> 2.5 molecules of ATP (3 molecules of ATP in older textbooks) 1 molecule of FADH2 ------> 1.5 molecules of ATP (2 molecules of ATP in older textbooks)

20. Regulation of the TCA cycle by respiratory control The flow of intermediates through the TCA cycle is regulated by the demand for ATP. If energy demand is high as indicated by low [ATP]:[ADP] and [NADH]:[NAD+] there will be a high flow of intermediates through the TCA cycle to produce the required energy.

21. TCA cycle as a source of biosynthetic precursors • The TCA cycle in addition to generating energy rich • molecules also has a role in generating important • precursors for: • Synthesis of some amino acids • Synthesis of glucose • Synthesis of proteins • Synthesis of nucleic acids • Syntheis of fats • Synthesis of pophyrins (used to make haem for haemoglobin).