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Biochemistry pt. 1


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Biochemistry pt. 1

  1. 1. Fundamentals and Societal Applications
  2. 2. Biochemistry is the study of the structure, composition, and chemical reactions of substances in living systems.
  3. 3.  The importance of biochemistry can be seen from the fact that it is used in many daily activities.  It is used in clinical diagnosis, manufacture of various biological products, treatment of diseases, in nutrition, agriculture etc.  The study of biochemistry helps one understand the actual chemical concepts of biology.  That is the functioning of various body processes and physiology by uses of biomolecules.
  4. 4. Biochemistry is a valuable subject in medicine without which there would have been no such advancement in the field.  Physiology: Biochemistry helps one understand the biochemical changes and related physiological alteration in the body. Pathology of any disease is studied through biochemical changes.  Pathology: Based on the symptoms described by the patient, the physician can get a clue on the biochemical change and the associated disorder. For example, if a patient complains about stiffness in small joints, then the physician may predict it to be gout and get confirmed by evaluating uric acid levels in the blood. As uric acid accumulation in blood results in gout.  Nutrition deficiency: In the present scenario, many people rely on taking multivitamin & minerals for better health. The function and role of the vitamin in the body are described only by biochemistry.  Hormonal deficiency: There are many disorders due to hormonal imbalance in especially women and children. The formation, role of hormones in the normal body function is taught in biochemistry by which the physician can understand the concerned problem during treatment.
  5. 5. Almost all the diseases or disorders have some biochemical involvement. So the diagnosis of any clinical condition is easily possible by biochemical estimations.  Kidney function test: For example in kidney disorders, other chemotherapy treatment etc urine test help understand the extent of excretion of drugs or other metabolites, the change in pH, the color of urine etc.  Blood test: In diabetes, biochemical analytical test for blood glucose level (above 150mg/ deciliter helps one understand the severity of diabetes disorder. Another biochemical test for ketones bodies in urine also indicates the stage of diabetes. The appearance of ketone bodies or ketone urea is mostly the last stage of diabetes.  Liver function tests help understand the type of disease or damage to the liver, the effect of any medication on liver etc.  Serum cholesterol test: Evaluation of blood cholesterol level and other lipoproteins helps understand the proneness of the patient to cardiovascular diseases.
  6. 6. In agriculture, biochemistry plays a valuable role in farming, fishery, poultry, sericulture, beekeeping etc.  Prevent diseases: It helps for prevention, treatment of diseases and also increases the production or yield.  Enhance growth: Biochemistry gives an idea of how the use of fertilizers can increase plant growth, their yield, quality of food etc.  Enhance Yield: Some hormones promote growth, while other promote flowering, fruit formation etc. In fisheries, use of substances to promote fish growth, their reproduction etc can be understood.  Adulteration: Even the composition of food material produced, their alteration or adulteration for example in honey can be found by biochemical tests. Biochemistry tests help prevent contamination.  Biochemical tests for the pesticide residues or other toxic waste in plant, food grain and soil can be evaluated. Hence during import and export of food grains a biochemical check of the toxic residues is done to fix the quality.  In animal husbandry, the quality of milk can be checked by biochemical tests. It also helps diagnose any disease condition in animals and birds.  In fisheries, the water quality is regularly monitored by biochemical tests. Any drastic change in water chemistry & composition of fishery ponds can lead to the vast death of fishes and prawns, hence the tests are done on regular basis to see salt content (calcium content), pH, accumulation of waste due to not changing water for long etc.
  7. 7. In nutrition, biochemistry describes the food chemistry. For maintenance of health, optimum intake of many biochemicals like macro, micronutrients, vitamins, minerals, essential fatty acids & water is necessary.  Food chemistry gives an idea of what we eat, i.e. it’ s components like carbohydrates, proteins, fats,etc. and also the possible physiological alteration due to their deficiency.  The role of nutrients: Due to biochemistry the importance of vitamins, minerals, essential fatty acids, their contribution to health were known. Hence there is a frequent recommendation for inclusion of essential amino-acids, cod liver oil, salmon fish oil etc. by physicians and other health and fitness experts.  The nutrients value of food material can also be determined by biochemical tests.  The physician can prescribe to limit usage of certain food like excess sugar for diabetics, excess oil for heart & lung problem prone patients etc. As these carbohydrate and fat diets can inhibit the recovery rate from said disorder. This knowledge is due to their idea of food chemistry and related
  8. 8. In a pharmacy, many drugs are stored for regular dispensing.  Drug Constitution: Biochemistry gives an idea of the constitution of the drug, its chances of degradation with varying temperature etc. How modification in the medicinal chemistry helps improve efficiency, minimize side effects etc.  The half-life: This is a test done on biochemical drugs to know how long a drug is stable when kept at so and so temperature.  Drug storage: The storage condition required can be estimated by the biochemical test.For example many enzymes, hormones are stored for dispensing. These get deteriorated over time due to temperature or oxidation, contamination and also due to improper storage.  Drug metabolism: It also gives an idea of how drug molecules are metabolized by many biochemical reactions in presence of enzymes. This helps to avoid drugs which have a poor metabolism or those with excessive side effects from being prescribed or dispensed to the patient.  Biochemical tests: These tests helps fix the specific half-life or date of expiry of drugs.
  9. 9. Biochemistry of plants gave way to the breakthrough of how food is synthesized in them and the reason why they are autotrophs i.e. not dependent on other living beings for food. Biochemistry in plants describes  1. Photosynthesis: This describes how carbohydrates are synthesized by use of sunlight, CO2, and water in the green leaves of plants. It goes on to explain about different complex enzymes involved in the process to combine the energy of sun within the molecules H2O+ CO2 in the form of carbohydrates.  2. Respiration: By use of above photosynthesis pathway, plants leave out Oxygen while taking up Carbon dioxide from the air.  3. Different sugars: Biochemistry defines different types of carbohydrates formed in plants like trioses (3 carbon sugars i.e. glyceraldehyde), tetroses (4), pentoses (5), hexoses (6= glucose), heptuloses (7) etc. Heptuloses are the carbohydrates which go on to form the nucleic acids i.e deoxyribonucliec acid (DNA), ribonucleic acid (RNA).  4. Plants secondary metabolites: Biochemistry also describes how the plant products like gums, tannins, alkaloids, resins, enzymes, phytohormones are formed inside the plants.  5. Other functions: It also describes how plants fruits get ripened, how plant seed germinates, the respiration process inside the plant cell, how proteins and amino acids are formed on rough endoplasmic reticulum and fats are formed on smooth ER.
  10. 10. In Biology… • Living organisms must work to stay alive, to grow and to reproduce • All living organisms have the ability to produce energy and to channel it into biological work • Living organisms carry out energy transductions, conversions of one form of energy to another form
  11. 11. • Modern organisms use the chemical energy in fuels (carbonhydrates, lipids) to bring about the synthesis of complex macromolecules from simple precursors • They also convert the chemical energy into concentration gradients and electrical gradients, into motion and heat, and, in a few organisms into light (fireflies, some deep-sea fishes)
  12. 12. • Biological energy transductions obey the same physical laws that govern all other natural processes • Bioenergetics is the quantitative study of the energy transductions that occur in living cells and of the nature and function of the chemical process underlying these transductions
  13. 13. The branch of physical chemistry known as thermodynamics is concerned with the study of the transformations of energy. That concern might seem remote from chemistry, let alone biology; indeed, thermodynamics was originally formulated by physicists and engineers interested in the efficiency of steam engines. However, thermodynamics has proved to be of immense importance in both chemistry and biology. Not only does it deal with the energy output of chemical reactions but it also helps to answer questions that lie right at the heart of biochemistry, such as how energy flows in biological cells and how large molecules assemble into complex structures like the cell.
  14. 14. • The first law of thermodynamics, also known as Law of Conservation of Energy, states that energy can neither be created nor destroyed; energy can only be transferred or changed from one form to another. For example, turning on a light would seem to produce energy; however, it is electrical energy that is converted. • A way of expressing the first law of thermodynamics is that any change in the internal energy (∆E) of a system is given by the sum of the heat (q) that flows across its boundaries and the work (w) done on the system by the surroundings: ΔE=q+w
  15. 15. • This law says that there are two kinds of processes, heat and work, that can lead to a change in the internal energy of a system. Since both heat and work can be measured and quantified, this is the same as saying that any change in the energy of a system must result in a corresponding change in the energy of the surroundings outside the system. In other words, energy cannot be created or destroyed. If heat flows into a system or the surroundings do work on it, the internal energy increases and the sign of q and w are positive. Conversely, heat flow out of the system or work done by the system (on the surroundings) will be at the expense of the internal energy, and q and w will therefore be negative.
  16. 16. • The second law of thermodynamics says that the entropy of any isolated system always increases. Isolated systems spontaneously evolve towards thermal equilibrium—the state of maximum entropy of the system. More simply put: the entropy of the universe (the ultimate isolated system) only increases and never decreases. • A simple way to think of the second law of thermodynamics is that a room, if not cleaned and tidied, will invariably become more messy and disorderly with time - regardless of how careful one is to keep it clean. When the room is cleaned, its entropy decreases, but the effort to clean it has resulted in an increase in entropy outside the room that exceeds the entropy lost.
  17. 17. The third law of thermodynamics states that the entropy of a system approaches a constant value as the temperature approaches absolute zero. The entropy of a system at absolute zero is typically zero, and in all cases is determined only by the number of different ground states it has. Specifically, the entropy of a pure crystalline substance (perfect order) at absolute zero temperature is zero. This statement holds true if the perfect crystal has only one state with minimum energy.
  18. 18. Living organisms and the second law • The reacting system is the collection of matter that is undergoing a particular chemical or physical process; it may be an organism, a cell, or two reacting compounds. The reacting system and its surroundings together constitute the universe. In the laboratory, some chemical or physical processes can be carried out in isolated or closed systems, in which no material or energy is exchanged with the surroundings. Living cells and organisms, however, are open systems, exchanging both material and energy with their surroundings; living systems are never at equilibrium with their surroundings, and the constant transactions between system and surroundings explain how organisms can create order within themselves while operating within the second law of thermodynamics.
  19. 19. Three thermodynamic quantities that describe the energy changes occurring in a chemical reaction 1.Gibbs free energy, G, expresses the amount of energy capable of doing work during a reaction at constant temperature and pressure. When a reaction proceeds with the release of free energy (that is, when the system changes so as to possess less free energy), the free-energy change, G, has a negative value and the reaction is said to be exergonic. In endergonic reactions, the system gains free energy and G is positive. The units of ΔG are expressed in joules/mole or calories/mole (1 calorie: 4.184 J)
  20. 20. 2. Enthalpy, Δ H, is the heat content of the reacting system. It reflects the number and kinds of chemical bonds in the reactants and products. • When a chemical reaction releases heat, it is said to be exothermic; the heat content of the products is less than that of the reactants and Δ H has, by convention, a negative value. • Reacting systems that take up heat from their surroundings are endothermic and have positive values of Δ H. The units of ΔH are expressed in joules/mole or calories/mole (1 calorie: 4.184 J)
  21. 21. Entropy, S, is a quantitative expression for the randomness or disorder in a system When the products of a reaction are less complex and more disordered than the reactants, the reaction is said to proceed with a gain in entropy. *Units of entropy are expressed in joules/mole x K Relationship of entropy and enthalpy expressed in: Δ G = Δ H - T Δ S
  22. 22. Relationship of free energy, entropy, enthalpy expressed in: Δ G = Δ H - T Δ S Δ G = change in Gibbs free energy of the reacting system Δ H =change in enthalpy of the system/total energy of the system Δ T = absolute temperature Δ S =change in entropy of the system. • Δ S has a positive sign when entropy increases • Δ H, has a negative sign when heat is released by the system to its surroundings. • Either of these conditions, which are typical of favorable processes, tend to make G negative. In fact, G of a spontaneously reacting system is always negative.
  23. 23. • The second law of thermodynamics states that the entropy of the universe increases during all chemical and physical processes • Does not require that the entropy increase take place in the reacting system itself. • The order produced within cells as they grow and divide is more than compensated for by the disorder they create in their surroundings in the course of growth and division. • In short, living organisms preserve their internal order by taking from the surroundings free energy in the form of nutrients or sunlight, and returning to their surroundings an equal amount of energy as heat and entropy.
  24. 24. Cells Require Free Sources of Energy • Cells are isothermal systems—they function at essentially constant temperature (they also function at constant pressure). Heat flow is not a source of energy for cells, because heat can do work only as it passes to a zone or object at a lower temperature. The energy that cells can and must use is free energy, described by the Gibbs free-energy function G, which allows prediction of the direction of chemical reactions, their exact equilibrium position, and the amount of work they can in theory perform at constant temperature and pressure. Heterotrophic cells acquire free energy from nutrient molecules, and photosynthetic cells acquire it from absorbed solar radiation. Both kinds of cells transform this free energy into ATP and other energy-rich compounds capable of providing energy for biological work at constant temperature.
  25. 25. Relationship of Standard Free Energy Change and Equilibrium Constant • The composition of a reacting system tends to continue changing until equilibrium is reached. At the equilibrium the rates of the forward and revers reactions are equal and no further change occurs in the system. The Keq is defined by the molar concentrations of products and reactants at equilibrium • aA+bB cC+ dD • [C]c [D]d • Keq = ------------------ • [A]a [B]b • Where [A], [B], [C], and [D] are the molar concentrations of the reaction components at the point of equilibrium.
  26. 26. • When a reacting system is not at equilibrium, the tendency to move toward the equilibrium represents a driving force. The magnitude of this driving force is expressed as free energy change (ΔG). • • Under standard conditions (250C), when reactants and products are initially at the 1 M concentrations the force driving the system toward equilibrium is defined as the standard free energy change (ΔG0)
  27. 27. • By this definiation, standart state for reactions involves [H+] = 1M or pH=0. • However most biochemical reactions occur in well- buffered aqueous solutions near pH=7 • For convenience of calculations, biochemists define a different standard state in which the concentration of [H+] is 10-7 M , and for reactions that involve Mg2+ (available in most reactions involving ATP), its concentration in solution is commonly taken to be constant at 1mM •
  28. 28. • Physical constants based on this biochemical standard state are called standard transformed constants and written as ΔG'0 and K'eq to distinguish them from the untransformed constants which are used by chemists.
  29. 29. • ΔG'0 is the difference between the free energy content of the products and the free energy contents of the reactants under standard conditions • ΔG'0 = ΔG'0 products– ΔG'0 reactives) • When ΔG'0 is negative, the products contain less free energy than the reactants and the reaction will proceed spontaneously under standard conditions • When ΔG'0 is positive, the products contain more free energy than the reactants and the reaction will tend to go in the revers direction under standard conditions
  30. 30. • Each chemical reaction has a characteristic standard free energy change which may be positive, negative or zero depending on the equilibrium constant of the reaction • ΔG'0 tell us in which direction and how far a given reaction must go to reach equilibrium when the initial concentration of each component is 1M, the pH is 7, the temparature is 250C. • Thus ΔG'0 is a constant; a characteristic for a given reaction
  31. 31. • Actual free energy change (ΔG) is a function of reactant and product concentrations and of the temparature prevailing during the reaction which will not necessarily match the standard conditions as defined before
  32. 32. • ΔG of any reaction proceeding spontaneously toward its equilibrium is always negative, become less negative as the reaction proceeds, and is zero at he point of equilibrium, indicating that no more work can be done by the reaction • ΔG and ΔG'0 for a reaction like that • A+B C+D • is written as • [C] [D] • ΔG=ΔG'0 + RTln • [A] [B]
  33. 33. • An example: • A+B C+D • Reaction is taking place at the standard temparature and pressure • But the concentrations of A,B,C and D are not equal and none of them at the 1M concentration • In order to determine actual ΔG under these non- standard concentrations as the reaction proceeds from left to right, we enter the actual concentrations of A,B,C and D in this equation.
  34. 34. • Rest of the terms in the equation (R,T, ΔG'0 ) are standard values • When the reaction is at equilibrium there is no force driving the reaction in either direction and ΔG is zero, thus equation reduces to • [C] [D] • 0= ΔG'0 + RTln • [A] [B] • ΔG'0 = -RT lnK'eq ΔG'0
  35. 35. • The criteria for spontaneity of a reaction is the value of ΔG not ΔG'0 • Standard free energy changes are additive. • In the case of two sequential chemical reactions, • A B ΔG'0 1 • B C ΔG'0 2
  36. 36. • Since the two reactions are sequential, we can write the overall reaction as • A C ΔG'0 total • The ΔG'0 values of sequential reactions are additive. • ΔG'0 total = ΔG'0 1 + ΔG'0 2
  37. 37. • A B ΔG'0 1 • B C ΔG'0 2 • Sum: A C ΔG'0 1 + ΔG'0 2 • This principle of bioenergetics explains how a thermodynamically unfavorable (endergonic) reaction can be driven in the forward direction by coupling it to a highly exergonic reaction through a common intermediate
  38. 38. • The main rule in biochemical reactions in living organisms: • All endergonic reactions are coupled to an exergonic reaction. There is an energy cycle in cells that links anabolic and catabolic reactions.
  39. 39. Glycolysis • Glykys = Sweet, Lysis = splitting • During this process one molecule of glucose (6 carbon molecule) is degraded into two molecules of pyruvate (three carbon molecule). • Free energy released in this process is stored as 2 molecules of ATP, and 2 molecules of NADH. • Glucose + 2NAD+ = 2Pyruvate + 2NADH + 2H+ d= -146 kJ/mol • 2ADP + 2Pi = 2ATP + 2H2O dGo = 2X(30.5 kJ/mol) = 61 kJ/mol • ----------------------------------------------------- • dGo (overall) = -146+61 = -85 kJ/mol • In standard condition glycolysis is an exergonic reaction which tends to be irreversible because of negative dGo.
  40. 40. → It is also called as Embden-Meyerhof Pathway (EMP) → it is defined as the sequence of reactions converting glucose or glycogen to pyruvate or lactate with production of ATP. → Enzymes takes place in cytosomal fraction of the cell. → major pathway in tissues lacking mitochondria like erythrocytes, cornea, lens etc. → it is essential for brain which is dependent in glucose for energy. → under anaerobic condition = glu + 2ADP + 2iP ----- 2 Lactate + 2ATP
  41. 41. Glucose + 6O2 = 6CO2 + 6H2O dGo= -2840 kJ/mol Glucose + 2NAD+ = 2Pyruvate + 2NADH + 2H+ dGo = -146 kJ/mol 5.2% of total free energy that can be released by glucose is released in glycolysis. Fate of Glucose in Living Systems
  42. 42. •Glycolysis was the very first biochemistry or oldest biochemistry studied. •It is the first metabolic pathway discovered. •Louis Pasture 1854-1864: Fermentation is caused by microorganism. Pastuer’s effect: Aerobic growth requires less glucose than anaerobic condition. •Buchner; 1897: Reactions of glycolysis can be carried out in cell- free yeast extract. •Harden and Young 1905: 1: inorganic phosphate is required for fermentation. 2: yeast extract could be separated in small molecular weight essential coenzymes or what they called Co- zymase and bigger molecules called enzymes or zymase. •1940: with the efforts of many workers, complete pathways for glycolysis was established. History of Glycolysis
  43. 43. Net Gain of Glycolysis Basically in the process of glycolysis, the following are invested: Which will yield
  44. 44. • There are 10 enzyme-catalyzed reactions in glycolysis. There are two stages • Stage 1: (Reactions 1-5) A preparatory stage in which glucose is phosphorylated, converted to fructose which is again forphorylated and cleaved into two molecules of glyceraldehyde-3-phosphate. In this phase there is an investment of two molecules of ATP. • Stage 2: (Reactions 6-10) The two molecules of glyceraldehyde-3- phosphate are converted to pyruvate with concomitant generation of four ATP molecules and two molecules of NADH. Thus there is a net gain of two ATP molecules per molecule of Glucose in glycolysis. Importance of phosphorylated intermediates: 1. Possession of negative charge which inhibit their diffusion through membrane. 2. Conservation of free energy in high energy phosphate bond. 3. Facilitation of catalysis.
  45. 45. 1. Hexokinase reaction: Phosphorylation of hexoses (mainly glucose) I. This enzyme is present in most cells. In liver Glucokinase is the main hexokinase (both ISOENZYMES) which prefers glucose as substrate. II. It requires Mg-ATP complex as substrate. Un- complexed ATP is a potent competitive inhibitor of this enzyme. III. Enzyme catalyses the reaction by proximity effect; bringing the two substrate in close proximity. IV. This enzyme undergoes large conformational change upon binding with Glucose. It is inhibited allosterically by G6P.
  46. 46. Diagram of Hexokinase reaction
  47. 47. Difference between Hexokinase & Glucokinase
  48. 48. 2. Phosphoglucose Isomerase or Phosphohexose Isomerase: Isomerization of G6P to Fructose 6 phosphate. I. This enzyme catalyzes the reversible isomerization of G6P (an aldohexose) to F6P (a ketohexose). II. This enzyme requires Mg ++ for its activity. III.It is specific for G6P and F6P.
  49. 49. 3. Phosphofructokinase-1 Reaction: Transfer of phosphoryl group from ATP to C-1 of F6P to produce Fructose 1,6 bisphosphate. I. This step is an important irreversible, regulatory step. II. The enzyme Phosphofructokinase-1 is one of the most complex regulatory enzymes, with various allosteric inhibitors and activators. III. ATP is an allosteric inhibitor, and Fructose 2,6 biphosphate is an activator of this enzyme. IV. ADP and AMP also activate PFK-1 whereas citrate is an inhibitor.
  50. 50. Aldolase Reaction: Cleavage of Fructose 1,6 bisphosphate into glyceraldehyde 3 phosphate (an aldose) and dihydroxy acetone phosphate (a ketose). I. This enzyme catalyses the cleavage of F1,6 biphosphate by aldol condensation mechanism. II. As shown below, the standard free energy change is positive in the forward direction, meaning it requires energy. Since the product of this reaction are depleted very fast in the cells, this reaction is driven in forward direction by the later two reactions.
  51. 51. 5. Triose phosphate mutase reaction: Conversion of Dihydroxyacetone phosphate to glyceraldehyde 3 Phosphate. I. This a reversible reaction catalysed by acid-base catalysis in which Histidine-95 and Glutamate -165 of the enzyme are involved.
  52. 52. 6. Glyceraldehyde-3-phosphate dehydrogenase reaction (GAPDH): Conversion of GAP to Bisphosphoglycerate. I. This is the first reaction of energy yielding step. Oxidation of aldehyde derives the formation of a high energy acyl phosphate derivative. II. An inorganic phosphate is incorporated in this reaction without any expense of ATP. III.NAD+ is the cofactor in this reaction which acts as an oxidizing agent. The free energy released in the oxidation reaction is used in the formation of acylphosphate.
  53. 53. The mechanism of GAPDH reaction: Evidence for the mechanism; I. Iodoacetate inhibits this reaction, indicating the involvement of Cysine residue of enzyme. II. Tritium from GAP is transferred to NAD, indicating transfer of hydide ion in oxidation reaction. III. 32P exchanges with PO4 - - indicating acyl enzyme intermediate. Steps in reaction mechanism: 1. Glceraldehyde- 3- phosphate (GAP) binding to the enzyme. 2. Nucleophilic attack by SH group (sulfhydril group) on CHO group forming a thiohemiacytal. 3. Direct transfer of hydride to NAD+ leading to the formation of thioester. Energy of this oxidation is conserved in synthesis of thioester and NADH. 4. Another molecule of NAD+ replaces NADH from enzyme site. 5. Nucleophilic attack on thioester by PO4 – - to form 1,3 bisphosphoglycerate
  54. 54. 1. Hexokinase reaction: Phosphorylation of hexoses (mainly glucose) I. This enzyme is present in most cells. In liver Glucokinase is the main hexokinase (both ISOENZYMES) which prefers glucose as substrate. II. It requires Mg-ATP complex as substrate. Un- complexed ATP is a potent competitive inhibitor of this enzyme. III. Enzyme catalyses the reaction by proximity effect; bringing the two substrate in close proximity. IV. This enzyme undergoes large conformational change upon binding with Glucose. It is inhibited allosterically by G6P.
  55. 55. 7. Phosphoglycerate kinase Reaction: Transfer of phosphoryl group fron 1,3 bisphosphoglycerate to ADP generating ATP. I. The name of this enzyme indicates its function for reverse reaction. II. It catalyses the formation by proximity effect. ADP-Mg bind on one domain and 1,3BPG binds on the other and a conformational change brings them together similar to hexokinase. III. This reaction and the 6th step are coupled reaction generating ATP from the energy released by oxidation of 3-phosphoglyceraldehyde. IV. This step generates ATP by SUBSTRATE-LEVEL PHOSPHORYLATION.
  56. 56. 8. Phosphoglycerate Mutase Reaction: Conversion of 3- phosphoglycerate to 2-phosphoglycerate (2-PG). I. In active form, the phosphoglycerate mutase is phosphorylated at His-179. II. There is transfer of the phosphoryl group frm enzyme to 3-PG, generating enzyme bound 2,3-biphosphoglycerate intermediate. This compound has been observed occasionally in reaction mixture. III. In the last step of reaction the phosphoryl group from the C-3 of the intermediate is transferred to the enzyme and 2-PG is released. IV. In most cells 2,3BPG is present in trace amount, but in erythrocytes it is present in significant amount. There it regulates oxygen affinity to hemoglobin.
  57. 57. 9. Enolase Reaction: Dehydration of 2- phosphoglycerate (2-PG) to phosphoenolpyruvate (PEP). I. Dehydration of 2-PG by this reaction increases the standard free enrgy change of hydrolysis of phosphoanhydride bond. II. Mechanism: Rapid extraction of proton from C-2 position by a general base on enzyme, generating a carbanion. The abstracted proton is readily exchanges with solvent. III.The second rate limiting step involves elimination of OH group generating PEP
  58. 58. 10. Pyruvate Kinase Reaction: Transfer of phosphoryl group from PEP to ADP generating ATP and Pyruvate. I. This is the second substrate level phosphorylation reaction of glycolysis. II. This enzyme couple the free enrgy of PEP hydrolysis to the synthesis of ATP III.This enzyme requires Mg++ and K+
  59. 59. • A tautomer is a separate type isomer by an organic compound that has the property that it can quickly change their isomeric form by chemical reaction called tautomerization. Typically, this occurs as the migration of hydrogen atoms (protons) by an exchange of one single bond with a double bond.
  60. 60. From one molecule of Glucose: 1Gl+2ATP+2NAD++ 4ADP+ 4Pi = 2pyruvate+2NADH+4ATP+ 2ADP+ 2Pi After balancing: 1Gl + 2NAD++ 2ADP + 2Pi = 2pyruvate+2ATP + 2NADH 2 molecules of ATP generated can directly be used for doing work or synthesis. The 2 NADH molecules are oxidized in mitochondria under aerobic condition and the free energy released is enough to synthesize 6 molecules of ATP by oxidative phosphorylation. Under the aerobic condition, pyruvate is catabolized further in mitochondria through pyruvate dehydrogenase and cytric acid cycle where all the carbon atoms are oxidized to CO2. The free energy released is used in the synthesis of ATP, NADH and FADH2. Under anaerobic condition: Pyruvate is converted to Lactate in homolactic fermentation or in ethanol in alcohalic fermentation.
  61. 61. 1. Hexokinase reaction: Phosphorylation of hexoses (mainly glucose) I. This enzyme is present in most cells. In liver Glucokinase is the main hexokinase (both ISOENZYMES) which prefers glucose as substrate. II. It requires Mg-ATP complex as substrate. Un- complexed ATP is a potent competitive inhibitor of this enzyme. III. Enzyme catalyses the reaction by proximity effect; bringing the two substrate in close proximity. IV. This enzyme undergoes large conformational change upon binding with Glucose. It is inhibited allosterically by G6P.
  62. 62. Energetics of Glycolysis Pathway ATP FORMED: 1. Gly-3-PO4--- 1,3 Bisphosphoglycerate = 6 ATP 2. 1,3 Bisphosphoglycerate-3-Phosphoglycerate = 2 ATP 3. Phosphoenolpyruvate-- Enol pyruvate = 2 ATP ATP CONSUMED: 4. Glucose---- Glucose-6-PO4 = 1 ATP 5. Fru-6-PO4---- Fru-1,6 bisphosphate = 1ATP ----------------------- Net ATP synthesized 10 – 2 = 8 ATP
  63. 63. 1. Insulin stimulate Hexokinase & Glucokinase by converting glucose to glu-6-PO4 2. Insulin stimulate Phosphofructokinase converting fru-6-PO4 to Fru-1,6 bisphosphate 3. Glucagon stimulate liver glu-6-PO4 by converting glu-6-PO4 to glucose & fru-1,6- bisphosphate. 4. Fru-1,6- bisphosphate is converted to fru-6- PO4
  64. 64. 1. Iodoacetate inhibit Gly-3-PO4 dehydrogenase involved in gly-3-PO4 to 1,3-bisphosphoglycerate 2. Arsenate inhibit sysnthesis of ATP in the conversion of 1,3 bisphosphoglycerate to 3-phosphoglycerate. 3. Fluoride inhibit enolase in conversion of 2-Phosphoglycerate to phosphoglycerate
  65. 65. In an anaerobic condition or in the need of sudden need of high amount of ATP, glycolysis is the main source for generation of ATP. NAD+ is one of the crucial cofactor required for GAPDH reaction. In order to regenerate NAD+ from the reduced form (NADH), this reaction takes place in muscle cells. Lactate dehydrogenase (LDH) reduces pyruvate to lactate using NADH and thereby oxidizing it to NAD+ . Other than regenerating NAD+ for running GAPDH reaction, LDH reaction is a waste of energy, and its product lactic acid brings the pH lower and causes fatigue.
  66. 66. Glycolysis can generate sudden burst of ATP without oxygen, using glucose and glycogen storage of muscle and liver. NAD+ is regenerated by lactic fermentation to carry out GAPDH reaction of glycolysis.
  67. 67. Alcoholic fermentation: Microorganisms and yeast convert pyruvate to alcohol and carobon dioxide to regenerate NAD+ for glycolysis (step 6, GAPDH). It is a two step process: 1. Pyruvate decarboxylase (PDC) reaction: This enzyme is Mg++- dependent and requires an enzyme- bound cofactor, thiamine pyrophosphate (TPP). In this reaction a molecule of CO2 is released producing acetaldehyde. 2. Alcohal dehydrogenase reaction: Acetaldehyde is reduced to ethanol using NADH as reducing power, thus regenerating NAD+ .
  68. 68. I. Nucleophilic attack on cabonyl gp carbon of pyruvate by TPP anion. II. Departure of CO2 leaving the carbanion-TPP adduct. III. Protonation of carbanion IV. Release of acetaldehyde following regeneration of TPP
  69. 69. Two types controls for metabolic reactions: a) Substrate limited : When concentrations of reactant and products in the cell are near equilibrium, then it is the availability of substrate which decides the rate of reaction. b) Enzyme-limited: When concentration of substrate and products are far away from the equilibrium, then it is activity of enzyme that decides the rate of reaction. These reactions are the one which control the flux of the overall pathway. There are three steps in glycolysis that have enzymes which regulate the flux of glycolysis. I. The hexokinase (HK) II. The phoshofructokinase (PFK) III. The pyruvate kinase
  70. 70. Its activity is controlled by a complex allosteric regulation. This reaction commits the cells to channel glucose to glycolysis. ATP is the end product of glycolysis as well as it is substrate for PFK- 1. In presence of high concentration of ATP, ATP binds to inhibition site of PFK, and thereby decreases the activity of enzyme. AMP, ADP and Fructose 2, 6 biphosphate act as allosteric activators of this enzyme. Activation of enzyme by AMP overcomes the inhibitory effect of ATP. Two other enzymatic activities are involved in the regulation of PFK. a) Adnylate kinase: It readily equilibrates 2 ADP molecules to one ATP and 1 AMP: 2ADP = ATP + AMP, K = [ATP][AMP] / [ADP] = 0.44 Any decrease in ATP and increase in ADP results in an increase in AMP concentration, which activates PFK. b) Fructose 1,6-bisphosphatase (FBPase): It catalyzes conversion of FBP to Fructose 6-phosphate, thus reverting back the PFK reaction.
  71. 71. Substrate cycle or futile cycle: In order to control the flux of glycolysis and to have better regulation, cells have FBPase which keeps degrading the product of PFK reaction (FBP) to its substrate (F-6-P). This is called substrate cycle. This is a futile exercise where, cells invest an ATP to produce FBP which is hydrolysed back to F6-P by FBPase. This is a price cells pay to keep glycolysis in check. AMP acts as a potent inhibitor of FBPase. Thus the rate of glycolysis can be increased many fold by AMP as it activates PFK and at the same time it inhibits FBPase activity. Hexokinase: It is allosterically inhibited by its product Glucose 6 phosphate. In liver Glucokinase is inhibited by Fructose 6 Phosphate. Uncomplexed ATP acts as a competitive inhibitor of this enzyme. Pyruvate Kinase: It is allosterically inhibited by ATP. ATP binding to the inhibitor site of pyruvate kinase decxreases its ability to bing to phosphoenol pyruvate (PEP) the substrate.It is also inhibited by Acetyl coenzyme A and long chain fatty acid.
  72. 72. The citric acid cycle is the central metabolic hub of the cell.  It is the final common pathway for the oxidation of fuel molecule such as amino acids, fatty acids, and carbohydrates. In eukaryotes, the reactions of the citric acid cycle take place inside mitochondria, in contrast with those of glycolysis, which take place in the cytosol.
  73. 73. The citric acid cycle (Krebs cycle, tricarboxylic acid cycle) includes a series of oxidation-reduction reactions in mitochondria that result in the oxidation of an acetyl group to two molecules of carbon dioxide and reduce the coenzymes that are reoxidized through the electron transport chain, linked to the formation of ATP.
  74. 74. A four- carbon compound (oxaloacetate) condenses with a two-carbon acetyl unit to yield a six-carbon tricarboxylic acid (citrate). An isomer of citrate is then oxidatively decarboxylated. The resulting five-carbon compound (α-ketoglutarate) also is oxidatively decarboxylated to yield a four carbon compound (succinate). Oxaloacetate is then regenerated from succinate. Two carbon atoms enter the cycle as an acetyl unit and two carbon atoms leave the cycle in the form of two molecules of carbon dioxide.
  75. 75. o Three hydride ions (hence, six electrons) are transferred to three molecules of nicotinamide adenine dinucleotide (NAD+), whereas one pair of hydrogen atoms (hence, two electrons) are transferred to one molecule of flavin adenine dinucleotide (FAD) . oThe function of the citric acid cycle is the harvesting of high- energy electrons from carbon fuels.
  76. 76. Oxygen is required for the citric acid cycle indirectly in as much as it is the electron acceptor at the end of the electron-transport chain, necessary to regenerate NAD+ and FAD.
  77. 77. o The citric acid cycle itself neither generates a large amount of ATP nor includes oxygen as a reactant. oInstead, the citric acid cycle removes electrons from acetyl CoA and uses these electrons to form NADH and FADH2 . oIn oxidative phosphorylation, electrons released in the reoxidation of NADH and FADH2 flow through a series of membrane proteins (referred to as the electron-transport chain) to generate a proton gradient across the membrane oThe citric acid cycle, in conjunction with oxidative phosphorylation, provides the vast majority of energy used by aerobic cells in human beings, greater than 95%.
  78. 78. •The four-carbon molecule, oxaloacetate that initiates the first step in the citric acid cycle is regenerated at the end of one passage through the cycle. •The oxaloacetate acts catalytically: it participates in the oxidation of the acetyl group but is itself regenerated. •Thus, one molecule of oxaloacetate is capable of participating in the oxidation of many acetyl molecules
  79. 79. Step-1 Formation of Citrate- The citric acid cycle begins with the condensation of a four-carbon unit, oxaloacetate, and a two-carbon unit, the acetyl group of acetyl CoA. Oxaloacetate reacts with acetyl CoA and H2O to yield citrate and CoA. This reaction, which is an aldol condensation followed by a hydrolysis, is catalyzed by citrate synthase.
  80. 80. Oxaloacetate first condenses with acetyl CoA to form citryl CoA, which is then hydrolyzed to citrate and CoA..
  81. 81. Citrate is isomerized into isocitrate to enable the six-carbon unit to undergo oxidative decarboxylation. The isomerization of citrate is accomplished by a dehydration step followed by a hydration step. The result is an interchange of a hydrogen atom and a hydroxyl group. The enzyme catalyzing both steps is called Aconitase because cis-aconitate is an intermediate.
  82. 82. Aconitase is an iron-sulfur protein, or nonheme iron protein. It contains four iron atoms that are not incorporated as part of a heme group.
  83. 83. •The poison Fluoroacetate is toxic, because fluoroacetyl-CoA condenses with oxaloacetate to form fluorocitrate, which inhibits Aconitase, causing citrate to accumulate. •The mode of inhibition is suicidal inhibition
  84. 84. •Isocitrate undergoes dehydrogenation catalyzed by isocitrate dehydrogenase to form, initially, Oxalo succinate, which remains enzyme-bound and undergoes decarboxylation to α -ketoglutarate. • The decarboxylation requires Mg++ or Mn++ ions. •There are three isoenzymes of isocitrate dehydrogenase. •One, which uses NAD+, is found only in mitochondria. •The other two use NADP+ and are found in mitochondria and the cytosol.
  85. 85. Respiratory chain-linked oxidation of isocitrate proceeds almost completely through the NAD+-dependent enzyme.
  86. 86. The conversion of isocitrate into α- ketoglutarate is followed by a second oxidative decarboxylation reaction, the formation of Succinyl CoA from α-ketoglutarate.
  87. 87. • α-Ketoglutarate undergoes oxidative decarboxylation in a reaction catalyzed by a multi-enzyme complex similar to that involved in the oxidative decarboxylation of pyruvate. •The α--ketoglutarate dehydrogenase complex requires the same cofactors as the pyruvate dehydrogenase complex—thiamine diphosphate, lipoate, NAD+, FAD, and CoA— and results in the formation of succinyl-CoA.
  88. 88. •The equilibrium of this reaction is so much in favor of succinyl-CoA formation that it must be considered to be physiologically unidirectional. •As in the case of pyruvate oxidation, arsenite inhibits the reaction, causing the substrate, α -ketoglutarate, to accumulate.
  89. 89. •Succinyl CoA is an energy-rich thioester compound •The cleavage of the thioester bond of succinyl CoA is coupled to the phosphorylation of a purine nucleoside diphosphate, usually GDP. •This reaction is catalyzed by succinyl CoA synthetase (succinate thiokinase).
  90. 90. oThis is the only example in the citric acid cycle of substrate level phosphorylation. o Tissues in which gluconeogenesis occurs (the liver and kidney) contain two isoenzymes of succinate thiokinase, one specific for GDP and the other for ADP
  91. 91. •The GTP formed is used for the decarboxylation of oxaloacetate to phosphoenolpyruvate in gluconeogenesis, and provides a regulatory link between citric acid cycle activity and the withdrawal of oxaloacetate for gluconeogenesis. Nongluconeogenic tissues have only the isoenzyme that uses ADP.
  92. 92. •The first dehydrogenation reaction, forming fumarate, is catalyzed by succinate dehydrogenase, which is bound to the inner surface of the inner mitochondrial membrane. •The enzyme contains FAD and iron-sulfur (Fe:S) protein, and directly reduces ubiquinone in the electron transport chain.
  93. 93. Fumarase (fumarate hydratase) catalyzes the addition of water across the double bond of fumarate, yielding malate.
  94. 94. •Malate is converted to oxaloacetate by malate dehydrogenase, a reaction requiring NAD+. •Although the equilibrium of this reaction strongly favors malate, the net flux is to oxaloacetate because of the continual removal of oxaloacetate (to form citrate, as a substrate for gluconeogenesis, or to undergo transamination to aspartate) and also the continual reoxidation of NADH. .
  95. 95. •As a result of oxidations catalyzed by the dehydrogenases of the citric acid cycle, three molecules of NADH and one of FADH2 are produced for each molecule of acetyl-CoA catabolized in one turn of the cycle. • These reducing equivalents are transferred to the respiratory chain, where reoxidation of each NADH results in formation of 3, and 2 ATP of FADH2. •Consequently, 11 high-transfer-potential phosphoryl groups are generated when the electron-transport chain oxidizes 3 molecules of NADH and 1 molecule of FADH2, • In addition, 1 ATP (or GTP) is formed by substrate-level phosphorylation catalyzed by succinate thiokinase. .
  96. 96. •1 acetate unit generates approximately 12 molecules of ATP. • In dramatic contrast, only 2 molecules of ATP are generated per molecule of glucose (which generates 2 molecules of acetyl CoA) by anaerobic glycolysis. •Molecular oxygen does not participate directly in the citric acid cycle. •However, the cycle operates only under aerobic conditions because NAD+ and FAD can be regenerated in the mitochondrion only by the transfer of electrons to molecular oxygen.
  97. 97. •Regulation of the TCA cycle like that of glycolysis occurs at both the level of entry of substrates into the cycle as well as at the key reactions of the cycle. •Fuel enters the TCA cycle primarily as acetyl- CoA. The generation of acetyl-CoA from carbohydrates is, therefore, a major control point of the cycle. •This is the reaction catalyzed by the PDH complex.
  98. 98. 1) Regulation of PDH Complex a) Allosteric modification-PDH complex is inhibited by acetyl- CoA and NADH and activated by non-acetylated CoA (CoASH) and NAD+. b) Covalent modification-The pyruvate dehydrogenase activities of the PDH complex are regulated by their state of phosphorylation. This modification is carried out by a specific kinase (PDH kinase) and the phosphates are removed by a specific phosphatase (PDH phosphatase). PDH kinase is activated by NADH and acetyl-CoA and inhibited by pyruvate, ADP, CoASH, Ca2+ and Mg2+. The PDH phosphatase, in contrast, is activated by Mg2+ and Ca2+
  99. 99. 2) Regulation of TCA cycle enzymes The most likely sites for regulations are the nonequilibrium reactions catalyzed citrate synthase, isocitrate dehydrogenase, and α- ketoglutarate dehydrogenase. The dehydrogenases are activated by Ca2+, which increases in concentration during muscular contraction and secretion, when there is increased energy demand.
  100. 100. a) Citrate synthase- There is allosteric inhibition of citrate synthase by ATP and long- chain fatty acyl-CoA. b) Isocitrate dehydrogenase- is allosterically stimulated by ADP, which enhances the enzyme's affinity for substrates. In contrast, NADH inhibits iso-citrate dehydrogenase by directly displacing NAD+. ATP, too, is inhibitory.
  101. 101. a) α-ketoglutarate dehydrogenase -α- Ketoglutarate dehydrogenase is inhibited by succinyl CoA and NADH. In addition, α-ketoglutarate dehydrogenase is inhibited by a high energy charge. Thus, the rate of the cycle is reduced when the cell has a high level of ATP. d) Succinate dehydrogenase is inhibited by oxaloacetate, and the availability of oxaloacetate, as controlled by malate dehydrogenase, depends on the [NADH]/[NAD+] ratio.
  102. 102. •Since three reactions of the TCA cycle as well as PDH utilize NAD+ as co-factor, the cellular ratio of NAD+/NADH has a major impact on the flux of carbon through the TCA cycle. •The activity of TCA cycle is immediately dependent on the supply of NAD+, which in turn, because of the tight coupling between oxidation and phosphorylation, is dependent on the availability of ADP and hence, ultimately on the rate of utilization of ATP in chemical and physical work. •Thus, respiratory control via the respiratory chain and oxidative phosphorylation primarily regulates citric acid cycle activity.
  103. 103. Excess of ATP depicts energy rich state of the cell, hence TCA cycle is inhibited while reverse occurs when the cell is in a low energy state with excess of ADP.
  104. 104. •The citric acid cycle is not only a pathway for oxidation of two-carbon units, but is also a major pathway for interconversion of metabolites arising from transamination and deamination of amino acids, and providing the substrates for amino acid synthesis by transamination, as well as for gluconeogenesis and fatty acid synthesis. •Because it functions in both oxidative and synthetic processes, it is amphibolic.
  105. 105. •The citric acid cycle is the final common pathway for the oxidation of carbohydrate, lipid, and protein because glucose, fatty acids, and most amino acids are metabolized to acetyl-CoA or intermediates of the cycle. •The function of the citric acid cycle is the harvesting of high-energy electrons from carbon fuels. • 1 acetate unit generates approximately 12 molecules of ATP per turn of the cycle.
  106. 106. As a major metabolic hub of the cell, the citric acid cycle also provides intermediates for biosynthesis of various compounds. i) Role in Gluconeogenesis- All the intermediates of the cycle are potentially glucogenic, since they can give rise to oxaloacetate, and hence net production of glucose (in the liver and kidney, the organs that carry out gluconeogenesis.
  107. 107. The key enzyme that catalyzes net transfer out of the cycle into gluconeogenesis is phospho-enol-pyruvate carboxy kinase, which catalyzes the decarboxylation of oxaloacetate to phosphoenolpyruvate, with GTP acting as the phosphate donor.
  108. 108. Since the transamination reactions are reversible, the cycle also serves as a source of carbon skeletons for the synthesis of some amino acids like Alanine, aspartate, Asparagine Glutamate , glutamine etc.
  109. 109. Aspartic acid is a precursor of Asparagine, Lysine, Methionine, Threonine and Isoleucine. These amino acid except Asparagine are essential amino acids, they are synthesized only in plants.
  110. 110. iii) Role in fatty acid synthesis- Acetyl-CoA, formed from pyruvate by the action of pyruvate dehydrogenase, is the major substrate for long-chain fatty acid synthesis . Acetyl-CoA is made available in the cytosol from citrate synthesized in the mitochondrion, transported into the cytosol, and cleaved in a reaction catalyzed by ATP- citrate lyase.
  111. 111. Citrate is transported out of the mitochondrion when Aconitase is saturated with its substrate. This ensures that citrate is used for fatty acid synthesis only when there is an adequate amount to ensure continued activity of the cycle Acetyl co A can also be used for the synthesis of cholesterol, steroids etc.
  112. 112. Succinyl co A condenses with amino acid Glycine to form Alpha amino beta keto Adipic acid, which is the first step of haem biosynthesis.
  113. 113. Glutamate and Aspartate derived from TCA cycle are utilized for the synthesis of purines and pyrimidines.
  114. 114. •Riboflavin, in the form of flavin adenine dinucleotide (FAD), a cofactor for succinate dehydrogenase •Niacin, in the form of nicotinamide adenine dinucleotide (NAD), the electron acceptor for isocitrate dehydrogenase, α- ketoglutarate dehydrogenase, and malate dehydrogenase; •Thiamine(vitamin B1) , as thiamine pyro phosphate, the coenzyme for decarboxylation in the α -ketoglutarate dehydrogenase reaction; •Pantothenic acid, as part of coenzyme A such as acetyl-CoA and succinyl-CoA and •Biotin- in CO2 fixation reaction to compensate oxaloacetate concentration.
  115. 115. •Anaplerosis is the act of replenishing TCA cycle intermediates that have been extracted for biosynthesis (in what are called cataplerotic reactions). •The TCA Cycle is a hub of metabolism, with central importance in both energy production and biosynthesis. •Therefore, it is crucial for the cell to regulate concentrations of TCA Cycle metabolites in the mitochondria. •Anaplerotic flux must balance cataplerotic flux in order to retain homeostasis of cellular metabolism
  116. 116. 1) Formation of oxaloacetate from pyruvate In case oxaloacetate is converted into amino acids for protein synthesis or used for gluconeogenesis and, subsequently, the energy needs of the cell rise. The citric acid cycle will operate to a reduced extent unless new oxaloacetate is formed, because acetyl CoA cannot enter the cycle unless it condenses with oxaloacetate. Even though oxaloacetate is recycled, a minimal level must be maintained to allow the cycle to function.
  117. 117. Oxaloacetate is formed by – a)Carboxylation of pyruvate, by pyruvate carboxylase b)Through formation of malate from pyruvate by Malic enzyme c)From malate to oxaloacetate by malate dehydrogenase Mammals lack the enzymes for the net conversion of acetyl CoA into oxaloacetate or any other citric acid cycle intermediate.
  118. 118. 2) Formation of oxaloacetate from Aspartate- Oxaloacetate can also be formed from Aspartate by transamination reaction. 3) Formation of Alpha keto glutarate- Alpha ketoglutarate can be formed from Glutamate dehydrogenase or from transamination reactions. 4) Formation of Succinyl co A – Succinyl co A can be produced from the oxidation of odd chain fatty acid and from the metabolism of methionine and isoleucine (through carboxylation of Propionyl co A to Methyl malonyl co A and then Succinyl co A)
  119. 119. •fats burn in the flame of carbohydrates means fats can only be oxidized in the presence of carbohydrates. •Acetyl co A represents fat component, since the major source is fatty acid oxidation. •Acetyl co A is completely oxidized in the TCA cycle in the presence of oxaloacetate.
  120. 120. Pyruvate is mainly used up for Anaplerotic reactions to compensate for oxaloacetate concentration. Thus without carbohydrates (Pyruvate), there would be no Anaplerotic reactions to replenish the TCA- cycle components. With a diet of fats only, the acetyl CoA from fatty acid degradation would not get oxidized and build up due to non functioning of TCA cycle. Thus fats can burn only in the flame of carbohydrates.
  121. 121. • Bacteria are capable of growth on fatty acids and lipids. Lipids are part of the membranes of living organisms and if available (usually because the organism that was using them dies) can be used as a food source. Lipids are large molecules and cannot be transported across the membrane. A class of extracellular enzymes called lipases are responsible for the breakdown of lipids.
  122. 122. • Lipases attack the bond between the glycerol molecule oxygen and the fatty acid. Phospholipids are attacked by phospholipases. There are four classes of phospholipases given different names depending upon the bond they cleave. Phospholipases are not particular about their substrate and will attack a glycerol ester linkage containing any length fatty acid attached to it. The result of this digestion is a hydrophillic head molecule, glycerol and fatty acids of various chain lengths. The head can be one of several small organic molecules that are funneled into the TCA cycle by one or two reactions that we won't cover here. Glycerol is converted into 3-Phosphoglycerate (depending upon the action of phospholipase C or phospholipase D) and eventually pyruvate via glycolysis. This leaves the fatty acids to deal with.
  123. 123. • Fatty acids are degraded by a four step process called b-oxidation. The fatty acid is first activated by the addition of Coenzyme-A to the end. This activation requires energy in the form of ATP, but is only performed once per fatty acid degraded. The b carbon (see figure) is then oxidized from CH2 to C=O (a ketone) by three reactions. (This is where the pathway gets its name.) The oxidized b group is now susceptible to attack. An enzyme called b- ketothiolase splits the fatty acid into acetyl-CoA and adds another Coenzyme-A to the previously oxidized b group on the fatty acid.
  124. 124. • The fatty acid is now two carbons shorter and an Acetyl-CoA has been generated which can be fed into the TCA cycle. The smaller fatty acid moves through the b-oxidation pathway again, producing another Acetyl-CoA and shrinking by 2 carbons. By performing successive rounds of beta oxidation on a fatty acid, it is possible to convert it completely to Acetyl-CoA. The perceptive reader might notice that for fatty acids with odd numbers of carbons, the final reaction will yield acetyl-CoA and Coenzyme-A hooked to a three carbon fatty acid (propionyl-CoA). Propionyl-CoA is handled differently by different bacteria. In E. coli it is converted into pyruvate.
  125. 125. When comparing catabolism of fats and sugars two points jump out. Reuse of components - Whenever possible, the cell will reuse a carrier, cofactor or enzyme. For example in fatty acid breakdown, Coenzyme-A plays a major role and the electron carriers FAD and NAD are used. Funneling - Cells also try to reuse common pathways. A given substrate will be converted into a common metabolite and then funneled into an already existing pathway. Fatty acids are broken down into Acetyl-Coenzyme-A and this is fed into the TCA cycle.
  126. 126. • Oxidative phosphorylation is a highly efficient method of producing large amounts of ATP, the basic unit of energy for metabolic processes. During this process electrons are exchanged between molecules, which creates a chemical gradient that allows for the production of ATP. The most vital part of this process is the electron transport chain, which produces more ATP than any other part of cellular respiration.
  127. 127. The electron transport chain is the final component of aerobic respiration and is the only part of glucose metabolism that uses atmospheric oxygen. Electron transport is a series of redox reactions that resemble a relay race. Electrons are passed rapidly from one component to the next to the endpoint of the chain, where the electrons reduce molecular oxygen, producing water. This requirement for oxygen in the final stages of the chain can be seen in the overall equation for cellular respiration, which requires both glucose and oxygen.
  128. 128. • A complex is a structure consisting of a central atom, molecule, or protein weakly connected to surrounding atoms, molecules, or proteins. The electron transport chain is an aggregation of four of these complexes (labeled I through IV), together with associated mobile electron carriers. The electron transport chain is present in multiple copies in the inner mitochondrial membrane of eukaryotes and the plasma membrane of prokaryotes. See next slide for image
  129. 129. The electron transport chain The electron transport chain is a series of electron transporters embedded in the inner mitochondrial membrane that shuttles electrons from NADH and FADH2 to molecular oxygen. In the process, protons are pumped from the mitochondrial matrix to the intermembrane space, and oxygen is reduced to form water.
  130. 130. To start, two electrons are carried to the first complex aboard NADH. Complex I is composed of flavin mononucleotide (FMN) and an enzyme containing iron-sulfur (Fe-S). FMN, which is derived from vitamin B2 (also called riboflavin), is one of several prosthetic groups or co-factors in the electron transport chain. A prosthetic group is a non-protein molecule required for the activity of a protein. Prosthetic groups can be organic or inorganic and are non-peptide molecules bound to a protein that facilitate its function. Prosthetic groups include co-enzymes, which are the prosthetic groups of enzymes. The enzyme in complex I is NADH dehydrogenase, a very large protein containing 45 amino acid chains. Complex I can pump four hydrogen ions across the membrane from the matrix into the intermembrane space; it is in this way that the hydrogen ion gradient is established and maintained between the two compartments separated by the inner mitochondrial membrane.
  131. 131. Complex II directly receives FADH2, which does not pass through complex I. The compound connecting the first and second complexes to the third is ubiquinone (Q). The Q molecule is lipid soluble and freely moves through the hydrophobic core of the membrane. Once it is reduced to QH2, ubiquinone delivers its electrons to the next complex in the electron transport chain. Q receives the electrons derived from NADH from complex I and the electrons derived from FADH2 from complex II, including succinate dehydrogenase. This enzyme and FADH2 form a small complex that delivers electrons directly to the electron transport chain, bypassing the first complex. Since these electrons bypass, and thus do not energize, the proton pump in the first complex, fewer ATP molecules are made from the FADH2 electrons. The number of ATP molecules ultimately obtained is directly proportional to the number of protons pumped across the inner mitochondrial membrane.
  132. 132. The third complex is composed of cytochrome b, another Fe-S protein, Rieske center (2Fe-2S center), and cytochrome c proteins; this complex is also called cytochrome oxidoreductase. Cytochrome proteins have a prosthetic heme group. The heme molecule is similar to the heme in hemoglobin, but it carries electrons, not oxygen. As a result, the iron ion at its core is reduced and oxidized as it passes the electrons, fluctuating between different oxidation states: Fe2+ (reduced) and Fe3+ (oxidized). The heme molecules in the cytochromes have slightly different characteristics due to the effects of the different proteins binding them, which makes each complex. Complex III pumps protons through the membrane and passes its electrons to cytochrome c for transport to the fourth complex of proteins and enzymes. Cytochrome c is the acceptor of electrons from Q; however, whereas Q carries pairs of electrons, cytochrome c can accept only one at a time.
  133. 133. The fourth complex is composed of cytochrome proteins c, a, and a3. This complex contains two heme groups (one in each of the cytochromes a and a3) and three copper ions (a pair of CuA and one CuB in cytochrome a3). The cytochromes hold an oxygen molecule very tightly between the iron and copper ions until the oxygen is completely reduced. The reduced oxygen then picks up two hydrogen ions from the surrounding medium to produce water (H2O). The removal of the hydrogen ions from the system also contributes to the ion gradient used in the process of chemiosmosis.
  134. 134. •Oxidative phosphorylation is the metabolic pathway in which electrons are transferred from electron donors to electron acceptors in redox reactions; this series of reactions releases energy which is used to form ATP. •There are four protein complexes (labeled complex I-IV) in the electron transport chain, which are involved in moving electrons from NADH and FADH2 to molecular oxygen. •Complex I establishes the hydrogen ion gradient by pumping four hydrogen ions across the membrane from the matrix into the intermembrane space. •Complex II receives FADH2 , which bypasses complex I, and delivers electrons directly to the electron transport chain. •Ubiquinone (Q) accepts the electrons from both complex I and complex II and delivers them to complex III. •Complex III pumps protons through the membrane and passes its electrons to cytochrome c for transport to the fourth complex of proteins and enzymes. •Complex IV reduces oxygen; the reduced oxygen then picks up two hydrogen ions from the surrounding medium to make water.
  135. 135. ATP synthase is one of the wonders of the molecular world. ATP synthase is an enzyme, a molecular motor, an ion pump, and another molecular motor all wrapped together in one amazing nanoscale machine. It plays an indispensable role in our cells, building most of the ATP that powers our cellular processes. The mechanism by which it performs this task is a real surprise.
  136. 136. ATP synthesis is composed of two rotary motors, each powered by a different fuel. The motor at the top, termed F0, an electric motor. It is embedded in a membrane (shown schematically as a gray stripe here), and is powered by the flow of hydrogen ions across the membrane. As the protons flow through the motor, they turn a circular rotor (shown in blue). This rotor is connected to the second motor, termed F1. The F1 motor is a chemical motor, powered by ATP. The two motors are connected together by a stator, shown on the right, so that when F0 turns, F1 turns too.
  137. 137. So why have two motors connected together? The trick is that one motor can force the other motor to turn, and in this way, change the motor into a generator. This is what happens in our cells: the F0 motor uses the power from a proton gradient to force the F1 motor to generate ATP. In our cells, food is broken down and used to pump hydrogen ions across the mitochondrial membrane. The F0 portion of ATP synthase allows these ions to flow back, turning the rotor in the process. As the rotor turns, it turns the axle and the F1 motor becomes a generator, creating ATP as it turns.
  138. 138. Large, complex molecular machines like ATP synthase pose difficult problems for structural scientists, so the structures of these machines are often determined in parts. The picture shown here is a composite of four different structures, combining structures determined by X-ray crystallography and NMR spectroscopy.
  139. 139. When operating as a generator, it uses the power of rotational motion to build ATP, or when operating as a motor, it breaks down ATP to spin the axle the opposite direction. The synthesis of ATP requires several steps, including the binding of ADP and phosphate, the formation of the new phosphate- phosphate bond, and release of ATP. As the axle turns, it forces the motor into three different conformations that assist these difficult steps. Two states are shown here. The one on the left shows a conformation that assists the binding of ADP, and the one on the right shows a conformation that has been forced open to release ATP. Notice how the oddly-shaped axle forces the change in conformation. See next slide for figure
  140. 140. In this picture, we are looking down the axis of rotation, as if we where looking down at the top of the picture on the first page. The rotor is composed of 12 identical protein chains, colored blue here, and the ion pump is a single chain, colored red. The pump has an arginine amino acid that hands off a hydrogen ion to aspartates on the rotor. Aspartate amino acids typically have a negative charge, but since the rotor is surrounded by membrane lipids, this would be very unfavorable. So, the rotor only turns when the aspartates have a hydrogen attached, neutralizing their charge. Hydrogen ions take a convoluted path through the F0 motor, turning the rotor in the process. They are gathered by a chain of amino acids in the pump, and transferred to the arginine. The arginine passes the hydrogen to the rotor, which turns all the way around. Then the hydrogen is offloaded by other amino acids on the pump, and finally passed to the opposite side of the membrane. The exact path of the hydrogen ions through the pump is still a matter of intense study. See next slide for figure
  141. 141. PHOTOSYNTHESIS In 1780 Joseph Priestly discovered photosynthesis: “…plants can “restore air which has been injured by the burning of candles.” “…the air would neither extinguish a candle, nor was it all inconvenient to a mouse which I put into it.”
  142. 142. •Photosynthesis provides essentially all free energy in biological systems by converting solar energy into chemical energy. •Carbohydrates are formed from light- driven reactions that collectively appear deceptively simple: CO2 + H2O + light  CnH2nOn + O2
  143. 143. • Photosynthesis occurs in specialized organelles called chloroplasts:
  144. 144. TEM of Chloroplast from Corn
  145. 145. • Photosynthesis consists of two sets of reactions: the LIGHT and DARK reactions. • The LIGHT-driven reactions are the primary events of photosynthesis; these occur in the thylakoid membrane. • The DARK reactions occur in the stroma.
  146. 146. •The light-reactions of photosynthesis generate high-energy electrons that are used to form NADPH. •On their way to NADPH, these high- energy electrons flow through a membrane-bound “electron transport” pathway, generating a proton motive potential (Δp) from which ATP is made.
  147. 147. Chlorophyll a:
  148. 148. Sunlight spectrum, Chlorophyll a and Bacteriochlorophyll a
  149. 149. • Light-harvesting Pigments help absorb wavelengths of light in the region of the spectrum where chlorophyll pigments do not absorb light. • Photons absorbed by these pigments also transfer energy to chlorophyll. β-carotene helps protect plants from photochemical reactions, expecially those involving oxygen; (it serves as an “antioxidant.”)
  150. 150. The majority of chlorophyll molecules in a photosynthetic unit do not get involved in photosynthesis directly. Rather, they transfer their absorbed energy to a reaction center complex. C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C
  151. 151. Light energy absorbed by antenna chlorophyll molecules and auxillary pigments is transferred to specialized reaction centers. This energy transfer (often called “resonance energy transfer” or “exciton hopping”) is very efficient, approaching 100%!
  152. 152. X-ray Structure of Photosystem II (S. elongatus)
  153. 153. X-ray Structure of Photosystem II (S. elongatus)
  154. 154. Mn cluster in the OEC Schematic mechanism of oxygen generation in PSII:
  155. 155. Herbicides that inhibit Photosystem II by blocking the transfer of electrons to QH2:
  156. 156. Electron Flow through PSII: Electron Flow through Cytochrome bf: Plastocyanin:
  157. 157. • Photosystem I:
  158. 158. Photosystem I: 700nm photon
  159. 159. Biochem 3070 - Photosynthesis Ferredoxin Structure Stryer,, Biochemistry, 5th ed.
  160. 160. “Z-Scheme” of Photosynthesis:
  161. 161. Andre Jagendorf and Ernest Uribe’s classic experiment, showing the production of ATP from an artificially induced proton gradient across isolated chloroplasts in the absence of any light! (First Reported in 1965)
  162. 162. Stroma Lumen (Thylakoid Space)
  163. 163. Stroma Lumen Inner-membrane Space Matrix
  164. 164. What about CO2? CO2 + H2O + light  CnH2nOn + O2
  165. 165. Melvin Calvin discovered the CO2 acceptor by using C- 14 radioactive labeled carbon dioxide: He discovered that the reactions of CO2 fixation are almost identical to reactions of the phosphate shunt:
  166. 166. The Calvin Cycle: From light-driven reactions
  167. 167. Ribulose 1,5-bisphosphate carboxylase/oxygenase [“rubisco”]: Rubisco consitutes more than 16% of all protein in chloroplasts. It is the most abundant protein in the entire biosphere!
  168. 168. Proposed mechanism for Rubisco:
  169. 169. References • index.php?page=Leaf%3A+How+does+ATP+Synthase+Make+ATP%3F • • textbook/cellular-respiration-7/oxidative-phosphorylation-76/electron- transport-chain-362-11588/ • •
  170. 170. • •
  171. 171. Biosynthesis and Metabolic Regulation • Cell Signaling and Metabolism • Pentose Phosphate Pathway • Regulation of Blood Glucose
  172. 172. Cell Signaling and Metabolism Cells typically communicate using chemical signals. These chemical signals, which are proteins or other molecules produced by a sending cell, are often secreted from the cell and released into the extracellular space. There, they can float – like messages in a bottle – over to neighboring cells. Not all cells can “hear” a particular chemical message. In order to detect a signal (that is, to be a target cell), a neighbor cell must have the right receptor for that signal. When a signaling molecule binds to its receptor, it alters the shape or activity of the receptor, triggering a change inside of the cell. Signaling molecules are often called ligands, a general term for molecules that bind specifically to other molecules (such as receptors).
  173. 173. The message carried by a ligand is often relayed through a chain of chemical messengers inside the cell. Ultimately, it leads to a change in the cell, such as alteration in the activity of a gene or even the induction of a whole process, such as cell division. Thus, the original intercellular (between-cells) signal is converted into an intracellular (within-cell) signal that triggers a response.
  174. 174. Forms of signalling Cell-cell signalling involves the transmission of a signal from a sending cell to a receiving cell. However, not all sending and receiving cells are next-door neighbors, nor do all cell pairs exchange signals in the same way. There are four basic categories of chemical signalling found in multicellular organisms: • Paracrine signalling • Autocrine signalling • Endocrine signalling • Signalling by direct contact
  175. 175. Paracrine Signalling Often, cells that are near one another communicate through the release of chemical messengers (ligands that can diffuse through the space between the cells). This type of signaling, in which cells communicate over relatively short distances, is known as paracrine signaling. Paracrine signaling allows cells to locally coordinate activities with their neighbors. Although they're used in many different tissues and contexts, paracrine signals are especially important during development, when they allow one group of cells to tell a neighboring group of cells what cellular identity to take on.
  176. 176. In order for paracrine factors to successfully induce a response in the receiving cell, that cell must have the appropriate receptors available on the cell membrane to receive the signals, also known as being competent. Additionally, the responding cell must also have the ability to be mechanistically induced.
  177. 177. Autocrine Signalling In autocrine signaling, a cell signals to itself, releasing a ligand that binds to receptors on its own surface (or, depending on the type of signal, to receptors inside of the cell). This may seem like an odd thing for a cell to do, but autocrine signaling plays an important role in many processes. For instance, autocrine signaling is important during development, helping cells take on and reinforce their correct identities. From a medical standpoint, autocrine signaling is important in cancer and is thought to play a key role in metastasis (the spread of cancer from its original site to other parts of the body). In many cases, a signal may have both autocrine and paracrine effects, binding to the sending cell as well as other similar cells in the area.
  178. 178. Tumor development is a complex process that requires cell division, growth, and survival. One approach used by tumors to upregulate growth and survival is through autocrine production of growth and survival factors. Autocrine signaling plays critical roles in cancer activation and also in providing self- sustaining growth signals to tumors.
  179. 179. Endocrine Signalling When cells need to transmit signals over long distances, they often use the circulatory system as a distribution network for the messages they send. In long-distance endocrine signaling, signals are produced by specialized cells and released into the bloodstream, which carries them to target cells in distant parts of the body. Signals that are produced in one part of the body and travel through the circulation to reach far-away targets are known as hormones. In humans, endocrine glands that release hormones include the thyroid, the hypothalamus, and the pituitary, as well as the gonads (testes and ovaries) and the pancreas. Each endocrine gland releases one or more types of hormones, many of which are master regulators of development and physiology.
  180. 180. For example, the pituitary releases growth hormone (GH), which promotes growth, particularly of the skeleton and cartilage. Like most hormones, GH affects many different types of cells throughout the body. However, cartilage cells provide one example of how GH functions: it binds to receptors on the surface of these cells and encourages them to divide.
  181. 181. Signalling by Direct Contact Gap junctions in animals and plasmodesmata in plants are tiny channels that directly connect neighboring cells. These water-filled channels allow small signaling molecules, called intracellular mediators, to diffuse between the two cells. Small molecules, such as calcium ions, are able to move between cells, but large molecules like proteins and DNA cannot fit through the channels without special assistance. The transfer of signaling molecules transmits the current state of one cell to its neighbor. This allows a group of cells to coordinate their response to a signal that only one of them may have received. In plants, there are plasmodesmata between almost all cells, making the entire plant into one giant network. In another form of direct signaling, two cells may bind to one another because they carry complementary proteins on their surfaces. When the proteins bind to one another, this interaction changes the shape of one or both proteins, transmitting a signal. This kind of signaling is especially important in the immune system, where immune cells use cell-surface markers to recognize “self” cells (the body's own cells) and cells infected by pathogens.
  182. 182. Activity 1: Answer the following. 1. This is the general term for molecules that bind specifically to other molecules. 2. The message carried by a ligand is often relayed through __________ inside the cell. 3. In _____________, signals are produced by specialized cells and released into the bloodstream, which carries them to target cells in distant parts of the body. 4. These are water-filled channels that allow small signaling molecules to diffuse between the two cells. 5. It is thought to be a key role in metastasis.
  183. 183. Pentose Phosphate Pathway
  184. 184. Similarly to some of the processes in cellular respiration, the molecules that go through the pentose phosphate pathway are mostly made of carbon. The easiest way to understand this pathway is to follow the carbon. The breakdown of the simple sugar, glucose, in glycolysis provides the first 6-carbon molecule required for the pentose phosphate pathway. During the first step of glycolysis, glucose is transformed by the addition of a phosphate group, generating glucose-6-phosphate, another 6-carbon molecule. The pentose phosphate pathway can use any available molecules of glucose-6-phosphate, whether they are produced by glycolysis or other methods.
  185. 185. Oxidative PhaseStep 1: Glucose-6-phosphate is oxidized to form lactone. NADPH is produced as a byproduct of this reaction as NADP+​ is reduced as glucose-6-phosphate is oxidized. Following the oxidation of glucose-6-phosphate, another reaction, catalyzed by a different enzyme, uses water to form 6-phosphogluconate, the linear product. NADPH is similar in structure and function as the high energy electron shuttle, NADH, mentioned in the cellular respiration articles. NADPH has an added phosphate group and is used in the cell to donate its electrons, just like NADH. Once NADPH has donated its electrons it is said to be oxidized (oxidation = loss of electrons) and is now symbolized as, NADP+. NADPH is often used in reactions that build molecules and occurs in a high concentration in the cell, so that it is readily available for these types of reactions. Step 2: Next, a carbon is removed (cleaved) and CO2 is released. Once again, the electrons released from this cleavage is used to reduce NADP+ to NADPH. This new 5-carbon molecule is called ribulose-5-phosphate.
  186. 186. The “oxidative” word of this phase comes from the process of oxidation. Oxidation is the breakdown of a molecule as it loses at least one of its electrons.
  187. 187. Non-Oxidative Phase Step 3: Ribulose-5-phosphate can be converted into two different 5-carbon molecules. One is the sugar used to make up DNA and RNA called, ribose-5- phosphate and this is the molecule we will focus on. Ribulose-5-phosphate isn’t being divided because the carbon count is the same in the next step. Step 4: The rest of the cycle is now made up of different options that depend on the cell’s needs. The ribose-5-phosphate from step 3 is combined with another molecule of ribose-5-phosphate to make one, 10-carbon atom. Excess ribose-5-phosphate, which may not needed for nucleotide biosynthesis, is converted into other sugars that can be used by the cell for metabolism. The 10-carbon atom is interconverted to create a 3-carbon molecule and a 7-carbon molecule. The 3-carbon product can be shipped over to glycolysis if it needs. That being said, recall that we can also work our way back up to another molecule in this phase. So that 3-carbon molecule could also be shipped over from glycolysis and transformed into ribose-5-phosphate for DNA and RNA production.
  188. 188. Step 5: The 3-carbon molecule and the 7-carbon molecule, from the interconversion above in step 4, interconvert again to make a new 4-carbon molecule and 6- carbon molecule. The 4-carbon molecule is a precursor for amino acids, while the 6-carbon molecule can be used in glycolysis. The same reversal of steps in option 4 can happen here as well. The pentose phosphate pathway takes place in the cytosol of the cell, the same location as glycolysis. The two most important products from this process are the ribose-5-phosphate sugar used to make DNA and RNA, and the NADPH molecules which help with building other molecules.
  189. 189. In summary Oxidative phase: • -1 H2O • +2 NADPH • +1 CO2 Non-oxidative phase: • Ribose-5-phosphate for DNA/RNA building (also produced in the oxidative phase) Consider the following NADPH is readily available to donate its electrons in the cell because it occurs in such high concentration. Aside from helping build molecules, what kind of benefit is this really for the cell? NADPH is able to donate its electrons to compounds that fight dangerous oxygen molecules. These compounds are called antioxidants and you’ve probably heard about them being in some foods. Antioxidants donate electrons to neutralize dangerous oxygen radicals (super reactive oxygen molecules). Once they have given away their electrons, antioxidants need to quickly reload in case there are more oxygen radicals. NADPH is able to give antioxidants their constant flow of electrons to fight oxygen crime.
  190. 190. Activity: Write the correct order of the Pentose Phosphate Pathway. __ Ribulose-5-phosphate can be converted into two different 5-carbon molecules. __ Glucose-6-phosphate is oxidized to form lactone. NADPH is produced as a byproduct of this reaction as NADP+​ is reduced as glucose-6-phosphate is oxidized. __ The pentose phosphate pathway takes place in the cytosol of the cell, the same location as glycolysis. __ A carbon is removed (cleaved) and CO2 is released. Once again, the electrons released from this cleavage is used to reduce NADP+ to NADPH. __ The 10-carbon atom is interconverted to create a 3- carbon molecule and a 7-carbon molecule.
  191. 191. Regulation of Blood Glucose • Gluconeogenesis • Glycogen Metabolism • Hormonal Control
  192. 192. Gluconeogenesis
  193. 193. Step 1 - the first irreversible step: We start with the two pyruvate molecules that came from a non-carbohydrate source. Pyruvate (3 carbons) is combined with bicarbonate to create oxaloacetate (4 carbons). This reaction requires ATP. Another GTP is then used to transform oxaloacetate into phosphoenolpyruvate, a 3-carbon molecule with a phosphate group attached. Steps 1 through 4 will occur twice, each time with a pyruvate molecule. We need enough carbons to eventually get a 6 carbon molecule. As we mentioned before, detours are required in gluconeogenesis to get around the irreversible reactions of the glycolysis pathway. The reactions in step 1, converting pyruvate into oxaloacetatete, and oxaloacetate into phosphoenolpyruvate, are the first detour.
  194. 194. Step 2: A hydroxyl group is used to change the 3-carbon molecule, preparing for a phosphate group to be transferred to another carbon in the molecule. Step 3: ATP is used to add another phosphate group to the 3-carbon molecule. Step 4: The high energy electrons carried by NADH are used to remove one of the phosphate groups. Once NADH has lost its high energy electrons (a process which is called “oxidation”) it’s called NAD^+.
  195. 195. Glycogen Metabolism
  196. 196. Glycogen degradation and synthesis are relatively simple biochemical processes. Glycogen degradation consists of three steps: (1) the release of glucose 1- phosphate from glycogen, (2) the remodeling of the glycogen substrate to permit further degradation, and (3) the conversion of glucose 1-phosphate into glucose 6-phosphate for further metabolism. The glucose 6-phosphate derived from the breakdown of glycogen has three fates: (1) It is the initial substrate for glycolysis, (2) it can be processed by the pentose phosphate pathway to yield NAPDH and ribose derivatives; and (3) it can be converted into free glucose for release into the bloodstream. This conversion takes place mainly in the liver and to a lesser extent in the intestines and kidneys.
  197. 197. Glycogen synthesis requires an activated form of glucose, uridine diphosphate glucose (UDP-glucose), which is formed by the reaction of UTP and glucose 1-phosphate. UDP-glucose is added to the nonreducing end of glycogen molecules. As is the case for glycogen degradation, the glycogen molecule must be remodeled for continued synthesis. The regulation of these processes is quite complex. Several enzymes taking part in glycogen metabolism allosterically respond to metabolites that signal the energy needs of the cell. These allosteric responses allow the adjustment of enzyme activity to meet the needs of the cell in which the enzymes are expressed. Glycogen metabolism is also regulated by hormonally stimulated cascades that lead to the reversible phosphorylation of enzymes, which alters their kinetic properties. Regulation by hormones allows glygogen metabolism to adjust to the needs of the entire organism. By both these mechanisms, glycogen degradation is integrated with glycogen synthesis. We will first examine the metabolism, followed by enzyme regulation and then the elaborate integration of control mechanisms.
  198. 198. Hormonal Control
  199. 199. Regulation of Blood Glucose Levels: Insulin and Glucagon Cells of the body require nutrients in order to function. These nutrients are obtained through feeding. In order to manage nutrient intake, storing excess intake, and utilizing reserves when necessary, the body uses hormones to moderate energy stores. Insulin is produced by the beta cells of the pancreas, which are stimulated to release insulin as blood glucose levels rise (for example, after a meal is consumed). Insulin lowers blood glucose levels by enhancing the rate of glucose uptake and utilization by target cells, which use glucose for ATP production. It also stimulates the liver to convert glucose to glycogen, which is then stored by cells for later use. As insulin binds to its target cell via insulin receptors and signal transduction, it triggers the cell to incorporate glucose transport proteins into its membrane. This allows glucose to enter the cell, where it can be used as an energy source. These actions mediated by insulin cause blood glucose concentrations to fall, called a hypoglycemic, or "low sugar" effect, which inhibits further insulin release from beta cells through a negative feedback loop.
  200. 200. Impaired insulin function can lead to a condition called diabetes mellitus, which has many effects on the body . It can be caused by low levels of insulin production by the beta cells of the pancreas, or by reduced sensitivity of tissue cells to insulin. This prevents glucose from being absorbed by cells, causing high levels of blood glucose, or hyperglycemia (high sugar). High blood glucose levels make it difficult for the kidneys to recover all the glucose from nascent urine, resulting in glucose being lost in urine. High glucose levels also result in less water being reabsorbed by the kidneys, causing high amounts of urine to be produced; this may result in dehydration. Over time, high blood glucose levels can cause nerve damage to the eyes and peripheral body tissues, as well as damage to the kidneys and cardiovascular system. Oversecretion of insulin can cause hypoglycemia, low blood glucose levels. This causes insufficient glucose availability to cells, often leading to muscle weakness. It can sometimes cause unconsciousness or death if left untreated.
  201. 201. Hyperglycemia
  202. 202. Regulation of Blood Glucose Levels: Thyroid Hormones The basal metabolic rate, which is the amount of calories required by the body at rest, is determined by two hormones produced by the thyroid gland: thyroxine, also known as tetraiodothyronine or T4, and triiodothyronine, also known as T3. T3 and T4 release from the thyroid gland are stimulated by thyroid-stimulating hormone (TSH), which is produced by the anterior pituitary. These hormones affect nearly every cell in the body except for the adult brain, uterus, testes, blood cells, and spleen. They are transported across the plasma membrane of target cells where they bind to receptors on the mitochondria, resulting in increased ATP production. In the nucleus, T3and T4activate genes involved in energy production and glucose oxidation. This results in increased rates of metabolism and body heat production. This is known as the hormone's calorigenic effect. Disorders can arise from both the underproduction and overproduction of thyroid hormones. hypothyroidism, underproduction of the thyroid hormones, can cause a low metabolic rate leading to weight gain, sensitivity to cold, and reduced mental activity, among other symptoms. In children, hypothyroidism can cause cretinism, which can lead to mental retardation and growth defects. Hyperthyroidism, the overproduction of thyroid hormones, can lead to an increased metabolic rate, which may cause weight loss, excess heat production, sweating, and an increased heart rate.
  203. 203. Activity: Identify the following. 1. They release from the thyroid gland and stimulated by thyroid-stimulating hormone (TSH), which is produced by the anterior pituitary. 2. A readily mobilized storage form of glucose. 3. Impaired insulin function can lead to a condition called __________, which has many effects on the body. 4. It allows glycogen metabolism to adjust to the needs of the entire organism. 5. It makes difficult for the kidneys to recover all the glucose from nascent urine, resulting in glucose being lost in urine.
  204. 204. References: • textbook/the-endocrine-system-37/regulation-of-body-processes- 212/hormonal-regulation-of-metabolism-799-12035/ • • blood-glucose • metabolism/a/glycolysis-and-gluconeogenesis • metabolism/a/pentose-phosphate-pathway • thway.pdf • of-cell-signaling/a/introduction-to-cell-signaling
  205. 205. ANSWERS: Activity 1 1. Ligands 2. A chain of chemical messengers 3. Endocrine signalling 4. Intracellular mediators 5. Autocrine signalling Activity 2: 3, 1, 5, 2, 4 Activity 3: 1. tetraiodothyronine or T4, and triiodothyronine, also known as T3 2. Glycogen 3. diabetes mellitus 4. regulation by hormones 5. High blood glucose levels