Lec05 tc acycle


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Lec05 tc acycle

  1. 1. Berg • Tymoczko • Stryer Biochemistry Sixth Edition Chapter 17: The Citric Acid Cycle Copyright © 2007 by W. H. Freeman and Company
  2. 2. The Citric Acid Cycle = Krebs Cycle = Three Carboyxlic Acid Cycle Most mols enter the cycle as Acetyl-CoA There are three stages – Acetyl-CoA production – Acetyl-CoA oxidation – Electron transfer Its distinguishing characteristics are: – The use of oxygen as the ultimate electron acceptor – The complete oxidation of organic substrates to CO2 and H2O – The conservation of much of the free energy as ATP The reactions of the citric acid cycle occur in the mitochondrial matrix, in contrast with glycolysis. An overview of the citric acid cycle Reactions of the citric acid cycle
  3. 3. The Oxidative Decarboxylation of Pyruvate 1. The condensation of Acetyl-CoA and oxaloacetate to form citrate 2. Isomerization of citrate 3. Oxidation of isocitrate 4. Oxidation of -KG to succinyl-CoA 5. Conversion of succinyl-CoA to succinate 6. Oxidation of succinate to fumarate 7. Hydration of fumarate to malate 8. Oxidation of malate to oxalacetate
  4. 4. The citric acid cycle Citric acid cycle (also called the Krebs cycle, TCA ) oxidizes Acetyl CoA to CO2 + H2O Acetyl CoA Most mols enter the TCA cycle as Acetyl CoA. The cycle provides intermediates for biosynthesis. So, catabolism of proteins, fats and carbohydrates occurs in the 3 stages of cellular respiration. Stage I  oxidation of f.a, Glc, some a.a yields Acetyl CoA Stage II  oxidation of acetyl groups via the TCA cycle includes 4 steps in which electrons are abstracted. Stage III  Electrons carried by NADH and FADH2 are funnelled into a chain of mitochondrial electron carriers-- respiratory chain- ultimately reducing O2 to H2O. This electron flow drives the synthesis of ATP, in the process of oxidative phosphorylation.
  5. 5. Cycles distinguishing characteristics are:• The use of oxygen as the ultimate electron acceptor.• The complete oxidation of organic substrates to CO2 and H2O.• The conservation of much of the free energy as ATP.The reactions of the citric acid cycle occur inside mitochondria,in contrast with those of glycolysis, which take place in thecytosol.
  6. 6. An overview of the citric acid cycle:• An overview of TCA cycle – A 4C compound (oxaloacetate) condenses with a acetyl unit to yield a 6C tricarboxylic acid (citrate). – An isomer of citrate is then oxidatively decarboxylated. – The resulting 5C (a-ketoglutarate) is oxidatively decarboxylated to yield a 4C compound (succinate). – Oxaloacetate is then regenerated from succinate.• Reactions of the TCA cycle
  7. 7. The oxidative decarboxylation of pyruvate. • This is done by a multi-enzyme complex located in the mitochondrial matrix. Pyruvate dehydrogenase complex • Pyruvate  Acetyl CoA – a major fuel of the citric acid cycle – irreversible reaction. • That means we cannot make pyruvate from Acetyl CoA and also explains why glucose can not be formed from Acetyl CoA in gluconeogenesis.
  8. 8. PD complex 5 cofactors are involved in PD complex... All of which are coenzyme derived from vits. The regulation of this enzyme complex also shows how a combination of covalent modification and allosteric regulation results in precisely regulated flux through a metabolic step. Finally, the pyruvate dehydrogenase complex is the prototype for 2 other important enzyme complexes that we’ll cover later. – -ketoglutarate dehydrogenase  TCA cycle – -ketoacid dehydrogenase  a.a degradation
  9. 9. Reactions of PD complexStep 1. Pyruvate reacts with the bound TPP of E1, undergoing deCO2 to form the Ohethyl derivative.Step 2. The transfer of 2e- and the acetyl group from TPP to E2 to form acetyl thioester of the reduced lipoyl group.Step 3. Transesterification -SH group of CoA replaces the SH group of E2 to yield AcetylCoA.Step 4. E3 promotes transfer of 2H atoms from E2 to the FAD of E3 restoring the oxidized form of the lypoyllysyl group of E2.Step 5. The reduced FADH2 ON E3 TRANSFERS HYDRIDE ION TO NAD.
  10. 10. PyruvateDehydrogenase
  11. 11. OXIDATIVE DECARBOXYLATION OF PYRUVATE PDC is regulated by 2 mechanism. 1. Product inhibition – Inhibited by Acetyl CoA – High concentrations of NADH 2. Covalent modification: PDC exists in 2 forms: 1. Active  nonphosphorylated 2. Inactive  phosphorylated form. Phosphorylated and nonphosphorylated PDC can be interconverted by 2 separate enzymes. 1. A kinase 2. A phosphotase
  12. 12. 8 STEPS IN THE TCA CYCLE1) The condensation of acetylCoA and oxaloacetate to form citrate – The reaction uses an intermediate of the TCA cycle OA and produces another intermediate of the cycle (citrate). Thus, the entry of acetylCoA into the Krebs cycle does not lead to the net production or consumption of cycle intermediates.A refresher on enzyme nomenclature • Synthases: catalyze condensation reactions in which no ATP, GTP is required as an energy source. • Synthetases: also catalyze condensation reactions but this name implies that ATP or GTP is used for the synthetic reaction.Citrate syntase is inhibited by ATP, NADH, succinyl CoA derivatives offatty acids.
  13. 13. Citrate, in addition to being an intermediate in the TCAcycle, has other functions: Provides a source of AcetylCoA for fa synthesis. Citrate inhibits PFK, the rate limiting step in glycolysis, and activates Acetyl-CoA carboxylase, the rate limiting enzyme for fa synthesis.
  14. 14. 2) ISOMERIZATION OF CITRATE:Citrate is isomerized to isocitrate by a dehydrationstep followed by a hydration step. Cis-aconitateoccurs as an enzyme-bound intermediate.
  15. 15. 3) OXIDATION OF ISOCITRATE:Isocitrate dehydrogenase catalyzes the irreversibleoxidadite deCO2 of isocitrate yielding the first of 3NADH mols produced by the cycle and CO2. – Enzyme is activated by ADP. Elevated levels of mitochondrial ADP signals a need for the generation of more high-energy phosphate (ATP). – The enzyme is inhibited by ATP and NADH, which are increased when the cell has abundant energy.
  16. 16. 4) OXIDATION OF -KG TO SUCCINYLCOA– Irreversible reaction.– Enzyme: –KGDC. It is similar to PDC reaction.– It also has 3 enzymes (analogous to E1, E2, E3) and 5 cofactors. (TPP, lipoic acid, FAD, NAD, and CoA)– Enzyme is inhibited by ATP, GTP, NADH, and succinylCoA– Enzyme is not regulated by phosphorylation/dephosphorylation reactions as described for PDC, 2nd CO2, and 2nd NADH are produced.
  17. 17. 5) CONVERSION OF SUCCINYL COA TO SUCCINATE:– This reaction is coupled to the phosphorylation of GDP to GTP. The energy content of GTP is the same as that of ATP, because 2 nucleotides are interconvertible by the nucleoside diphosphate kinase reaction.– This is an example of substrate -level phosphorylation in which the ATP production is coupled to the conversion of substrate to product, rather than resulting from respiratory-chain.
  18. 18. 6) OXIDATION OF SUCCINATE TO FUMARATE: – FAD rather than NAD is the e-acceptor, since the reducing power of succinate is not sufficient to reduce NAD+. Malonate, a dicarboxylic acid that is a structural analog of succinate, competitively inhibits succinate dehydrogenase.
  21. 21. STOICHIOMETRY OF THE CYCLESummary of the reactions:1. Two carbon atoms enter the cycle as acetyl CoA and leave as CO2.2. The TCA cycle does not involve the net consumption or production of OA or any other intermediate of the cycle.3. Four pairs of e- are transferred during one turn of the cycle: 3 pairs of e- reducing NAD+ to NADH and one pair reducing FAD to FADH2.ATP FORMATION IN THE AEROBIC OXIDATION OF AMOLECULE OF GLC VIA GLYCOLYSIS, THE PDCREACTION AND THE TCA CYCLE:
  22. 22. CITRIC ACID CYCLE COMPONENTS ARE IMPORTANT BIOSYNTHETIC INTERMEDIATES. The TCA cycle is an amphibolic pathway, meaning it serves in both catabolic and anabolic processes. – It also provides precursors for many biosynthetic pathways. – But if this is the case , we have to replace the ones used for the biosynthesis of some molecules. – Those reactions which replenish TCA acid cycle intermediates are called anaplerotic reactions. – Under normal circumstances there is a dynamic balance between the reactions by which the cycle intermediates are used and those by which they are replenished by the anaplerotic reactions. – So that the concentrations of the citric acid cycle intermediates remain almost constant. Given the number of biosynthetic products synthesized from the TCA cycle intermediates, this cycle serves a critical role apart from its role in energy yielding metabolism.
  23. 23. ATP Formation in the Aerobic Oxidation of GLC viaGlycolysis, the PDC Reaction, and the TCA Cycle #ATP #ATP REACTION or coenz formedGLC  G-G-P -ATP -1F-G-P  F-1,6-biP -ATP -1G-3-P  1, 3 bisP Glycerate 2 NADH 61, 3 bis.Gly  3 P Gly 2 ATP 2PEP P 2 ATP 2Pyru  AcetylCoA 2 NADH 6Iso.  αKG 2 NADH 6αKG  Succ. 2 NADH 6Succinyl  Succinate 2 GTP 2Succinate  Fumarate 2 FADH2 4Malate OA 2NADH 6
  24. 24. ANAPLEROTIC REACTIONSREACTION TISSUE, ORGANS 1) 2) 3) 4) They are all reversible. When TCA needs OA, pyruvate is carboxylated to OA. Free energy is required to attach CO2 to pyruvate comes from ATP. Carboxylation of pyruvate also requires, like in other carboxylation reactions BIOTIN, which is a prosthetic group of pyruvate carboxylase.
  25. 25. THREE ENZYMESOF THE TCA CYCLE ARE REGULATED  The TCA cycle is under tight regulation. 3 factors are important for the rate of flux through the cycle. 1. Substrate availability 2. Product inhibition 3. Allosteric feedback inhibition of early enzymes by later intermediates in the cycle.
  26. 26.  There are 3 irreversible steps in the cycle, therefore potential sites for control. Those are catalyzed by : – Citrate synthase – Isocitrate dehydrogenase – -KG dehydrogenase. Each can become a rate limiting step under certain circumstances. When acetyl CoA and OA are available or not , citrate formation increase or decrease. NADH increases (a product of the oxidation of isocitrate and -KG) , NADH/[NAD] increases, those dehydrogenase reactions are severely inhibited.
  27. 27. The TCAcycle is asource ofbiosyntheticprecursors
  28. 28. Regulation ofCarbohydrateMetabolism
  29. 29. The disruption of pyruvate metabolism is the causeof beriberi and heavy metal poisoning TPP deficiency causes beriberi Hg, Ar, and Pb have high affinity for -SH Lipoic acid is one of the cofactors in PDC PDC becomes inactive when lipoic acid is bound to heavy metals. CNS solely depends on Glc metabolism therefore effected by heavy metal poisoning.
  30. 30. The glyoxylate cycle permits AcetylCoAto be incorparated into carbohydrates:  The glyoxylate cycle , a modification of the TCA cycle, is a biosnthetic pathway that leads to the formation of glucose from AcetylCoA.  It occurs in : – Plants – Bacteria – Yeast – Not in Animals
  31. 31. glyoxylate cycle The glyoxylate cycle is especially active in oily seed plants. The glyoxylate cycle can be regarded as a shunt within the TCA cycle.The 6C intermediate isocitrate, rather thanundergoing decarboxylation, is converted to the 4Cmol succinate and 2C mol glyoxylate in a reactioncatalyzed by isocitrate lyase, the first of the 2enzymes in this cycle.
  32. 32. More about the glyoxylate cycle In plants, the enzymes of the glyoxylate cycle are in the membrane bound organelles and called glyoxysomes. Glyoxylate enzymes are not present in animal cells, thus animals can not sustain growth on acetylCoA or 2C mols, such as acetate. Role of the glyoxylate cycle: • 4C and 6C compounds are made from 2C compounds • Glucose is made from acetate • It is also essential reaction sequence for seedlings of fat storing in plants. TCA and Glyoxylate cycles are coordinately regulated.