1. The Tricarboxylic Acid Cycle (The Citric Acid Cycle) Babylon university College of pharmacy Department of clinical and scientific laboratory 3 rd class – biochemistry Second semester Dr. Abdulhussien M. K. Aljebory

2. The Citric Acid Cycle – Also called Tricarboxylic Acid Cycle (TCA) or Krebs Cycle. Three names for the same thing. – Cellular respiration and intermediates for biosynthesis. – Conversion of pyruvate to activated acetate – Reactions of the citric acid cycle – Anaplerotic reactions to regenerate the acceptor – Regulation of the citric acid cycle – Conversion of acetate to carbohydrate precursors in the glyoxylate cycle Key topics: To Know

3. Discovered CAC in Pigeon Flight Muscle

4. Products from One Turn of the Cycle

5. Net Effect of the Citric Acid Cycle Acetyl-CoA + 3NAD + + FAD + GDP + P i + 2 H 2 O 2CO 2 +3NADH + FADH 2 + GTP + CoA + 3H + Carbons of acetyl groups in acetyl-CoA are oxidized to CO 2 Electrons from this process reduce NAD + and FAD One GTP is formed per cycle, this can be converted to ATP Intermediates in the cycle are not depleted

6. Krebs in mitochondrial matrix Mitochondrion – Outer membrane very permeable Space between membranes called intermembrane space – Inner membrane (cristae) Permeable to pyruvate, Impermeable to fatty acids, NAD, etc – Matrix is inside inner membrane

8. Glucose Glucose-6- phosphate Pyruvate Glycogen Ribose, NADPH Pentose phosphate pathway Synthesis of glycogen Degradation of glycogen Glycolysis Gluconeogenesis LactateEthanol Acetyl Co A Fatty Acids Amino Acids The citric acid cycle is the final common pathway for the oxidation of fuel molecules — amino acids, fatty acids, and carbohydrates. Most fuel molecules enter the cycle as acetyl coenzyme A.

9. Three main pathways for energy production: 1- Glycolysis 2- Citric acid cycle 3- Oxidative-Phosphorylation Certain pathways are involved in both breakdown and buildup of molecules these pathways are called amphibolic. The citric acid cycle is an example of this. Eight successive reaction steps. The six carbon citrate is formed from two carbon acetyl- CoA and four carbon oxaloacetate. Oxidation of citrate yields CO2 and regenerates oxaloacetate. The energy released is captured in the reduced coenzymes NADH and FADH2.

10. An Overview of the Citric Acid Cycle A four-carbon oxaloacetate condenses with a two-carbon acetyl unit to yield a six-carbon citrate. An isomer of citrate is oxidatively decarboxylated and five-carbon  -ketoglutarate is formed.  -ketoglutarate is oxidatively decarboxylated to yield a four-carbon succinate. Oxaloacetate is then regenerated from succinate. Two carbon atoms (acetyl CoA) enter the cycle and two carbon atoms leave the cycle in the form of two molecules of carbon dioxide. Three hydride ions (six electrons) are transferred to three molecules of NAD +, one pair of hydrogen atoms (two electrons) is transferred to one molecule of FAD.

11. 1. Citrate Synthase Citrate formed from acetyl CoA and oxaloacetate Only cycle reaction with C-C bond formation Addition of C 2 unit (acetyl) to the keto double bond of C 4 acid, oxaloacetate, to produce C 6 compound, citrate citrate synthase

12. 2. Aconitase Elimination of H 2 O from citrate to form C=C bond of cis-aconitate Stereospecific addition of H 2 O to cis-aconitate to form isocitrate aconitase

13. 3. Isocitrate Dehydrogenase Oxidative decarboxylation of isocitrate to a-ketoglutarate (a metabolically irreversible reaction) One of four oxidation-reduction reactions of the cycle Hydride ion from the C-2 of isocitrate is transferred to NAD + to form NADH Oxalosuccinate is decarboxylated to a-ketoglutarate isocitrate dehydrogenase

14. 4. The  -Ketoglutarate Dehydrogenase Complex Similar to pyruvate dehydrogenase complex Same coenzymes, identical mechanisms E 1 - a-ketoglutarate dehydrogenase. E 2 – dihydrolipoyl succinyltransferase. E 3 - dihydrolipoyl dehydrogenase (with FAD)  -ketoglutarate dehydrogenase

15. 5. Succinyl-CoA Synthetase Free energy in thioester bond of succinyl CoA is conserved as GTP or ATP in higher animals (or ATP in plants, some bacteria) Substrate level phosphorylation reaction HS- + GTP + ADP GDP + ATP Succinyl-CoA Synthetase

16. Complex of several polypeptides, an FAD prosthetic group and iron-sulfur clusters Embedded in the inner mitochondrial membrane Electrons are transferred from succinate to FAD and then to ubiquinone (Q) in electron transport chain Dehydrogenation is stereospecific; only the trans isomer is formed 6. The Succinate Dehydrogenase Complex Succinate Dehydrogenase

17. 7. Fumarase Stereospecific trans addition of water to the double bond of fumarate to form L-malate Only the L isomer of malate is formed Fumarase

18. 8. Malate Dehydrogenase Malate Dehydrogenase Malate is oxidized to form oxaloacetate.

19. Stoichiometry of the Citric Acid Cycle  Two carbon atoms enter the cycle in the form of acetyl CoA.  Two carbon atoms leave the cycle in the form of CO 2.  Four pairs of hydrogen atoms leave the cycle in four oxidation reactions (three molecules of NAD + one molecule of FAD are reduced).  One molecule of GTP, is formed.  Two molecules of water are consumed.  9 ATP (2.5 ATP per NADH, and 1.5 ATP per FADH 2 ) are produced during oxidative phosphorylation.  1 ATP is directly formed in the citric acid cycle.  1 acetyl CoA generates approximately 10 molecules of ATP.

20. Regulation of the Citric Acid Cycle Pathway controlled by: (1) Allosteric modulators (2) Covalent modification of cycle enzymes (3) Supply of acetyl CoA (pyruvate dehydrogenase complex) Three enzymes have regulatory properties - citrate synthase (is allosterically inhibited by NADH, ATP, succinyl CoA, citrate – feedback inhibition) - isocitrate dehydrogenase (allosteric effectors: (+) ADP; (-) NADH, ATP. Bacterial ICDH can be covalently modified by kinase/phosphatase) -  -ketoglutarate dehydrogenase complex (inhibition by ATP, succinyl CoA and NADH

21. Regulation of the TCA Cycle Again, 3 reactions are the key sites Citrate synthase - ATP, NADH and succinyl-CoA inhibit Isocitrate dehydrogenase - ATP inhibits, ADP and NAD + activate   -Ketoglutarate dehydrogenase - NADH and succinyl-CoA inhibit, AMP activates Also note pyruvate dehydrogenase: ATP, NADH, acetyl-CoA inhibit, NAD +, CoA activate

22. NADH, ATP, succinyl CoA, citrate - Regulation of the citric acid cycle

23. Krebs Cycle is a Source of Biosynthetic Precursors Phosphoenol- pyruvate Glucose The citric acid cycle provides intermediates for biosyntheses

24. Net From Kreb’s Oxidative process – 3 NADH – FADH 2 – GTP X 2 per glucose – 6 NADH – 2 FADH 2 – 2 GTP All ultimately turned into ATP (oxidative phosphorylation)

25. Pentose Phosphate Pathway Also known as: Pentose shunt Hexose monophosphate shunt Phosphogluconate pathway It occurs in the cytosol.

26. One fate of G6P is the pentose pathway.

27. It’s a shunt

28. What does the pentose phosphate pathway achieve? The pathway yields reducing potential in the form of NADPH to be used in anabolic reactions requiring electrons. The pathway yields ribose 5-phosphate. – Nucleotide biosynthesis leading to: DNA RNA Various cofactors (CoA, FAD, SAM, NAD + /NADP + ).

31. The pentose pathway can be divided into two phases. Non- oxidative interconv ersion of sugars

32. Glycogen Metabolism

33. Glycogen is a polymer of glucose residues linked by   (1  4) glycosidic bonds, mainly   (1  6) glycosidic bonds, at branch points. Glycogen chains & branches are longer than shown. Glucose is stored as glycogen predominantly in liver and muscle cells.

34. Glycogen Phosphorylase catalyzes phosphorolytic cleavage of the  (1  4) glycosidic linkages of glycogen, releasing glucose-1-phosphate as reaction product. glycogen (n residues) + P i  glycogen (n–1 residues) + glucose-1-phosphate This phosphorolysis may be compared to hydrolysis: Hydrolysis: R-O-R' + HOH  R-OH + R'-OH Phosphorolysis: R-O-R' + HO-PO 3 2-  R-OH + R'-O-PO 3 2- Glycogen catabolism (breakdown):

35. Pyridoxal phosphate (PLP), a derivative of vitamin B 6, serves as prosthetic group for Glycogen Phosphorylase.

36. Pyridoxal phosphate (PLP) is held at the active site by a Schiff base linkage, formed by reaction of the aldehyde of PLP with the  -amino group of a lysine residue. In contrast to its role in other enzymes, the phosphate of PLP is involved in acid/base catalysis by Phosphorylase.

37. Debranching enzyme has 2 independent active sites, consisting of residues in different segments of a single polypeptide chain:  The transferase of the debranching enzyme transfers 3 glucose residues from a 4-residue limit branch to the end of another branch, diminishing the limit branch to a single glucose residue.  The  (1  6) glucosidase moiety of the debranching enzyme then catalyzes hydrolysis of the  (1  6) linkage, yielding free glucose. This is a minor fraction of glucose released from glycogen.  View an animationanimation The major product of glycogen breakdown is glucose-1-phosphate, from Phosphorylase activity.

38. Phosphoglucomutase catalyzes the reversible reaction: glucose-1-phosphate  glucose-6-phosphate A serine OH at the active site donates & accepts P i. The bisphosphate is not released. Phosphoglycerate Mutase has a similar mechanism, but instead uses His for P i transfer.

39. Glucose-6-phosphate may enter Glycolysis or (mainly in liver) be dephosphorylated for release to the blood. Liver Glucose-6-phosphatase catalyzes the following, essential to the liver's role in maintaining blood glucose: glucose-6-phosphate + H 2 O  glucose + P i Most other tissues lack this enzyme.

40. Uridine diphosphate glucose (UDP-glucose) is the immediate precursor for glycogen synthesis. As glucose residues are added to glycogen, UDP-glucose is the substrate and UDP is released as a reaction product. Nucleotide diphosphate sugars are precursors also for synthesis of other complex carbohydrates, including oligosaccharide chains of glycoproteins, etc. Glycogen synthesis

41. Regulation by covalent modification (phosphorylation): The hormones glucagon and epinephrine activate G-protein coupled receptors to trigger cAMP cascades.  Both hormones are produced in response to low blood sugar.  Glucagon, which is synthesized by  -cells of the pancreas, activates cAMP formation in liver.  Epinephrine activates cAMP formation in muscle.

42. The cAMP cascade results in phosphorylation of a serine hydroxyl of Glycogen Phosphorylase, which promotes transition to the active (relaxed) state. The phosphorylated enzyme is less sensitive to allosteric inhibitors. Thus, even if cellular ATP & glucose-6-phosphate are high, Phosphorylase will be active. The glucose-1-phosphate produced from glycogen in liver may be converted to free glucose for release to the blood. With this hormone-activated regulation, the needs of the organism take precedence over needs of the cell.

43. The cAMP cascade induced in liver by glucagon or epinephrine has the opposite effect on glycogen synthesis. Glycogen Synthase is phosphorylated by Protein Kinase A as well as by Phosphorylase Kinase. Phosphorylation of Glycogen Synthase promotes the "b" (less active) conformation. The cAMP cascade thus inhibits glycogen synthesis. Instead of being converted to glycogen, glucose-1-P in liver may be converted to glucose-6-P, and dephosphorylated for release to the blood.

44. Insulin, produced in response to high blood glucose, triggers a separate signal cascade that leads to activation of Phosphoprotein Phosphatase. This phosphatase catalyzes removal of regulatory phosphate residues from Phosphorylase, Phosphorylase Kinase, & Glycogen Synthase enzymes. Thus insulin antagonizes effects of the cAMP cascade induced by glucagon & epinephrine.

45. Ca ++ also regulates glycogen breakdown in muscle. During activation of contraction in skeletal muscle, Ca ++ is released from the sarcoplasmic reticulum to promote actin/myosin interactions. The released Ca ++ also activates Phosphorylase Kinase, which in muscle includes calmodulin as its  subunit. Phosphorylase Kinase is partly activated by binding of Ca ++ to this subunit.

47. Answer: Most of the Glycogenin is found associated with glycogen particles (branched glycogen chains) in the cytoplasm. Glycogen Synthase then catalyzes elongation of glycogen chains initiated by Glycogenin. Question: Where would you expect to find Glycogenin within a cell?

48. Question: How would you nutritionally treat deficiency of liver Glycogen Synthase?  Frequent meals of complex carbohydrates (avoiding simple sugars that would lead to a rapid rise in blood glucose)  Meals high in protein to provide substrates for gluconeogenesis.