1. Metabolism: Basic Concepts and Design

2. Roadmap of Metabolic Pathways

3. Metabolism Metabolism – reactions occurring in a living system that produce and consume the energy needed for the organism to exist. Metabolic pathways. Metabolic reactions. High Energy bonds in compounds. Thermodynamics of reactions.

4. Metabolism Metabolism - the entire network of chemical reactions carried out by living cells Metabolites - small molecule intermediates in the degradation and synthesis of biopolymers Catabolic reactions/Catabolism - degrade biomolecules to create smaller molecules and energy Anabolic reactions/ Anabolism - synthesize biomolecules for cell maintenance, growth and reproduction Amphibolism- Reactions which are both catabolic and anabolic in nature.

5. Glucose Metabolism Breakdown to small molecules and energy.

6. Catabolism: Purpose and Pathway  Purposes: Trap the energy of the biomolecules in the form of ATP. Generate the substances ( precursors) required for the synthesis of complex molecules.

7. Catabolism: Purpose and Pathway  Catabolism occurs in three stages: I.Catabolism of complex molecules into their building blocks:  Polysaccharides are broken down to monosaccharides, lipids to free fatty acids and glycerol, proteins to amino acids. II. Formation of simple intermediates:  The building blocks produced in stage 1 are degraded to simple intermediates such as pyruvate and acetyle CoA.  These intermediates are not readily identifiable as carbohydrates, lipids or proteins.  A small quantity of energy ( as ATP) is captured in stage 2.

8. Catabolism: Purpose and Pathway III. Final Oxidation of acetyl CoA :  Acetyl CoA is completely oxidized to CO 2, liberating NADH and FADH 2 that finally get oxidized to release large quantity of energy ( as ATP).  Krebs cycle( or citric acid cycle) is the common metabolic pathway involved in the final oxidation of all energy-rich molecules.  This pathway accepts the carbon compounds ( pyruvate, succinate etc.) derived from carbohydrates, lipids or proteins.

9. Stage 1 Stage 2 Stage 3 Fig. The three stages of catabolism ( ETC- Electron Transport Chain)

10. Anabolism: Purpose and Factors  Purpose:  For the synthesis of a large variety of complex molecules, the starting materials are very few.  These include pyruvate, acetyle CoA and the intermediates of citric acid cycle.  Factors:  Availability of precursors  The supply of energy( as ATP or GTP) and reducing equivalents( as NADPH+H + )

11. Intermediary Metabolism: Refers to the entire range of catabolic and anabolic reactions, not involving nucleic acids. Energy Metabolism: Deals with the metabolic pathways concerned with the storage and liberation of energy.

12. Catabolism and Anabolism Catabolism Anabolism degradative synthetic oxidativereductive energy producingenergy requiring (exergonic) (endergonic) makes pool moleculesuses pool molecules produces NADH & uses NADPH almost NADPH exclusively

14. A thermodynamically unfavorable reaction can be driven by a favorable reaction Two criteria: 1.The individual reaction must be specific. 2.The entire set of reaction that constitute the pathway must be thermodynamically favored.

15. Free Energy of Coupled Reactions ADP + Pi --- >ATP 1,3-bisphosphoglycerate --- > 3-phosphoglycerate + Pi 1,3-bisphosphoglycerate + ADP ---- > 3-phosphoglycerate + ATP  G o' = -49.4 kJ/mol  G o' = +30.5 kJ/mol  G o' = -18.9 kJ/mol

16. Free Energy of Coupled Reactions ADP + Pi --- >ATP 1,3-bisphosphoglycerate --- > 3-phosphoglycerate + Pi 1,3-bisphosphoglycerate + ADP ---- > 3-phosphoglycerate + ATP  G o' = -49.4 kJ/mol  G o' = +30.5 kJ/mol  G o' = -18.9 kJ/mol

17. ATP is the Universal Currency of Free Energy In Biological Systems Metabolism is facilitated by the use of a common energy currency, ATP. Part of the free energy derived from the oxidation of foodstuffs and from light is transformed into this highly accessible molecule which acts as the free energy donor in most energy requiring processes such as motion, active transport or biosynthesis. ATP Hydrolysis is Exergonic  ATP is an energy rich molecule because its triphosphate units contains two phosphoanhydride bonds.  A large amount of free energy is liberated when ATP is hydrolyzed to ADP and P i or when ATP is hydrolysed to AMP and PP i.

18. Adenosine Nucleotides Components of an energy system.

19. ATP+H 2 O ADP+P i ATP+H 2 O AMP+P i  G o' = -30.5 kJ/mol  G o' = -45.6 kJ/mol

20. Other ATP uses ATP can also be used to make other NTPs with nominal energy exchange using a nucleoside diphosphate kinase. ATP + NDP ADP + NTP Other involvement of ATP: 1. Phosphate transfer to make high energy bond: Glutamine synthesis uses P from ATP Glu + ATP —> γ-PGlu + ADP, then NH 3 displaces P to give Gln

21. Other ATP uses 2. PEP transfers P to make ATP: Enol-P (PEP) + ADP —> Pyr + ATP 3. Nucleotide transfer to make high energy bond: AMP from ATP combines with a fatty acid in making AcylSCoA catalyzed by acylSCoA synthetase (acyl thiokinase) during fatty acid activation. FA + ATP —> acyl-AMP + PPi, then CoASH displaces AMP to give acyl-SCoA

22. Driving Forces behind the Energy of ATP Hydrolysis 1.Resonance stabilization 2.Charge repulsion of oxygens of triphosphate unit of ATP 3.Number of charges on oxygens. 4.Stabilization due to hydration

23. Phosphate Resonance pKas of phosphoric acid: 2.1, 6.9 and 12.3

24. Phosphoryle Transfer Potential Is An Important Form of Cellular Energy Transformation ATP is not only the compound with a high phosphoryl transfer potential. Some compounds – PEP,1,3-BPG and creatine phosphate have a higher phosphoryl potential than that of ATP. PEP can transfer phosphoryl group to ADP to form ATP, which is one of the ways in which ATP is generated in the breakdown of sugars. It is significant ATP has a phosphoryl-transfer potential that is intermediate among the biologically important phosphorylated molecules- enables ATP to function efficiently as a carrier of phosphoryl groups.

25. Other High Energy Molecules

26.  G o' of Hydrolysis

28. The Oxidation of Carbon Fuels Is an Important Source of Cellular Energy Motion, active transport, signal amplification and biosynthesis can take place only if ATP is continually regerated from ADP. The generation of ATP is one of the primary roles of catabolism. The carbon in fuel molecules such as glucose and fats is oxidized to CO 2 and the energy released is used to regenerate ATP from ADP and P i. In aerobic organism the ultimate electron acceptor in the oxidation of carbon is O 2 and the oxidation product is CO 2. Fuel molecules are more complex than single carbon compounds but when a fuel is oxidized the oxidation takes place one carbon at a time.

29. ATP Use Synthesis

30. Aerobic Oxidation Oxidative phosphorylation does not occur without electron transport.

31. Oxidation States Oxidation of triacylglycerols affords more energy than do carbohydrates.

32. Sources of Energy

33. Ion Gradients Across Membranes Provide An Important Form Of Cellular Energy That can be Coupled To ATP Synthesis Electrochemical potential is an effective means of storing free energy. The electrochemical potential of ion gradients across membranes produced by the oxidation of fuel molecules or by photosynthesis, powers the synthesis of ATP in cells. In animals proton gradients generated by the oxidation of carbon fuels account for more than 90% of ATP generation. This process is called oxidative phosphorylation. ATP hydrolysis can then be used to form ion gradient of different types and functions.

34. Mitochondria Oxidation and electron transport Oxidative phosphorylation

35. Energy From Foodstuffs Is Extracted In Three Stages 1.Large molecules in food are broken down into smaller units. 2.The numerous small molecules are degrated to a few simple units that play a central role in metabolism. 3.ATP is produced from complex oxidation of the acetyl unit of acetyl CoA.

37. Metabolic pathways contain many recurring motifs Metabolism appears intimidating because of sheer no. of reactants and reactions. Unifying themes such as common metabolites, reaction and regulatory schemes from a common evolutionary heritage make the comprehension of this complexity more manageable. ATP is an activated carrier of phosphoryl groups because phosphoryle transfer from ATP is an exergonic process. The use of activated carriers is a recurring motif in biochemistry.

38. 1.Activated carriers of electrons for fuel oxidation :  Fuel molecules transfer electrons to special carriers which are either pyrimidine nucleotides or flavins.

39. Oxidation by NAD + A typically NAD + oxidation is -OH to C=O

40. Oxidation by FAD A typically FAD oxidation is -CH 2 -CH 2 - to -CH=CH-

41. 2. An activated carrier of electrons for reductive biosynthesis:  NADPH is used almost exclusively for reductive biosynthesis  NADH is used primarily for the generation of ATP.

42. NAD + Nicotinamide Nucleotide AMP R = -PO 3 = for NADP + AMP = Adenine Nucleotide A two electron transfer agent

43. 3. An activated carrier of two carbon fragments:  Acyl groups are important constituent both in catabolism and in anabolism.

44. Thioesters

46. Activated Carriers NADH and FADH 2 serves as electron carriers for most metabolic pathways. They are vitamin cofactors and also nucleotides. NAD + and FAD are derivatives of niacin and riboflavin respectively. NAD + accepts one electrom and one H to become NADH in oxidation( dehydrogenation) of CHOH to –C=O. FAD is reduced to FADH 2 by accepting 2 H in dehydrogenation of – CH-CH- to –C=C – Certain anabolic pathways, such as the pentose phosphate pathway and FA biosynthesis, use NADP + ⁄ NADPH instead of NAD + ⁄ NADH. The use of activated carriers illustrates two key aspects of metabolism.

47. 1.Firstly the kinetic stability of these molecules in the absence of specific catalysts is essential for their biological fuction because it enables enzymes to control the flow of free energy and reducing power. 2. Second, most interchanges of activated groups in metabolism are accomplished by a rather small set of carriers.

48. Carriers and Coenzymes

50. Types of metabolic reactions  The biochemical reactions are mainly of four types: I.Oxidation-reduction II.Ligation reaction III.Isomerization reaction IV.Group transfer V.Hydrolytic reaction VI.The addition of functional groups to double bonds or the removal of groups to form double bonds. These reactions are catalyzed by specific enzymes.

51. Krebs Cycle Oxidations Also, there are two oxidative decarboxylations in the Kreb’s Cycle (citric acid cycle).

52. Ligation with ATP

53. Isomerization

54. Group Transfer

55. Hydrolysis

56. Cleavage to form a Double Bond

58. Methods Employed To Study Metabolism: Experimental Approaches  The experimental approaches divided into three categories: 1.Use of whole organism or its components 2.Utility of metabolic probes 3.Application of isotopes. The actual methods employed may be either in vivo( in the living system) or in vitro(in the test tube) or more frequently, both

59. Experimental Approaches 1.Use of whole organism or its components : a.Whole organism :  Glucose tolerance test ( GTT), employed to measure the response of man( or other animals) towards carbohydrate metabolism is a good example of the use of whole organism. b. Isolated organs, tissues slices, whole cells, subcellular organelles, cell-free systems and recently purified components are frequently used to elucidate biochemical reactions and metabolic pathways.

60. Experimental Approaches 2. Utility of metabolic probes :  Two types of metabolic probes are commonly used : a.Metabolic inhibitors : I nhibitors of electron transport chain have been largely responsible to elucidate the sequence of electron carriers. b. Metabolic mutations : The inborn errors of metabolism in higher organism and the genetic manipulations in the microorganisms have also contributed a lot to the understanding of metabolisms.

61. Experimental Approaches 3. Application of isotopes:  By the use of isotopes, the molecules of the living system can be labeled without altering their chemical properties.  Application of isotopes in biochemistry has revolutionized the study of metabolism.

62. Energy Overview Energy distribution 1/3 2/3 nutrients ----> pool molecules ----> CO 2, H 2 O, NH 3  biomolecules

63. Pathways Metabolism includes all enzyme catalyzed reactions Metabolism can be subdivided into various areas: hexose shunt, electron transport, etc. The metabolism of the four major groups of biomolecules will be considered: Carbohydrates Lipids Amino Acids Nucleotides

64. Pathways Multiple-step pathways permit control of energy input and output Catabolic multi-step pathways provide energy in smaller stepwise amounts) Each enzyme in a multi-step pathway usually catalyzes only one single step in the pathway Control points occur in multistep pathways

65. Regulation Metabolism is highly regulated to permit organisms to respond to changing conditions Most pathways are irreversible Metabolism is regulated by controlling: 1.The amounts of enzymes 2.Their catalytic activities 3.The accessibility of substrate

66. Levels of Regulation 1.The amounts of enzymes:  Depends on both its rate of synthesis and its rate of degradation. E.g: In E.coli,the presense of lactose, induces within minutes a more than 50-fold increase in the rate of synthesis of b- galactosidase, an enzyme required for the breakdown of this disaccharide. 2. Controlling catalytic activity: a.Reversible allosteric control:  The first reaction in many biosynthetic pathways is allosterically inhibited by the ultimate product of the pathway.  The inhibition of aspertate transcarbamoylase by cytidine triphosphate.

67. b. Reversible covalent modification :  Glycogen phosphorylase, the enzyme catalyzing the breakdown of glycogen, a storage form of sugar, is activated by the phosphorylation of a particular serine residue when glucose is scare. c. Hormones coordinate metabolic relations between different tissues often by regulating the reversible modification of key enzymes.  Epinephrine triggers a signal transduction cascade in muscle, resulting in phosphorylation and activation of key enzymes and leading to the rapid degradation ogf glycogen to glucose which is then used to supply ATP for musce contraction.

68.  Many hormones act through intracellular messengers, such as cyclic AMP and calcium ion that coordinate the activities of many target proteins. d. Energy charge : Many reaction in metabolism are controlled by the energy status of the cell. ATP generatinf pathways are inhibited by a high energy charge, wheras ATP utilizing pathways are stimulated by a high energy charge.

69. Energy Charge of a Cell ATP + ½ ADP Energy Charge = ------------------------- ATP + ADP + AMP Limits are 0 and 1.0 If all is ATP, the energy charge = 1 If all is AMP, the energy charge = 0 ATP can be regenerated using adenylate kinase (this is a nucleoside monophosphate kinase): 2 ADP ATP + AMP

70. Rate vs Energy Charge

71. 3. Controlling the accessibility of substrate : a. Compartmentalization segretes opposed reaction: Fatty acid oxidation takes place in mitochodria, whereas fatty acid synthesis take place in the cytoplasm. b. Flux of substrate:  Glucose breakdown can take place in many cells only if insulin is present to promote the entry of glucose into the cell.  The transfer of substrate from one compartment of a cell to another ( cytoplasm to mitochondria).can serve as control point.