The Tricarboxylic Acid Cycle

TCA cycle overview
Glycolysis converts glucose to pyruvate
Produces 2 molecules of 2ATP per glucose
Large amounts of potential energy from glucose remains unused
Aerobic oxidation of pyruvate ensures that this energy is not lost
The TCA cycle is the final common pathway for the oxidation of fuel molecules such as amino acids, fatty acids and carbohydrates
The cycle is also an important source of precursors, not only for the storage forms of fuel, but also for the building blocks of many other molecules such as amino acids, nucleotides bases and sterols

1. BTN223: The Tricarboxylic Acid Cycle* * AKA Citric Acid Cycle *AKA Krebs Cycle Prof. Riaan den Haan Hans Adolf Krebs. Biochemist; born in Germany. Worked in Britain. His discovery in 1937 of the ‘ Krebs cycle ’ of chemical reactions was critical to the understanding of cell metabolism and earned him the 1953 Nobel Prize for Physiology or Medicine.

2. Aerobic cells use a metabolic “wheel”, the citric acid-/Krebs-/ tricarboxylic acid cycle to generate energy from pyruvate In eukaryotes the reactions of the citric acid cycle take place inside mitochondria TCA cycle - overview

3. Glycolysis converts glucose to pyruvate Produces 2 molecules of ATP per glucose Large amounts of potential energy from glucose remains unused Aerobic oxidation of pyruvate ensures that this energy is not lost The TCA cycle is the final common pathway for the oxidation of fuel molecules such as amino acids, fatty acids and carbohydrates The cycle is also an important source of precursors , not only for the storage forms of fuel, but also for the building blocks of many other molecules such as amino acids, nucleotide bases and sterols TCA Cycle - overview

4. The TCA cycle consists of a series of oxidation-reduction reactions in the mitochondria Under aerobic conditions pyruvate from glycolysis is oxidatively decarboxylated to acetyl-coenzyme A (acetyl- CoA ) Acetyl- CoA is oxidized to two molecules of carbon dioxide This involves a 24-electron oxidation process This oxidation process results in the reduction of coenzymes NAD +  NADH and FAD  FADH 2 , some ATP (or equivalent) is also produced The coenzymes are transported to the electron transport pathway, where they are oxidized to drive the synthesis of ATP The function of the TCA cycle is the harvesting of high-energy electrons from carbon fuels TCA Cycle - overview

5. The TCA cycle seems like a complicated way to oxidize acetate units to CO 2 – why? The oxidation of acetate to CO 2 is used to reduce NAD + to NADH But, this cannot happen in a single step because acetate can’t be directly oxidized into CO 2 Oxidation of an acetyl group to CO 2 requires a C-C cleavage This has to be done in an energetically favorable way under cellular conditions What Is the Chemical Logic of the TCA Cycle?

6. C-C cleavage reactions in biological systems can only occur in one of two ways: Between carbons Cs α and β to a carbonyl group α -cleavage of an α - hydroxyketone (between COO - and C-OH) What Is the Chemical Logic of the TCA Cycle?

7. Neither of these strategies suitable for acetate (or Acetyl- CoA ) No β-carbon No hydroxyl (OH) group Instead: acetate is condensed with oxaloacetate to form citrate Citrate contains a β-carbon and can undergo β-cleavage Therefore During the TCA cycle, 2 carbons flow into the cycle via acetyl- CoA Acetyl- CoA cannot be oxidized directly These 2 carbons condense with the final substrate of TCA cycle ( oxaloacetate ) to form a 6-carbon structure citrate Citrate can undergo oxidation This oxidation leads to the generation of 2 carbon dioxide molecules TCA cycle combines β-cleavage reaction with oxidation to form CO 2 , regenerate oxaloacetate and capture liberated energy in NAD and ATP What Is the Chemical Logic of the TCA Cycle?

8. Sources: glucose (through pyruvate), fatty acids, amino acids, ketone bodies Central role in citric acid cycle, oxidative phosphorylation , fatty acid metabolism, cholesterol biosynthesis Acetyl- CoA – entry point to the TCA

9. Glycolysis occurs in the cytoplasm and the TCA cycle and oxidative phosphorylation in the mitochondria Pyruvate must enter the mitochondria to enter the TCA cycle Pyruvate is then converted to acetyl- CoA by the process of o xidative decarboxylation via the pyruvate dehydrogenase enzyme complex Pyruvate conversion to Acetyl- CoA

10. Multi-enzyme complex (series of enzymes linked together), noncovalent assembly of three enzymes in this case – uses 5 co-enzymes Catalyzes oxidative decarboxylation of pyruvate to acetyl- CoA Combines a redox reaction (pyruvate donates electron, NAD + receives electron) and decarboxylation (loss of carbons in form of carbon dioxide) Pyruvate dehydrogenase

11. Large multi-enzyme complex Multiple copies of three enzymes: E1, E2, E3 E1: pyruvate dehydrogenase E2: dihydrolipoamide acetyltransferase E3: dihydrolipoamide dehydrogenase Inner core Icosahedral structure 60 copies of E2 12 copies of E3 binding protein (E3BP) that links E3 to E2 Periphery: 30 copies of E1 (tetramer of subunits a 2 b 2 ) 12 copies of E3 (homodimer) Pyruvate dehydrogenase

12. Pyruvate dehydrogenase Eukaryotic PDH is one of the largest multi-enzyme complexes known, ~50nM and ~9.5 mega-Daltons

13. Coenzyme = Cofactor which is loosely bound to the enzyme Prosthetic group = Cofactor which is tightly bound to the enzyme PDH: prosthetic groups and coenzymes Enzyme Abbreviated Prosthetic group Pyruvate dehydrogenase E1 Thiamine pyrophosphate (TPP) Dihydrolipoyl transacetylase E2 Lipoic Acid ( Lipoamide ) Dihydrolipoyl dehydrogenase E3 FAD Other Coenzymes Coenzyme A NADH and NADPH

14. Thiamine pyrophosphate (TPP) Prosthetic group on E1 Synthesized from Thiamine (Vitamin B1) Assists in the decarboxylation process The flavin coenzyme (FAD) Prosthetic group on E3 Synthesized from riboflavin (Vitamin B2) Involved in one or two electron transfer reactions The nicotinamine coenzyme (NAD) Synthesized from nicotinamide (Vitamin B3) NAD + /NADH carry out hydride transfer reactions Lipoic acid ( Lipoamide ) Prosthetic group on E2 Contains a disulfide and acts as an arm to transfer acetyl group Coenzyme A Synthesized from cysteamine , panthothenate and ATP In TCA cycle it carries the acetyl group in acetyl- CoA PDH: prosthetic groups and coenzymes

15. Pyruvate reacts with TPP and is decarboxylated (E1) Carbon dioxide is released Hydroxyethyl -TPP is formed Hydroxyethyl -TPP reacts with lipoamide (E1/E2) Hydroxyethyl group is transferred to lipoic acid ( lipoamide ) Oxidized to form acetyl dihydrolipoate (-lipoamide) Acetyl group is transferred to CoA to form acetyl- CoA and lipoamide-dithiol (reduced disulfide) (E2) The reduced lipoamide is reoxidized by FAD to form FADH 2 (E3) The FADH 2 is reoxidized by NAD + to form NADH PDH: mechanism

16. Thus the final products of the PDH complex reaction are Acetyl- CoA NADH CO 2 All other reactants are regenerated back to their original form PDH: mechanism

17. BTN223: The Tricarboxylic Acid Cycle* * AKA Citric Acid Cycle *AKA Krebs Cycle

18. Glycolysis converts glucose to pyruvate Pyruvate is fed into TCA cycle under aerobic conditions Pyruvate is converted to acetyl- CoA via the multi-enzyme pyruvate dehydrogenase Pyruvate dehydrogenase consists of 3 enzymes and needs 5 different coenzymes End products are CO 2 , acetyl- CoA , and NADH Recap https://www.youtube.com/watch?v=_cXVleFtzeE

19. First 4 reactions in TCA cycle ends in the production of succinyl-CoA and 2 molecules of CO 2 TCA cycle: Acetyl- CoA to Succinyl-CoA and the production of CO 2 Substrate Product Enzyme Other Oxaloacetate + Acetyl-CoA Citrate Citrate synthase Citrate Isocitrate Aconitase Isocitrate α - ketoglutarate Isocitrate dehydrogenase CO 2 α - ketoglutarate Succinyl -CoA α - ketoglutarate dehydrogenase CO 2

20. First reaction in TCA cycle is a synthase reaction - a new molecule is made but ATP is not used Oxaloacetate + Acetyl- CoA + H 2 O  Citrate + CoA Initiates TCA cycle Acetyl- CoA condenses with oxaloacetate to form citrate Citrate synthase is a dimer – each subunit binds oxaloacetate and acetyl- CoA Binding of oxaloacetate induces a conformational change which facilitates the binding of acetyl- CoA TCA-Cycle Reaction 1: Citrate Synthase

21. Reaction is irreversible due to large energy of forward reaction (large negative ∆G) NADH is an allosteric inhibitor NADH is a product of the TCA cycle TCA cycle will slow down if too much NADH Succinyl-CoA is an allosteric inhibitor Later intermediate in cycle Analog of acetyl- CoA Can bind citrate synthase but not react with oxaloacetate = inhibition of reaction Regulation of citrate synthase

22. Citrate is a poor substrate for oxidation Aconitase isomerizes citrate to yield isocitrate which has a secondary -OH, which can be readily oxidized Isomerization of citrate (tertiary alcohol) to isocitrate (secondary alcohol) Citrate  [H2O + cis-Aconitate ]  Isocitrate Aconitase contains an iron-sulfur center as a prosthetic group which facilitates the rearrangement reaction Dehydration reaction followed by a hydration with an aconitate intermediate Water is abstracted from citrate to yield aconitate (dehydration) H and OH are added back in opposite positions to produce isocitrate Citrate needs to become isocitrate so that in next step decarboxylation is possible TCA-Cycle Reaction 2: Aconitase

23. Oxidative decarboxylation of isocitrate yields α - ketoglutarate Isocitrate  α - ketoglutarate Oxidation of isocitrate linked to reduction of NAD +  NADH Isocitrate is oxidatively decarboxylated (carbon removed to yield CO 2 ) Isocitrate dehydrogenase is a link to the electron transport pathway through the production of NADH TCA-Cycle Reaction 3: Isocitrate dehydrogenase

24. Reaction is irreversible due to large energy of forward reaction (large negative ∆G) NADH and ATP are allosteric inhibitors (high energy) ADP is allosteric activator (low energy) Isocitrate dehydrogenase - regulation

25. A second oxidative decarboxylation α - ketoglutarate  succinyl-CoA Oxidative decarboxylation (oxidative removal of a carbon to yield CO 2 and NADH) Multi-enzyme complex similar to pyruvate dehydrogenase α - ketoglutarate dehydrogenase Dihydrolipoyl transsuccinylase Dihydrolipoyl dehydrogenase TCA-Cycle Reaction 4: α - Ketoglutarate Dehydrogenase

26. Reaction is identical to pyruvate dehydrogenase - structurally and mechanistically Instead of acetyl- CoA , succinyl-CoA (an analog ) is formed Five coenzymes used - TPP, CoASH , lipoic acid, NAD + , and FAD α - Ketoglutarate Dehydrogenase subunits and coenzymes

27. Last 4 reactions in TCA cycle produces GTP, FADH 2 and NADH Oxaloacetate is regenerated and cycle begins again TCA cycle: succinyl-CoA to oxaloacetate Substrate Product Enzyme Other Succinyl -CoA Succinate Succinyl -CoA synthetase GDP  GTP Succinate Fumarate Succinate dehydrogenase FAD  FADH 2 Fumarate Malate Fumarase Malate Oxaloacetate Malate dehydrogenase NAD +  NADH

28. A nucleoside triphosphate is made This is possible because succinyl-CoA is a high-energy intermediate The reaction removes the CoA group and yields succinate – energy used to drive the phosphorylation of GDP to GTP Substrate level phosphorylation – substrate rather than electron-transport chain provides the energy for phosphorylation GTP produced by mammals in this reaction is converted to ATP via nucleoside diphosphate kinase GTP + ADP  ATP + GDP TCA-Cycle Reaction 5: Succinyl-CoA synthetase

29. An FAD-dependent oxidation of a single bond to a double bond Succinate dehydrogenase catalyses the conversion of succinate to fumarate and reduction of FAD to FADH 2 Dehydrogenase – thus removal of hydrogen atoms This enzyme is bound to the inner mitochondrial membrane Is part of BOTH TCA cycle AND electron transport pathway The electrons transferred from succinate to FAD (to form FADH 2 ) are passed directly to ubiquinone (UQ) in the electron transport pathway Succinate dehydrogenase is a dimeric protein = two subunits, one large one small FAD covalently bound to larger subunit TCA-Cycle Reaction 6: Succinate dehydrogenase

30. Succinate dehydrogenase contains 3 different iron-sulfur clusters: These iron-sulfur clusters receive the electrons captured by FAD and pass them onto the electron transport chain TCA-Cycle Reaction 6: Succinate dehydrogenase

31. Hydration of fumarate to malate (addition of water) Hydration involves trans-addition of the elements of water across the double bond Prepares structure so it can donate electrons to NAD + in next reaction TCA-Cycle Reaction 7: Fumarase

32. Malate Dehydrogenase completes the cycle by oxidizing malate to oxaloacetate Hydrogen donated by malate to reduce NADH The carbon that gets oxidized is the one that received the -OH in the previous reaction This reaction is energetically expensive: ∆G o ' = +30 kJ/mol Thus the concentration of oxaloacetate in the mitochondrial matrix is quite low (though ∆G is close to 0 in cellular conditions) However, the malate dehydrogenase reaction is pulled forward by the favorable citrate synthase reaction TCA-Cycle Reaction 8: Malate dehydrogenase

33. TCA cycle involves a flow of carbon molecules in and out of the cycle. TCA cycle starts with the addition of 2-carbon molecules from acetyl- CoA to the TCA cycle intermediate oxaloacetate , which contains 4-carbons , to make a 6-carbon molecule citrate. The flow of carbon through the TCA cycle and conservation of energy of oxidation 6-carbon citrate is rearranged ( isocitrate ) 6-carbon isocitrate is decarboxylated (loses a carbon in form of carbon dioxide) to 5-carbon molecule ( α - ketoglutarate ) and then again to a 4-carbon molecule ( succinyl-CoA ) Next 4 molecules contain 4-carbons And then cycle starts again

34. The flow of carbon through the TCA cycle and conservation of energy of oxidation One acetate through the cycle produces two CO 2 , one ATP, four reduced coenzymes Energy released through oxidation of acetyl- CoA is conserved in the reduction of NAD + , FAD + and the synthesis of GTP which can be converted to ATP The TCA cycle is exergonic , with a net ΔGº' for one pass around the cycle of -40 kJ/mol The combination of glycolysis and TCA produce 12 reduced coenzymes, which can eventually produce over 32 molecules of ATP Two carbon molecules enter the cycle as acetyl- CoA and leave as two carbon dioxide molecules The carbonyl C of acetyl- CoA becomes CO 2 only in the second turn of the cycle (following entry of acetyl- CoA ) The methyl C of acetyl- CoA survives two cycles completely, but half of what's left exits the cycle on each turn after that

35. Citrate synthase : catalyzes the condensation of acetyl- CoA and oxaloacetate to yield citrate. Aconitase : isomerizes citrate to the easily oxidized isocitrate . Isocitrate dehydrogenase : oxidizes & decarboxylates isocitrate to form  - ketoglutarate . (1st NADH and CO 2 ).  - ketoglutarate dehydrogenase : oxidatively decarboxylates  - ketoglutarate to succinyl-CoA . (2nd NADH and CO 2 ). Succinyl-CoA synthetase converts succinyl-CoA to succinate . Forms GTP. Succinate dehydrogenase : catalyzes the oxidation of central single bond of succinate to a trans double bond, yielding fumarate and FADH 2 . Fumarase : catalyzes the hydration of the double bond to produce malate . Malate dehydrogenase : reforms oxaloacetate by oxidizing secondary OH group to ketone (3rd NADH). The function of the TCA cycle is the harvesting of high-energy electrons from carbon fuels Final products of citric acid cycle: 2 CO 2 molecules. 3 NADH, 1 FADH 2 , and 1 GTP Summary

36. You need to be able to draw a flow diagram of the citric acid cycle showing substrates, enzymes, coenzymes and products You don’t need to know the structures or mechanisms You need to be able to write a short sentence describing each reaction https://www.youtube.com/watch?v=_cXVleFtzeE

37. TCA cycle not only functions to convert carbon to electrons and energy, but also provides many intermediates for other biosynthetic pathways α- Ketoglutarate transaminated to make glutamate, which is used to make nucleotides, as well as arginine and proline Succinyl-CoA is used to make porphyrins (e.g. of a porphyrine is heme , found in hemoglobin) Fumarate is a precursor in production of aspartate which is used to make nucleotides, as well as other amino acids threonine , methionine , isoleucine and lysine TCA cycle provides many intermediates for other biosynthetic pathways

38. Oxaloacetate also precursor in aspartate production, can also be decarboxylated to form PEP (fed back into glycolysis) as well as aromatic amino acids Citrate exported from mitochondria and broken down to oxaloacetate and acetyl- CoA Acetyl- CoA functions in cytoplasm as precursor in fatty acid synthesis Oxaloacetate is reduced to malate , which is either transported back to mitochondria or decarboxylated to pyruvate TCA cycle provides many intermediates for other biosynthetic pathways

39. All 20 common amino acids can be made from metabolites derived from glycolysis & the TCA cycle (highlighted in orange). TCA cycle provides intermediates for aa synthesis

40. Regulation of the TCA cycle

41. TCA cycle lies between glycolysis and the electron transport chain Must be tightly controlled to prevent wasting metabolic energy in production of unnecessary ATP or to prevent energy shortage in cell TCA cycle also important producer of precursors for other pathways, which would also be effected if TCA cycle went uncontrolled Regulation occurs at 4 important points: Pyruvate converted to acetyl- CoA (not part of TCA cycle but input of acetyl- CoA is needed for cycle to occur) 3 enzymatic steps in the TCA cycle: citrate synthase , isocitrate dehydrogenase, α- ketoglutarate dehydrogenase Regulation of the TCA cycle

42. Changes in free energy of the reactions of TCA cycle indicates 3 irreversible steps – the key regulatory sites A step is irreversible when the free energy for the forward reaction is so large that it occurs spontaneously and is not in equilibrium with the reverse reaction = large negative free energy Citrate synthase Isocitrate dehydrogenase α - ketoglutatrate dehydrogenase complex Regulation of the TCA cycle

43. Pyruvate is an important metabolite Under aerobic conditions, pyruvate is converted to acetyl- CoA by pyruvate dehydrogenase This is an irreversible step, and must be tightly regulated Pyruvate dehydrogenase is allosterically regulated High concentrations of ATP, NADH, acetyl- CoA inhibit High concentrations of NAD + , CoA activate High concentrations of acetyl- CoA inhibits the transacetylase component of E2 High concentrations of NADH inhibits the dihydrolipoyl dehydrogenase component of E3 Regulation of pyruvate dehydrogenase

44. Pyruvate dehydrogenase kinase is a regulatory enzyme that is part of the pyruvate dehydrogenase complex in mammals Dehydrogenase kinase is allosterically activated by NADH and acetyl- CoA Pyruvate dehydrogenase is phosphorylated on E1 This blocks the first step of catalysis, the decarboxylation of pyruvate Pyruvate dehydrogenase phosphatase is associated with the dehydrogenase complex when NADH and acetyl- CoA levels are low Keeps E1 dephosphorylated and thus active Is inactivated by high NADH or acetyl- CoA Pyruvate dehydrogenase is regulated by phosphorylation

45. TCA cycle is regulated by feedback inhibition Feedback inhibition = products from the system inhibit enzymes in the same system The principle signals are acetyl- CoA , succinyl-CoA , ATP, ADP, AMP, NAD + and NADH. Regulation of the TCA cycle Enzyme Activators Inhibitors Pyruvate dehydrogenase NAD + , CoA Acetyl CoA, NADH, ATP Citrate synthase ATP, NADH, succinyl -CoA Isocitrate dehydrogenase NAD +, ADP ATP, NADH α - ketoglutarate dehydrogenase AMP NADH, succinyl -CoA