Bioenergetics and metabolism.pptx

BIOENERGETICS- THERMODYNAMIC PRINCIPLES, FREE ENERGY
METABOLISM - GLYCOLYSIS, CITRIC ACID CYCLE, OXIDATIVE PHOSPHORYLATION

1. BIOENERGETICS- THERMODYNAMIC PRINCIPLES, FREE ENERGY METABOLISM - GLYCOLYSIS, CITRIC ACID CYCLE, OXIDATIVE PHOSPHORYLATION Bioenergetics and metabolism

2. Bioenergetics Is a quantitative study of the energy transductions(changes of one form of energy into another) that occur in living cells and of the nature and functions of the chemical processes underlying these transductions.

3. Thermodynamic principles 1 st law- the energy is neither created nor destroyed, although it can be transformed from one form to another i.e. the total energy of a system, including surroundings, remains constant. Mathematically it can be expressed as- ∆U= ∆q- ∆w ∆U- is the change in internal energy ∆q- is the heat exchanged from the surroundings ∆w- is the work done by the system If ∆q is positive, heat has been transferred to the system, giving an increase in internal energy When ∆q is negative, heat has been transferred to the surroundings, giving a decrease in internal energy When ∆w is positive, work has been done by the system, giving a decrease in internal energy When ∆w is negative, work has been done by the surroundings, giving an increase in internal energy

4. Thermodynamic principles 2 nd law- the total entropy of a system must increase if a process is to occur spontaneously Mathematically, it can be expressed as ∆S≥ (∆Q/T) Where ∆S- is the changes in entropy of the system Entropy is unavailable form of energy and it is difficult to determine it, so a new thermodynamic term called free energy is defined

5. Free energy Free energy /Gibb's energy indicates the portion of the total energy of a system that is available for useful work. The change in free energy is denoted as ∆G Under constant temperature and pressure, the relationship between free energy change (∆G) of a reacting system and the change in entropy (∆S) is expressed by the following equation ∆G= ∆H-T∆S Where ∆H is the change in enthalpy and T is absolute temperature ∆H is the measure of change in heat content of reactants and products When chemical reaction releases heat, it is said to be exothermic The heat content of the product is less than that of the reactants and ∆H has, by convention a negative value Chemical reactions that take up heat from their surroundings are endothermic and have positive values of ∆H

6. Free energy The change in free energy ∆G, can be used to predict the direction of a reaction at constant temperature and pressure if, ∆G= negative Reaction proceeds spontaneously Loss of free energy Exergonic ∆G= positive Reaction proceeds only when free energy can be gained Endergonic ∆G= = 0 System is at equilibrium Both forward and reverse reactions occurs at equal levels

7. Free energy ∆G of the reaction A B depends on the concentration of reactant and product. At constant temperature and pressure, the following relation can be derived ∆G= ∆G°+RT ln [B]/[A] ∆G° is the standard free energy change R is the gas constant T is absolute temperature [A] and [B] are the actual concentrations of reactant and product

8. METABOLISM –GLYCOLYSIS, CITRIC ACID CYCLE, OXIDATIVE PHOSPHORYLATION

9. Metabolism All cell functions as biochemical factories Within the living cells, biomolecules are constantly being synthesized and transformed into some other biomolecules These synthesis and transformation constantly occur through enzyme catalyzed chemical reactions Together all the interconnected chemical reactions occurring within a cell are called metabolism(derived from the Greek word ‘for change’) Each of the chemical reactions results in the transformation of chemical compounds and the chemical compounds involved in this process are known as metabolites Majority of metabolic reactions do not occur in isolation but are always linked to some other reactions. It occurs in a series of linked chemical reactions called metabolic pathways in which one chemical transformed through a series of steps into another chemical. Metabolic pathways proceed in a stepwise manner, transforming substrates into end products through many specific chemical intermediates Each step of metabolic pathways is catalyzed by a specific enzymes

10. Metabolism Metabolic pathways can be Linear- ex. Glycolysis Cyclic- ex. Citric acid cycle Spiral- ex. Biosynthesis of fatty acids Flow of metabolites through metabolic pathway has a definite rate and direction Because the biomolecules within the cell are in continual state of degradation and resynthesis. This is called dynamic state of body constituents Metabolism serves 2 fundamentally different purposes- Generation of energy to derive vital functions The synthesis of biological molecules

11. Metabolism To achieve these metabolic pathways falls into 2 categories Anabolic pathways Catabolic pathways Involved in the synthesis of compounds and consume energy Endergonic in nature Ex. Synthesis of amino acids Synthesis of polypeptide from amino acids Involved in oxidative breakdown of larger complex molecules and release of energy Exergonic in nature Ex. When glucose is degraded to lactic acid in our skeletal muscle, energy is liberated Some pathways can be either anabolic or catabolic, depending on the energy condition i n the cell. They are referred as amphibolic pathways .they occurs at the ‘crossroads’ of Metabolism, acting as links between the anabolic and catabolic pathways Ex. Citric acid cycle

12. Respiration Living cells require an input of free energy. Energy is required for maintenance of highly organized structures, synthesis of cellular components, movements, generation of electric currents and for many other purposes Cells acquire free energy from the oxidation of organic compounds that are rich in potential energy. Since all organisms use organic compounds for source of energy, they must either make the organic compounds in a process like photosynthesis or they must obtain energy-containing organic compounds called food through diet Respiration is a metabolic process, in which free energy released from the oxidation of organic compounds is used in formation of ATP. The compounds that are oxidized during this process is called respiratory substrates, which may be carbohydrates, fats, proteins or organic acids Carbohydrates are more common respiratory substrates

13. Aerobic respiration Respiration may be oxygen- dependent(aerobic respiration ) and oxygen –independent (anaerobic respiration) Molecular O 2 serves as the terminal (or final) acceptor of electrons. In this mode of respiration, complete oxidation of the respiratory substrate occurs, and the end products formed are primarily CO 2 andH 2 O

14. Aerobic respiration Carbohydrates, fats and proteins can all be oxidized as fuel, but here processes have been described by taking glucose as a respiratory substrate. Oxidation of glucose in exergonic process During aerobic respiration when glucose is completely oxidized into CO 2 and water and energy is liberated. Part of this energy is used fir the synthesis of ATP. For each molecule of glucose degraded to CO 2 and water by respiration, the cell makes up ATP molecules each with free energy

15. Anaerobic respiration Not all organisms (or cells) require oxygen for oxidation of respiratory substrates. Anaerobic respiration and fermentation do not require oxygen for oxidation of respiratory substrates. Here compounds such as sulfate, nitrate, sulphur or fumarate other than O 2 serves as a terminal electron acceptor Both aerobic and anaerobic respiration involve electron transport system to establish an electrochemical proton gradient across a membrane and ATP synthesis occurs through both substrate- level phosphorylation and oxidative phosphrylation

16. Fermentation Does not involve electro transport system to establish an electrochemical proto gradient. It instead only used substrate-level phosphorylation to produce ATP The final electron acceptors are organic compounds often formed as a result of incomplete oxidation of organic substrates during the fermentation pathway itself

17. Aerobic respiration Enzyme catalyzed reactions during aerobic respiration can be grouped into 3 major processes Process name Place of occurrence Type of cell Glycolysis cytosol Of cells in all living organisms Citric acid cycle Within the mitochondrial matrix and in cytosol Eukaryotic cell and cytosol of prokaryotic cell Oxidative phosphorylation Inner mitochondrial membrane Eukaryotic cell and plasma membrane of prokaryotic cells

18. Intracellular location of major processes of aerobic respiration In eukaryotes glycolysis cytosol Citric acid cycle Mitochondrial matrix Oxidative phosphorylation Inner mitochondrial membrane In prokaryotes glycolysis cytosol Citric acid cycle cytosol Oxidative phosphorylation Plasma membrane

19. GLYCOLYSIS

20. Glycolysis Glycolysis(from the Greek ‘glykys’ meaning sweet and lysis meaning ‘splitting’) also known as Embden-meyerhof pathway, is the process in which one mole of glucose is partially oxidized into the 2 moles of pyruvate in a series of enzyme-catalyzed reactions. Glycolysis occurs in the cytosol of all cells. It is a unique pathway that occurs in both aerobic as well as anaerobic conditions and does not involve molecular oxygen i.e.. Oxygen independent

21. Glycolysis- preparatory phase

22. Glycolysis- pay off phase

23. Steps involved in glycolysis Phosphorylation Isomerization Phosphorylation Cleavage Isomerization Oxidation Formation of ATP(substrate- level phosphorylation) Moving of remaining phosphate ester linkage in 3-phosphoglycerate Removal of water from 2-phosphoglycerate Transfer of high energy phosphate group

24. Steps involved in glycolysis Step-1 phosphorylation Glucose is phosphorylated by ATP to form glucose-6-phosphate The negative charge of phosphate prevents the passage of the glucose-6-phosphate through the plasma membrane, trapping glucose inside the cell The irreversible reaction is catalyzed by hexokinase hexokinase present in all cells of all organisms. It requires divalent metal ions like Mg 2+ /Mn 2+ for activity Hepatocytes and β -cells of the pancreas also contain a form of hexokinase called glucokinase (hexokinase D) Hexokinase and glucokinase are isozymes

25. Steps involved in glycolysis Step- 2 isomerization A reversible rearrangement of chemical structure(isomerization) moves the carbonyl oxygen from carbon 1 to carbon 2, forming a ketose from an aldose sugar. Thus, the isomerization of glucose-6-phosphate to fructose-6-phosphate is a conversion of an aldose to a ketose This step is catalyzed by enzyme phosphoglucoisomerase

26. Steps involved in glycolysis Step-3 phosphorylation fructose-6-phosphate is phosphorylated by ATP to fructose-1,6-bisphosphate The prefix bis- in bisphosphate means that 2 separate monophosphate groups are present, whereas the prefix di- means that 2 phosphate groups are present and are connected by an anhydride bond . The irreversible reaction is catalyzed by an allosteric enzyme phosphofructokinase -1 (PFK-1)

27. Steps involved in glycolysis Step-4 cleavage The fructose-1,6-bisphosphate is cleaved to produce two three- carbon molecules- glyceraldehyde-3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP) This reaction is catalyzed by enzyme aldolase

28. Steps involved in glycolysis Step-5 isomerization Only one of the two triose phosphates formed by aldolase- glyceraldehyde-3-phosphate (G3P) can be directly degraded in the subsequent reaction step of glycolysis. However, the other product, dihydroxyacetone phosphate, is rapidly and reversibly converted into glyceraldehyde-3-phosphate by the enzyme triose phosphate isomerase.

29. Steps involved in glycolysis Step-6 oxidation The two molecules of glyceraldehyde-3-phosphate are oxidized Enzyme dehydrogenase catalyzes the conversion of glyceraldehyde-3-phosphate (G3P) into 1,3-bisphosphoglycerate (1,3-BPG) The reaction occurs in two steps – 1 st step- oxidation of aldehyde to a carboxylic acid by NAD+ 2 nd step- the phosphorylation of carboxylic acid by inorganic phosphate Iodoacetate is a potent inhibitor of glyceraldehyde-3-phosphate dehydrogenase because it forms a covalent derivative of the essential –SH group of the enzyme active site, rendering it inactive

30. Steps involved in glycolysis Step7- formation of ATP High energy phosphate group is transferred from 1,3-bisphosphoglycerate to ADP This step is catalyzed by enzyme phosphoglycerate kinase The formation of ATP is referred to as substrate level- phosphorylation because the phosphate donor, 1,3-bisphosphoglycerate , is a substrate with high phosphoryl-transfer potential Formation of ATP from ADP by direct transfer of phosphoryl group from a ‘high energy’ compound is termed as substrate-level phosphorylation

31. Steps involved in glycolysis Step-8 Moving of remaining phosphate ester linkage in 3-phosphoglycerate The remaining phosphate ester linkage in 3-phosphoglycerate, which has relatively low free energy of hydrolysis, is moved from carbon 3 to carbon 2 to form 2-phosphoglycerate

32. Steps involved in glycolysis Step-9 Removal of water from 2-phosphoglycerate The removal of water from 2-phosphoglycerate creates a high-energy eno phosphate linkage The enzyme catalyzing this step, enolase, is inhibited by fluoride

33. Steps involved in glycolysis Step-10 Transfer of high energy phosphate group The transfer of high energy phosphate group that was generated in step 9 to ADP from ATP. This is the last step in glycolysis is the irreversible transfer of the phosphoryl group from phosphoenolpyruvate to ADP is catalyzed by pyruvate kinase Pyruvate kinase requires K+ and either Mg 2+ or Mn 2+ Net reaction Glucose+ (2NAD + ) +(2ADP)+ 2HPO 4 2- 2pyruvate+ 2NADH+ 2ATP + 2H 2 O

34. REGULATION OF GLYCOLYSIS Rate of which the glycolytic pathway operated is operated primarily by allosteric regulation of 3 enzymes- hexokinase, phosphofructokinase-1 and pyruvate kinase The reaction catalyzed by these 3 enzymes are irreversible Allosteric effector glucose-6-phosphate inhibits the hexokinases A high AMP concentration activates phosphofructokinase-1 and pyruvate kinase . In contrast, a high ATP concentration inhibits both enzymes Citrate and acetyl-coA, which indicates that alternative energy sources are available, inhibit phosphofructokinase-1 and pyruvate kinase, respectively Finally fructose-2,6-bisphosphate stimulates glycolysis by activating phosphofructokinase-1 and fructose-1,6-bisphosphate activates pyruvate kinase

35. REGULATION OF GLYCOLYSIS Enzyme activator Inhibitor Hexokinase ------------------------------ Glucose-6-phosphate Phosphofructokinase -1 Fructose-2,6-bisphosphate,AMP Citrate, ATP Pyruvate kinase Fructose-1,6-bisphosphate,AMP Acetyl-coA, ATP

36. Outline of glycolysis

37. Overview of Regulation of glycolysis

38. Fate of carbon atoms of glucose

39. CITRIC ACID CYCLE

40. Citric acid cycle Also known as Kreb s cycle or tricarboxylic acid cycle was discovered by H.A. Krebs, a German born British biochemist, who received the noble prize in 1953 This cycle occurs in the matrix of mitochondria( cytosol in prokaryotes) The net result of citric acid cycle is that for each acetyl group entering the cycle as acetyl-coA, two molecules of CO 2 are produced

41. Steps involved in citric acid cycle Step-1 citric acid cycle begins with the condensation of an oxaloacetate(4 carbon unit), and the acetyl group of acetyl-coA (2 carbon unit) Oxaloacetate reacts with acetyl-coA and water to yield citrate and coenzyme A This reaction, which is an aldol condensation followed by a hydrolysis, is catalyzed by citrate synthase

42. Steps involved in citric acid cycle Step-2a and 2b An isomerization reaction, in which water is first removed and then added back, moves hydroxyl group from one carbon atom to its neighbor The enzyme catalyzing this step, aconitase (nonheme iron protein ) is the target site for the toxic compound fluoroacetate( used as a pesticide). Fluoroacetate blocks the citric acid cycle by its metabolic conversion of fluorocitrate, which is potent inhibitor of aconitase

43. Steps involved in citric acid cycle Step-3 Isocitrate is oxidized and decarboxylated to α - ketoglutarate ( also called oxoglutarate) In first of four oxidation steps in the cycle, the carbon carrying the hydroxyl group is converted to a carbonyl group The intermediate product is unstable, losing CO 2 while still bound to the enzyme The oxidative decarboxylation of isocitrate is catalyzed by isocitrate dehydrogenase

44. Steps involved in citric acid cycle Step-4 The second oxidative decarboxylation reaction results in formation of succinyl coA from α - ketoglutarate α - ketoglutarate dehydrogenase catalyzes this oxidative step and produces NADH, CO 2 and a high-energy thioester bond to coenzyme A

45. Steps involved in citric acid cycle Step-5 The cleavage of the thioester bond if succinyl-coA is coupled with the phosphorylation of an ADP/ GDP ( substrate level phosphorylation ). This step is catalyzed by succinyl-coA synthetase (succinate thiokinase) ATP and GTP are energetically equivalent. This is the only step in citric acid cycle that directly yield a compound with high phosphoryl transfer potential through a substrate-level phosphorylation Animal cells have 2 isozymes of succinyl-coA synthetase, one specific for ADP and the other for GDP. The GTP formed by succinyl-coA synthetase can donate its terminal phosphoryl group to ADP to form ATP, in a reversible reaction catalyzed by nucleoside diphosphate kinase. In the cells of plants, bacteria and some animal tissues, an ATP molecule forms directly by substrate-level phosphorylation

46. Steps involved in citric acid cycle Step-6 In the third oxidation step in the cycle, FAD removes 2 hydrogen atoms from succinate The enzyme catalyzing this step, succinate dehydrogenase, is strongly inhibited by malonate, a structural analog of succinate and a classical example for competitive inhibitor

47. Steps involved in citric acid cycle Step-7 The addition of water to fumarate places a hydroxyl group next to a carbonyl carbon Step-8 In the last four oxidation steps in the cycle, the carbon carrying the hydroxyl group is converted to a carbonyl group, regenerating the oxaloacetate needed for step1 NAD + linked malate dehydrogenase catalyzes the oxidation of malate to oxaloacetate Overall reaction –by taking 1 molecule of acetyl- coA Acetyl-coA+ 3NAD + +FAD+GDP/ADP+3H 2 O  2CO 2 +3NADH+FADH 2 +GTP/ATP+H 2 O

48. Regulation of citric acid cycle Is regulated at its 3 strongly exergonic steps catalyzed by enzymes citrate synthase, isocitrate dehydrogenase and α - ketoglutarate dehydrogenase The regulatory enzymes of the citric acid cycle seem to control flux primarily by 3 simple mechanisms- substrate availability, product inhibition, allosteric feedback inhibition of enzyme The TCA cycle can be limited by the availability of the citrate synthase substrates, acetyl-coA and oxaloacetate Enzyme isocitrate dehydrogenase is allosterically activated by ADP and inhibited by reaction product, NADH Enzyme α - ketoglutarate dehydrogenase catalyzes the rate-limiting step in TCA cycle It is allosterically inhibited by succinyl-coA and NADH, the products of the reaction that it catalyzes

49. Citric acid cycle

50. Regulation of citric acid cycle

51. Oxidative phosphorylation Most of the free energy released during the oxidation of glucose to CO 2 is retained in the reduced coenzymes NADH and FADH 2 , generated during glycolysis and citric acid cycle. Electrons are released from NADH and FADH 2 and eventually transferred to O 2 forming water The standard free energy change for these exergonic reactions are-52.6kcal/mol(NADH) and -43.14kcal/mol(FADH 2 ) The large amount of energy released during oxidation of NADH and FADH2 is used in the formation of ATP. For this reason, the term oxidative phosphorylation is used to describe this energy conversion process

52. Oxidative phosphorylation Electrons are transferred from NADH/ FADH 2 to O2 through a series of electron carriers present on the inner mitochondrial membrane(in case of prokaryotes, it is present in plasma membrane ) the process of electron transport begins when the hydride ion is removed from NADH and is converted into a proton and 2 electrons. Most of the proteins(electron carriers) involved are grouped into 4 large respiratory enzyme complexes, each containing transmembrane proteins that hold the complex. The electrons start with the very high and gradually lose it as they pass along the chain Each complex in the chain has a greater affinity for electrons i.e. reduction potential than its predecessor and electrons pass sequentially from one complex to another until they are finally transferred to oxygen, which has the highest affinity for electrons The 4 major respiratory enzyme complexes of electron transport chain in the inner mitochondrial membrane are- NADH-coenzyme Q reductase /NADH dehydrogenase (COMPLEX 1) Succinate-coenzyme Q reductase (COMPLEX 2) Coenzyme Q-cytochrome c reductase/ cytochrome bc1 complex (COMPLEX 3 ) Cytochrome c oxidase (COMPLEX 4) COMPLEX 1,2,3 appear to be associated in the supramolecular complex termed the respirosomes All the 4 multiprotein enzyme complexes which act as electron carriers comprise prosthetic group, such as flavin, heme, Fe-S centers and copper.

53. Prosthetic groups in each multiprotein enzyme complex Enzyme complex Prosthetic groups COMPLEX 1 (46 subunits) FMN, Fe-S COMPLEX 2 (4 subunits) FAD, Fe-S COMPLEX 3 ( 11 subunits) Heme, Fe-S COMPLEX 4 (13 subunits) Heme, Cu +

54. Oxidative phosphorylation