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1. PREPARED BY MARRAH CELLULAR RESPIRATION

2. METABOLISMS

3. definition Is the process through which living systems acquire and use free energy to carry out functions It requires highly coordinated cellular activity

4. Metabolism performs 4 functions Obtain energy for the cell Convert nutrients into macromolecules Assemble macromolecules into cellular structures Degrade macromolecules as required for biological function

5. Classes of living organisms Two major classes, autotrophs and heterotrophs Autotrophs -use atmospheric carbondioxide as a sole source for the synthesis of macromolecules. They use the sun for biosynthetic purposes. Can be chemolithotrophs, chemoautotrophs or photoautotrophs

6. Chemolithotrophs-obtain free energy via the oxidation of inorganic compounds like Fe, H2S Photoautotrophs- these obtain free energy from light photons through photosynthesis

7. Heterotrophs These basically feed on others They obtain energy by ingesting complex carbon containing compound(lipids, carbohydrates and proteins) They are also divided into two, Aerobes Anaerobes

8. Aerobes –live in the presence of oxygen which they use to oxidise organic nutrients Anaerobes – live in the absence of oxygen Obligate anaerobes are poisoned by oxygen Facultative anaerobes can survive in either conditions e.g. yeast

9. Types of metabolic pathways Catabolism-is the degradation pathway It generates energy Anabolism- biosynthesis of biomolecules such as nucleotides, proteins, lipids and polysaccharides from simple precursor molecule This process requires energy Biomolecules are composed of predominantly hydrogen, carbon and nitrogen molecules

10. Principles of metabolic pathways Metabolic pathways are irreversible -metabolic pathways are highly exergonic which gives the pathway direction. Two interconvertible metabolites must take different pathways which helps the two pathways to be regulated independently

11. Every metabolic pathway has a first committed step -this step or reaction is irreversible and highly exergonic which commits the intermediate it produces to continue down the pathway

12. All metabolic pathways are regulated The control of the metabolic flux of metabolites through a pathway is accomplished by regulating the rate determining step of the pathway which often is the first committed step of the pathway

13. Metabolic pathways in eukaryotes occur in specific cellular locations Compartmentalization of metabolic pathways This helps the metabolic pathways to occur in different locations and simultaneously Mitochondrion-citric acid cycle, oxidative phosphorylation, amino acid catabolism Cytosol-glycolysis, pentose phosphate pathway, fatty acid biosynthesis, gluconeogenesis Nucleus-DNA replication, RNA TRANSCRIPTION, RNA processing Lysosomes- enzymatic digestion of cellular components Golgi apparatus-post translational modification of membrane and secretory proteins, formation of plasma membranes and secretory vesicles

14. Flux - flow of material through a metabolic pathway which depends upon: (1) Supply of substrates (2) Removal of products (3) Pathway enzyme activities

15. Levels of Metabolism Regulation Nervous system. Endocrine system. Interaction between organs. Cell (membrane) level. Molecular level

16. Feedback inhibition Product of a pathway controls the rate of its own synthesis by inhibiting an early step (usually the first “committed” step (unique to the pathway) Feed-forward activation Metabolite early in the pathway activates an enzyme further down the pathway

17. Covalent modification for enzyme regulation Interconvertible enzyme activity can be rapidly and reversibly altered by covalent modification Protein kinases phosphorylate enzymes (+ ATP) Protein phosphatases remove phosphoryl groups

18. Stages of metabolism Catabolism Stage I . Breakdown of macromolecules (proteins, carbohydrates and lipids to respective building blocks. Stage II . Amino acids, fatty acids and glucose are oxidized to common metabolite (acetyl CoA) Stage III . Acetyl CoA is oxidized in citric acid cycle to CO 2 and water. As result reduced cofactor, NADH 2 and FADH 2 , are formed which give up their electrons. Electrons are transported via the tissue respiration chain and released energy is coupled directly to ATP synthesis

19. Anabolism can also be divided into stages, however the anabolic pathways are characterized by divergence . Monosaccharide synthesis begin with CO 2 , oxaloacetate, pyruvate or lactate. Amino acids are synthesized from acetyl CoA, pyruvate or keto acids of Krebs cycle. Fatty acids are constructed from acetyl CoA. On the next stage monosaccharides, amino acids and fatty acids are used for the synthesis of polysaccharides, proteins and fats . Catabolism is characterized by convergence of three major routs toward a final common pathway. Different proteins, fats and carbohydrates enter the same pathway – tricarboxylic acid cycle.

20. The chemistry of metabolism There are about 3000 reactions in human cell. All these reactions are divided into six categories: Oxidation-reduction reactions Group transfer reactions Hydrolysis reactions Nonhydrolytic cleavage reactions Isomerization and rearrangement reactions Bond formation reactions using energy from ATP

21. Glycolysis and the pyruvate dehydroganase

22. Learning Objectives Answer questions about carbohydrate digestion Demonstrate understanding of glucose transport Solve problems concerning glycolysis Interpret scenarios about galactose metabolism Explain information related to fructose metabolism Answer questions about pyruvate dehydrogenase

23. All cells can carry out glycolysis In a few tissues, most importantly red blood cells, glycolysis represents the only energy-yielding pathway available Glucose is the major monosaccharide that enters the pathway, but others such as galactose and fructose can also be used The first steps in glucose metabolism in any cell are transport across the membrane and phosphorylation by kinase enzymes inside the cell to prevent it from leaving via the transporter

24. CARBOHYDRATE DIGESTION Most of the carbohydrates in foods are in complex forms, such as starch (amylose and amylopectin) and the disaccharides sucrose and lactose Salivary amaylase randomly hydrolises the starch to dextrins Dextrins are hydrolysed in the stomach to the disaccharides maltose and isomaltose Disaccharides in the intestinal brush border complete the digestion process: Maltase cleaves maltose to 2 glucoses Isomaltase cleaves isomaltose to 2 glucoses Lactase cleaves lactose to glucose and galactose Sucrase cleaves sucrose to glucose and fructose Uptake of glucose into the mucosal cells is performed by the sodium/glucose transporter, an active transport system

25. GLUCOSE TRANSPORT Glucose entry into most cells is concentration driven and independent of sodium Four glucose transporters (GLUT) are known They have different affinities for glucose coinciding with their respective physiologic roles

26. Normal glucose concentration in peripheral blood is 4–6 mM (70–110 mg/dL) GLUT 1 and GLUT 3 mediate basal glucose uptake in most tissues, including brain, nerves, and red blood cells. Their high affinities for glucose ensure glucose entry even during periods of relative hypoglycemia GLUT 2 , a low-affinity transporter, is in hepatocytes. After a meal, portal blood from the intestine is rich in glucose. GLUT 2 captures the excess glucose primarily for storage. When the glucose concentration drops below the Km for the transporter, much of the remainder leaves the liver and enters the peripheral circulation. In the β-islet cells of the pancreas. GLUT-2, along with glucokinase, serves as the glucose sensor for insulin release. GLUT 4 is in adipose tissue and muscle and responds to the glucose concentration in peripheral blood. The rate of glucose transport in these two tissues is increased by insulin, which stimulates the movement of additional GLUT 4 transporters to the membrane by a mechanism involving exocytosis

27. glycolysis Glycolysis is a cytoplasmic pathway that converts glucose into two pyruvates, releasing a modest amount of energy captured in two substrate-level phosphorylations and one oxidation reaction If a cell has mitochondria and oxygen, glycolysis is aerobic. If either mitochondria or oxygen is lacking, glycolysis may occur anaerobically (erythrocytes, exercising skeletal muscle), although some of the available energy is lost

28. a. This is the first intracellular reaction of glycolysis (remember all reactions are in the cytoplasm). b. Requires an ATP (Mg). This is one of the investment reactions. c. The phosphorylation of glucose traps the glucose inside the cell. d. The reaction is considered irreversible. e. Hexokinase has a K m for glucose of less than 0.1 mM (high affinity). It is also inhibited by the product glucose-6-phosphate f. Liver hepatocytes and pancreatic β cells contain another enzyme Glucokinase . Glucokinase has a K m for glucose of about 10 mM (low affinity). Glucokinase is not inhibited by its product glucose-6-phosphate. Glucokinase is induced by insulin. The levels of glucokinase in the liver of untreated Type 1 diabetics are lower than normal. Hexokinase and glucokinase are isoenzymes. Therefore, irrespective of the isoenzyme catalyzing the reaction, the Keq, ΔG, and ΔG o for the reaction remain the same.

29. a. This reaction is readily reversible (not a controlling step) and functions in both glycolysis and gluconeogenesis. b. Conversion of an aldose to a ketose.

30. Reaction #3: 6-Phosphofructo-1-kinase (PFK-1) or Phosphofructokinase-1 . a. a. Reaction is the rate-limiting step of glycolysis. b. It is irreversible, and the committed step. It is an allosteric enzyme and also a major regulatory enzyme. c. We have invested our second ATP molecule.

31. Reaction #4: Aldolase . a. We now have two phosphorylated trioses. b. Only glyceraldehyde-3-phosphate is used in glycolysis. Therefore, dihydroxyacetone phosphate has to be converted into glyceraldehyde-3-phosphate. This occurs in the next step.

32. Reaction #5: Triose phosphate isomerase. a. Catalyzes the interconversion of dihydroxyacetone phosphate and glyceraldehyde-3-phosphate. b. Because of the interconversion, one glucose molecule can be converted to two glyceraldehyde-3-phosphate molecules.

33. Reaction # 6: Glyceraldehyde-3-phosphate dehydrogenase a. The enzyme oxidizes the number one carbon aldehyde and then adds a phosphate group. We have an acid anhydride in the product 1,3-bisphophoglycerate. Remember from bioenergetics that acid anhydrides are high-energy bonds. b. The phosphate on the number 3 carbon is not an high-energy bond c. We have used an NAD + for the oxidation reaction. The cell has limited amounts of NAD + , so somewhere along the line we have to regenerate it or glycolysis will stop. d. This reaction is a target for Arsenate (AsO 4 3- ). The arsenate resembles inorganic phosphate (Pi). In the presence of arsenate, the product of the reaction is 1-arseno-3-phosphoglycerate. This product is unstable and decomposes into arsenate and 3-phosphoglycerate with no ATP formation. After this step, glycolysis continues. e. The enzyme contains an essential thiol (cysteine-SH) group at the active site. Iodoacetic acid (ICH 2 COOH) is also an inhibitor of this reaction. It reacts with the active site SH group and inhibits the enzyme.

34. Reaction #7: Phosphoglycerate kinase a. This is the first step of energy production. b. This is referred to as substrate-level phosphorylation as opposed to oxidative phosphorylation that occurs in mitochondrial ATP production. d. We have recovered both ATP that were invested. Remember that each glucose gives 2 phosphoglycerate molecules.

35. Reaction # 8: Phosphoglycerate mutase .

36. a. Catalyzes the dehydration of 2-phosphoglycerate to form phosphoenolpyruvate (PEP). c. Recall from the bioenergetics lecture that PEP contains an high-energy bond. d. This reaction is inhibited by Fluoride. Reaction#9: Enolase

37. Reaction # 10: Pyruvate Kinase a. Catalyzes the transfer of the phosphate from PEP to ADP to generate ATP and 9 pyruvate. b. Again, this is substrate-level phosphorylation . c. This reaction completes that part of glycolysis that is common to both anaerobic and aerobic metabolism. d. Under aerobic conditions and the presence of mitochondria, pyruvate can enter the citric acid cycle. NAD + is regenerated by malate/aspartate shuttle or by α-glycerophosphate shuttle e. Under anaerobic conditions or in the absence of mitochondria, pyruvate is reduced to lactate via the following reaction and regenerate NAD + .

38. Anerobic Glycolysis When pyruvate cannot be oxidized within mitochondria for some reason (e.g., hypoxia, genetic defects in pyruvate dehydrogenase or citric acid cycle enzymes, genetic defects in electron transport chain), pyruvate is reduced to lactate by lactate dehydrogenase.

39. Clinical considerations : A. Lactic acidosis Characterized by high blood levels of lactate (generally greater than 5 mM, while normal levels are usually less than 1.2 mM). Blood pH can be less than 7.1 in severe cases. Most tissues can convert lactate to CO 2 and H 2 O through the TCA cycle. If the oxygen supply is inadequate, cells must rely on glycolysis for ATP production. A decrease in ATP enhances glycolysis at the level of PFK-1 and produces more pyruvate and hence lactate. Accumulation of plasma lactate may be secondary to tissue hypoxia (circulatory insufficiency due to shock, heart failure), severe anemia, mitochondrial enzyme defects and inhibitors of oxygen transport (carbon monoxide, cyanide), liver disease, and ethanol. Bicarbonate is usually administered to control acidosis associated with lactate overproduction. Dichloroacetate is used as a drug to treat lactic acidosis because it activates pyruvate dehydrogenase in mitochondria, which facilitates the conversion of pyruvate to acetyl CoA so that lactate production is decreased. Strenuous exercise leads to anerobic glycolysis. This causes overproduction of lactate.

40. Diabetes In diabetes, insulin activity is low (type 1 – no insulin; type 2 – insulin resistance). Consequently, glucagon levels increase. Thus, glucagon/insulin ratio is higher in diabetes. There is no change in glucose uptake in liver because this tissue does not express GLUT4, the insulin-responsive facilitative glucose transporter. In skeletal muscle and adipocytes, glucose uptake decreases because of the absence of insulin-dependent recruitment of GLUT4 into the plasma membrane. In liver, glucose is not metabolized effectively because:

41. This also results in an increase in gluconeogenesis, producing more glucose from gluconeogenic precursors alanine and glycerol. These precursers come from muscle and adipocytes as a result of increased protein and aminoacid breakdown and lipolysis to provide energy because of the decreased utility of glucose as the energy source.

42. Glucokinase deficiency: Mutations in glucokinase gene leading to inactivation of the enzyme are associated with a form of non-insulin-dependent diabetes mellitus (type 2) called Maturity Onset diabetes of the Young (MODY). Complete absence of the enzyme activity will lead to type 1 diabetes because of the lack of insulin secretion in response to blood glucose. This condition is associated with severe hyperglycemia. Mutations in the gene leading to increased activity of the enzyme will cause hyperinsulinemic hypoglycemia. Pyruvate kinase deficiency and hemolytic anemia 1. Genetic defect causing a 5-20% reduction in red cell pyruvate kinase levels. a. It's rare but the most common genetic disease associated with the glycolytic pathway. 2. Results in markedly lower ATP concentrations in erythrocytes. 3. Cells swell and lyse. 4. Reticulocytes are unaffected because these "immature" red cells contain mitochondria and are able to generate ATP through oxidative phosphorylation. 5. The levels of 2, 3-bisphosphoglycerate are high in erythrocytes. 6. No Heinz bodies 7. The 2nd most common genetic cause of hemolytic anemia, only next to glucose 6-phosphate dehydrogenase deficiency.

43. Other glycolytic enzyme defects Deficiencies in the activities of phosphofructokinase, phopshoglycerate kinase, phosphoglycerate mutase, and lactate dehydrogenase represent important genetic defects in glycolysis. All of these have certain common clinical features: exercise intolerance, myoglobinuria, hemolytic anemia, absence of lactate increase in forearm exercise test, and increased glycogen deposition in the liver and skeletal muscle.

44. Glycolysis also provides intermediates for other pathways In the liver, glycolysis is part of the process by which excess glucose is converted to fatty acids for storage

45. Important enzymes in glycolysis Hexokinase/glucokinase: glucose entering the cell is trapped by phosphorylation using ATP. Hexokinase is widely distributed in tissues, whereas glucokinase is found only in hepatocytes and pancreatic β-islet cells Phosphofructokinases (PFK-1 and PFK-2): PFK-1 is the rate-limiting enzyme and main control point in glycolysis. In this reaction, fructose 6-phosphate is phosphorylated to fructose 1,6-bisphosphate using ATP. PFK-1 is inhibited by ATP and citrate, and activated by AMP. Insulin stimulates and glucagon inhibits PFK-1 in hepatocytes by an indirect mechanism involving PFK-2 and fructose 2,6-bisphosphate

46. Insulin activates PFK-2 (via the tyrosine kinase receptor and activation of protein phosphatases), which converts a tiny amount of fructose 6-phosphate to fructose 2,6-bisphosphate (F2,6-BP). F2,6-BP activates PFK-1. Glucagon inhibits PFK-2 (via cAMP-dependent protein kinase A), lowering F2,6-BP and thereby inhibiting PFK-1

47. Glyceraldehyde 3-phosphate dehydrogenase : catalyzes an oxidation and addition of inorganic phosphate (Pi) to its substrate. This results in the production of a high-energy intermediate 1,3-bisphosphoglycerate and the reduction of NAD to NADH. If glycolysis is aerobic, the NADH can be reoxidized (indirectly) by the mitochondrial electron transport chain, providing energy for ATP synthesis by oxidative phosphorylation.

48. 3-Phosphoglycerate kinase : transfers the high-energy phosphate from 1,3-bisphosphoglycerate to ADP, forming ATP and 3-phosphoglycerate. This type of reaction in which ADP is directly phosphorylated to ATP using a high-energy intermediate is referred to as a substrate-level phosphorylation. In contrast to oxidative phosphorylation in mitochondria, substrate level phosphorylations are not dependent on oxygen, and are the only means of ATP generation in an anaerobic tissue. Pyruvate kinase : the last enzyme in aerobic glycolysis, it catalyzes a substrate-level phosphorylation of ADP using the high-energy substrate phosphoenolpyruvate (PEP). Pyruvate kinase is activated by fructose 1,6-bisphosphate from the PFK-1 reaction (feed-forward activation)

49. Lactate dehydrogenase : is used only in anaerobic glycolysis. It reoxidizes NADH to NAD, replenishing the oxidized coenzyme for glyceraldehyde 3-phosphate dehydrogenase. Without mitochondria and oxygen, glycolysis would stop when all the available NAD had been reduced to NADH. By reducing pyruvate to lactate and oxidizing NADH to NAD, lactate dehydrogenase prevents this potential problem from developing. In aerobic tissues, lactate does not normally form in significant amounts. However, when oxygenation is poor (skeletal muscle during strenuous exercise, myocardial infarction), most cellular ATP is generated by anaerobic glycolysis, and lactate production increases

50. Important Intermediates of Glycolysis Dihydroxyacetone phosphate (DHAP) is used in liver and adipose tissue for triglyceride synthesis. 1,3-Bisphosphoglycerate and phosphoenolpyruvate (PEP) are high-energy intermediates used to generate ATP by substrate-level phosphorylation Glycolysis Is Irreversible Three enzymes in the pathway catalyze reactions that are irreversible. When the liver produces glucose, different reactions and therefore different enzymes must be used at these three points: ● Glucokinase/hexokinase ● PFK-1 ● Pyruvate kinase

51. ATP Production and Electron Shuttles Anaerobic glycolysis yields 2 ATP/glucose by substrate-level phosphorylation Aerobic glycolysis yields these 2 ATP/glucose plus 2 NADH/glucose that can be utilized for ATP production in the mitochondria; however, the inner membrane is impermeable to NADH. Cytoplasmic NADH is reoxidized to NAD and delivers its electrons to one of two electron shuttles in the inner membrane In the malate shuttle, electrons are passed to mitochondrial NADH and then to the electron transport chain. In the glycerol phosphate shuttle, electrons are passed to mitochondrial FADH Important points; Cytoplasmic NADH oxidized using the malate shuttle produces a mitochondrial NADH and yields approximately 3 ATP by oxidative phosphorylation Cytoplasmic NADH oxidized by the glycerol phosphate shuttle produces a mitochondrial FADH2 and yields approximately 2 ATP by oxidative phosphorylation

52. Glycolysis in the Erythrocyte In red blood cells, anaerobic glycolysis represents the only pathway for ATP production, yielding a net 2 ATP/glucose Erythrocytes have bisphosphoglycerate mutase, which produces 2,3-bisphosphoglycerate(BPG) from 1,3-BPG in glycolysis. 2,3-BPG binds to the β-chains of hemoglobinA ( HbA ) and decreases its affinity for oxygen. The rightward shift in the curve is sufficient to allow unloading of oxygen in tissues, but still allows 100% saturation in the lungs. An abnormal increase in erythrocyte 2,3-BPG might shift the curve far enough so HbA is not fully saturated in the lungs. Although 2,3-BPG binds to HbA , it does not bind well to HbF (α2γ2), with the result that HbF has a higher affinity for oxygen than maternal HbA , allowing transplacental passage of oxygen from mother to fetus

53. We need to remember that galactose is a metabolite of lactose Lactose is broken down to one molecule of glucose and galactose GALACTOSE METABOLISM

54. There are no catabolic pathways to metabolize galactose , so the strategy is to convert galactose into a metabolite of glucose The first reaction in the galactose–glucose interconversion pathway is the phosphorylation of galactose to galactose 1-phosphate by galactokinase in the liver

55. Galactose 1-phosphate then acquires a uridyl group from uridine diphosphate glucose (UDP-glucose)

56. The products of this reaction, which is catalyzed by galactose 1-phosphate uridyl transferase, are UDP-galactose and glucose 1-phosphate The galactose moiety of UDP-galactose is then epimerized to glucose. The configuration of the hydroxyl group at carbon 4 is inverted by UDP-galactose 4-epimerase Note that UDP-glucose is not consumed in the conversion of galactose into glucose, because it is regenerated from UDP-galactose by the epimerase Finally, glucose 1-phosphate, formed from galactose, is isomerized to glucose 6-phosphate by phosphoglucomutase

57. Lactase deficiency Causes a condition known as Lactose intolerance , or hypolactasia Lactose is fermented in the colon to lactic acid, methane and hydrogen gas The gas produced creates the uncomfortable feeling of gut distention and the annoying problem of flatulence(bloating) Lactic acid and lactose are osmotically active, they draw water into the intestines which leads to diarrhea, dehydration Diagnosis is based on a positive hydrogen breath test after an oral lactose load. Best treatment is to avoid foods containing lactose, milk and milk products (except unpasteurized yogurt, which contains active Lactobacillus ) or by lactase pills.

58. Galactosemia important enzymes in galactose metabolism are: Galactokinase Galactose 1-phosphate uridyltransferase The disruption of galactose metabolism is referred to as galactosemia The most common form is called clasic galactosemia which is an inherited deficiency of Galactose 1-phosphate uridyltransferase

59. Cont …… symptoms manifest on day three of life and affected infants fail to thrive Vomiting and diarrhea occur after milk ingestion, liver cirrhosis, jaundice which does not resolve on phototherapy, severe bacterial infections, Failure to thrive, lethargy, hypotonia, and mental retardation are other common and apparent features Babies will also develop cataracts

60. mgt mandatory screening of newborns for galactosemia is recommended through a newborn heel prick test in the early weeks of life formulas containing galactose-free carbohydrates are given The life expectancy will then be normal with an appropriate diet.

61. cataracts A cataract is the clouding of the normally clear lens of the eye The eye lens has high concentrations of aldose reductase enzyme. In absence of galactose metabolizing enzymes, aldose reductase reduces galactose to galactitol

62. Galactitol is osmotically active leading to osmotic damage of the lens

63. FRUCTOSE METABOLISM

64. Fructose is found in honey and fruit and as part of the disaccharide sucrose (common table sugar) Sucrose is hydrolyzed by intestinal brush border sucrase, and the resulting monosaccharides, glucose and fructose, are absorbed into the portal blood The liver phosphorylates fructose and cleaves it into glyceraldehyde and DHAP The important enzymes are, Fructokinase Fructose 1-P aldolase ( aldolase B)

65. Fructose is found in honey and fruit and as part of the disaccharide sucrose (common table sugar) Sucrose is hydrolyzed by intestinal brush border sucrase, and the resulting monosaccharides, glucose and fructose, are absorbed into the portal blood The liver phosphorylates fructose and cleaves it into glyceraldehyde and DHAP The important enzymes are, Fructokinase Fructose 1-P aldolase ( aldolase B)

66. Hereditary Fructose Intolerance Hereditary fructose intolerance is an autosomal recessive disease (incidence of 1/20,000) due to a defect in the gene that encodes aldolase B in fructose metabolism There is accumulation of fructose 1-phosphate in hepatocytes and thereby sequestering of inorganic phosphate in this substance Eventually, the liver becomes damaged due to the accumulation of trapped fructose 1-phosphate Signs include lethargy, jaundice, vomiting, diarrhoea

67. Glycolysis The fate of pyruvate depends on oxygen availability. When oxygen is present, pyruvate is oxidized to acetyl-CoA which enters the Krebs cycle Without oxygen, pyruvate is reduced in order to oxidize NADH back to NAD +

68. CITRIC ACID CYCLE

69. also called the Krebs cycle or the tricarboxylic acid (TCA) cycle Takes place is in the mitochondria Although oxygen is not directly required in the cycle, the pathway will not occur anaerobically because NADH and FADH2 will accumulate if oxygen is not available for the electron transport chain

70. The primary function of the citric acid cycle is oxidation of acetyl-CoA to carbondioxide The energy released from this oxidation is saved as NADH, FADH2, and guanosine triphosphate (GTP) It does not represent a pathway for the net conversion of acetyl-CoA to citrate, to malate, or to any other intermediate of the cycle. The only fate of acetyl-CoA in this pathway is its oxidation to CO2

71. The cycle is central to the oxidation of any fuel that yields acetyl-CoA, including glucose, fatty acids, ketone bodies, ketogenic amino acids, and alcohol There is no hormonal control of the cycle, as activity is necessary irrespective of the fed or fasting state Control is exerted by the energy status of the cell through allosteric activation or deactivation. Many enzymes are subject to negative feedback

72. All the enzymes are in the matrix of the mitochondria except succinate dehydrogenase, which is in the inner membrane.

73. Key points: Isocitrate dehydrogenase, the major control enzyme, is inhibited by NADH and ATP and activated by ADP. α- Ketoglutarate dehydrogenase, like pyruvate dehydrogenase, is a multienzyme complex. It requires thiamine, lipoic acid, CoA, FAD, and NAD. Lack of thiamine slows oxidation of acetyl-CoA in the citric acid cycle. Succinyl -CoA synthetase (succinate thiokinase ) catalyzes a substrate-level phosphorylation of GDP to GTP. Succinate dehydrogenase is on the inner mitochondrial membrane, where it also functions as complex II of the electron transport chain. Citrate synthase condenses the incoming acetyl group with oxaloacetate to form citrate

74. Several intermediates of the cycle may serve other functions Citrate may leave the mitochondria (citrate shuttle) to deliver acetyl- CoA into the cytoplasm for fatty acid synthesis. Succinyl -CoA is a high-energy intermediate that can be used for heme synthesis and to activate ketone bodies in extrahepatic tissues. Malate can leave the mitochondria (malate shuttle) for gluconeogenesis

75. When intermediates are drawn out of the citric acid cycle, the cycle slows. Therefore when intermediates leave the cycle they must be replaced to ensure sufficient energy for the cell

76. • The cycle involves a sequence of compounds interrelated by oxidation-reduction and other reactions which finally produces CO2 and H2O. • It is the final common pathway of break down/catabolism of carbohydrates, fats and proteins. (Phase III of metabolism). • Acetyl-CoA derived mainly from oxidation of either glucose or β-oxidation of FA and partly from certain amino acids combines with oxaloacetic acid (OAA) to form citrate the first reaction of citric acid cycle.

77. In this reaction acetyl-CoA transfers its ‘acetyl-group’ (2-C) to OAA. By stepwise dehydrogenations and loss of two molecules of CO2, accompanied by internal re-arrangements, the citric acid is reconverted to OAA , which again starts the cycle by taking up another acetyl group from acetyl-CoA. A very small catalytic amount of OAA can bring about the complete oxidation of active-acetate Enzymes are located in mitochondrial matrix, either free or attached to the inner surface of the inner mitochondrial membrane, which facilitates the transfer of reducing equivalents to the adjacent enzymes of the respiratory chain.

78. The whole process is aerobic , requiring O2 as the final oxidant of the reducing equivalents. Absence of O2 (anoxia) or partial deficiency of O2 (hypoxia) causes total or partial inhibition of the cycle. The H atoms removed in the successive dehydro - genations are accepted by corresponding coenzymes. Reduced coenzymes transfer the reducing equivalents to electron-transport system, where oxidative phos - phorylation produces ATP molecules.

79. BIOMEDICAL IMPORTANCE OF CITRIC ACID CYCLE Final common pathway for carbohydrates, proteins and fats, through formation of 2-carbon unit acetyl-CoA. Acetyl-CoA is oxidised to CO2 and H2O giving out energy (CATABOLIC ROLE) Intermediates of TCA cycle play a major role in synthesis also like heme formation, formation of non-essential amino acids, FA synthesis, cholesterol and steroid synthesis- anabolic role.

80. ELECTRON TRANSPORT CHAIN AND OXIDATIVE PHOSPHORYLATION Although the value of ΔG should not be memorized, it does indicate the large amount of energy released by both reactions. The electron transport chain is a device to capture this energy in a form useful for doing work.

81. Electron Transport Chain The electron transport chain (ETC) is a series of membrane-bound electron carriers. -embedded in the mitochondrial inner membrane -electrons from NADH and FADH 2 are transferred to complexes of the ETC -each complex transfers the electrons to the next complex in the chain

82. Electron Transport Chain As the electrons are transferred, some electron energy is lost with each transfer. This energy is used to pump protons (H + ) across the membrane from the matrix to the inner membrane space. A proton gradient is established.

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84. Electron Transport Chain The higher negative charge in the matrix attracts the protons (H + ) back from the intermembrane space to the matrix. The accumulation of protons in the intermembrane space drives protons into the matrix via diffusion.

85. Electron Transport Chain Most protons move back to the matrix through ATP synthase . ATP synthase is a membrane-bound enzyme that uses the energy of the proton gradient to synthesize ATP from ADP + P i .

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88. Energy Yield of Respiration theoretical energy yields - 38 ATP per glucose for bacteria - 36 ATP per glucose for eukaryotes actual energy yield - 30 ATP per glucose for eukaryotes - reduced yield is due to “leaky” inner membrane and use of the proton gradient for purposes other than ATP synthesis

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