1. CZ5225: Modeling and Simulation in Biology Lecture 9: Biological Pathways I: Metabolic Pathways Prof. Chen Yu Zong Tel: 6874-6877 Email: yzchen@cz3.nus.edu.sg http://xin.cz3.nus.edu.sg Room 07-24, level 7, SOC1, NUS yzchen@cz3.nus.edu.sg http://xin.cz3.nus.edu.sgyzchen@cz3.nus.edu.sg http://xin.cz3.nus.edu.sg

2. 2 All metabolism may be thought of as the coupling of energy production and energy use. Some key concepts about metabolism

3. 3 Certain biochemical reactions occur spontaneously Net release of energy Others must be “forced” to occur coupling

4. 4 Energy and Chemical Reactions

5. 5 Lower activation E Specificity Activation Cofactors Modulators –Acidity –Temperature –Competitive inhibitors –Allosteric –Concentrations Enzymes speed biochemical reactions

6. 6

7. 7 Defined: –Equilibrium –Reversible Law of Mass Action

8. 8 Oxidation – reduction Hydrolysis – dehydration Addition – subtraction exchange Ligation Types of Enzymatic Reactions

9. 9 Pathways –Intermediates –Catabolic - energy –Anabolic - synthesis Cell Metabolism

10. 10 Metabolic Pathways Catabolic Pathways: Those that convert energy into biologically useful forms are called catabolic pathways Fuels (carbs & fats)  CO 2 + H 2 O + useful energy: catabolism Examples: degradation, pathways by which nutrients and cellular components are broken down for reuse or to generate energy

11. 11 Metabolic Pathways Anabolic Pathways: Those that require inputs of energy to proceed are called anabolic pathways Useful energy + small molecules  complex molecules: anabolism Biosynthesis, building up of biomolecules from simpler components Pathways that can be either anabolic or catabolic are referred to as amphibolic pathways

12. 12 Coupling favorable & unfavorable reactions A pathway must satisfy minimally two criteria: 1.Reaction must be specific, yielding only one particular product or set of products. Enzymes provide specificity 2.Whole set of reactions in a pathway must be thermodynamically favored. A reaction can occur spontaneously only if  G, the change in free energy, is negative 3. An important thermodynamic fact: the overall free energy change for a chemically coupled series of reactions is equal to the sum of the free-energy changes of the individual steps A  B + C  G 0’ = + 5 kcal mol -1 B  D  G 0’ = - 8 kcal mol -1 ******************************************* A  C + D  G 0’ = - 3 kcal mol -1

13. 13 Feedback inhibition Enzyme modulators No enzyme Enzyme isolation Energy availability - ATP Control of Metabolic Pathways

14. 14 ATP is the Universal Currency of Free Energy Metabolism is facilitated by the use of a common energy currency Part of the free energy derived from the oxidation of foodstuffs and from light is transformed into ATP - the energy currency A large amount of free energy is liberated when ATP is hydrolyzed to ADP & P i, or ATP to AMP & PP i ATP + H 2 O  ADP + P i  G 0’ = -7.3 kcal mol -1 ATP + H 2 O  AMP + PP i  G 0’ = -10.9 kcal mol -1 Under typical cellular conditions, the actual  G for these hydrolyses is approximately -12 kcal mol -1 ATP hydrolysis drives metabolism by shifting the equilibrium of coupled reactions: by a factor of approximately 10 8

15. 15 Structures of ATP, ADP,& AMP

16. 16 Structures of ATP, ADP,& AMP

17. 17 Coupled Reactions Involving ATP

18. 18 Coupled Reactions Involving ATP

19. 19 Coupled Reactions Involving ATP

20. 20 Glycolysis –Phosphorylation –Pyruvate Anaerobic respiration Lactate production 2 ATPs produced ATP Production

21. 21 Aerobic respiration In mitochondria Acetyl CoA and CO 2 Citric Acid Cycle Energy Produced –1 ATP –3 NADH –1 FADH Waste – 2 CO 2 s Pyruvate Metabolism

22. 22 Pyruvate Metabolism

23. 23 High energy electrons Energy transfer –ATP synthesized from ADP –H 2 O is a byproduct Electron Transport

24. 24 Electron Transport

25. 25 Complex Carbohydrates Glycogen catabolism –Liver storage –Muscle storage Glucose produced Biomolecules Catabolized to Make ATP

26. 26 Protein Catabolism Deamination Conversion –Glucose –Acetyl CoA

27. 27 Higher energy content Triglycerides to glycerol –Glycerol –Fatty acids –Ketone bodies - liver Lipid Catabolism

28. 28 Lipid Catabolism

29. 29 Stages of Catabolism from Foodstuffs Extraction of energy from foodstuffs can be divided into three stages

30. 30 Glycogen synthesis –Liver storage –Glucose to glycogen Gluconeogenesis –Amino acids –Glycerol –Lactate Synthetic (Anabolic) pathways

31. 31 Lipogenesis Acetyl Co A Glycerol Fatty acids Triglycerides

32. 32 Protein Synthesis 20 Amino acids DNA code sequence mRNA transcription processing Translation by ribosomes Chain (polymer) of amino acids

33. 33 Embden-Meyerhof Pathway (EM, glycolysis) Major pathway for the conversion of hexose sugars into pyruvate. It results in the formation of: -two NADH - two ATP

34. 34 (from Glyceraldehide-3-P to Pyruvate) Gain of 4 ATP

35. 35

36. 36 The Hexose Monophosphate (HM) Pathway (also known as oxidative pentose, OM, or pentose phosphate pathway) It provides all the key intermediates not provided by the EM pathway.

37. 37

38. 38 The Entner-Doudoroff Pathway It may be considered an alternate hexose monophsphate pathway. It provides a minimum of five of the critical biosynthetic intermediates: - glucose-6-P - triose phosphate - 3-phosphoglycerate - phosphoenol pyruvate (PEP) - pyruvate

39. 39 The Entner-Doudoroff Pathway It begins the same as the HM pathway up to phosphogluconic acid. Then, instead of being converted to pentose and carbon dioxide, it is dehydrated yielding 2-keto-3, dehydro, 6 phosphogluconic acid. pyruvateGlyceraldehyde-3-P The top half of the molecule of glucose

40. 40 The Entner-Doudoroff Pathway pyruvateGlyceraldehyde-3-P The top half of the molecule of glucose Both the EM and the ED pathway convert a glucose molecule to two molecules of pyruvate. In the EM pathway, pyruvate arises by the intermediate formation of glyceraldehyde-3-P. In the ED pathway, from the top half of the molecule of glucose.

41. 41

42. 42 Cyclic Metabolic Pathway

43. 43 Multiple Metabolic Pathways

44. 44 Multiple Metabolic Pathways

45. 45 Multiple Metabolic Pathways

46. 46 Post – Translational Protein Modification

47. 47 Metabolic Engineering Cells developed optimal use of their resources for their survival. Metabolic pathways are networks, regulated to optimally distribute their fluxes for best use of resources Metabolic engineering is to overcome the cellular regulation to produce product of our interest; or to create a new product that the host cells normally don’t need to produce.

48. 48 Scope of Metabolic Engineering Modify host cells, host multi-cellular organisms, or product Improved production, in selectivity or in quantity, of chemicals already produced by the host organism Extended substrate range for growth and product formation Addition of new catabolic activities for degradation of toxic chemicals Production of chemicals new to the host organism Modification of cell properties

49. 49 Methods of Metabolic Engineering Repeated mutations were necessary to create strains of the mold Penicillium chrysogenum which produce high titers of penicillin; that became the foundation of a commercial process and changed human health care. Radiation and chemical agents were employed by investigators to induce mutations in the microorganism.

50. 50 Methods of Metabolic Engineering Identify the target phenotype or trait Increase the frequency of occurrence of gene(s) that may confer the phenotype – Increase the mutation frequency in producing cells by Mutagen treatment (UV, X-ray, chemical mutagen) (Classical method) – Introduce additional gene(s) (that may already exist or absent in the host cell) known to give cells the desired properties (Genetic Engineering) – Introduce genetic element to inactivate or activate the gene by random insertion of extra sequence Identify the mutants (clones) that have the Desired trait. Two general means Screening Selection

51. 51 Methods of Metabolic Engineering

52. 52 Methods of Metabolic Engineering

53. 53 Methods of Metabolic Engineering

54. 54 Metabolic Engineering

55. 55 Metabolic Engineering

56. 56 Thermodynamics of Metabolic Pathways

57. 57 Thermodynamics of Metabolic Pathways Thermodynamics, as Related to Metabolism Reactions near equilibrium — Easily switch direction depending on relative concentrations of reactants and products Enzymes act to restore equilibrium Reactions far from equilibrium — Irreversible Enzymes act as dams — have insufficient activity to allow reaction to approach equilibrium; reactants build up; changes in activity of enzyme change flux

58. 58 Thermodynamics of Metabolic Pathways Three Major Implications of Thermodynamics for Metabolism Metabolic pathways are irreversible. Biological systems are governed by thermodynamics! For a process to be spontaneous ∆G must be negative Every metabolic pathway has a committed step. Usually the first irreversible step unique to a pathway. Usually an important site of regulation Catabolic and anabolic pathways differ