1. Copyright © 2011 Pearson Education Inc. Lecture prepared by Mindy Miller-Kittrell, University of Tennessee, Knoxville M I C R O B I O L O G Y WITH DISEASES BY TAXONOMY, THIRD EDITION Chapter 5 Microbial Metabolism

2. Copyright © 2011 Pearson Education Inc. Microbial Metabolism Metabolism =  The sum of all chemical reactions that take place in an organism  Ability of an organism to obtain, convert and utilize energy Metabolic pathway =  A sequence of chemical reactions in a cell in which the end product becomes the substrate for the next reaction  Enzymatically catalyzed

3. Copyright © 2011 Pearson Education Inc. Basic Chemical Reactions Underlying Metabolism  Catabolism and anabolism  Oxidation and reduction reactions  ATP production and energy storage  The roles of enzymes in metabolism

4. Copyright © 2011 Pearson Education Inc. Catabolism and Anabolism  Catabolism = breakdown reactions  complex molecules to simple  hydrolytic (use water)  energy-releasing (exergonic)  Anabolism = synthesis reactions  simple molecules to complex  dehydration (produce water)  energy-using (endergonic)

5. Copyright © 2011 Pearson Education Inc. Catabolism and Anabolism Catabolism provides the building blocks and energy (in the form of ATP) for anabolism.

6. Copyright © 2011 Pearson Education Inc. Oxidation and Reduction Reactions Oxidation-Reduction (Redox) reactions – Transfer electrons from one molecule to another – Oxidized molecule – Donates electron – occurs when electrons, and often protons, (hydrogen atoms) are released from a molecule – Reduced molecule – Accepts electron – occurs when electrons, and often protons, (hydrogen atoms) are accepted by a molecule – OILRIG (Oxidation Is Loss, Reduction Is Gain)

7. Copyright © 2011 Pearson Education Inc. Oxidation-Reduction (Redox) Reactions  The transfer of electrons from one molecule to another. These reactions occur simultaneously. Figure 5.2

8. Copyright © 2011 Pearson Education Inc. Oxidation-Reduction (Redox) Reactions  Reducing agent :  Causes the reduction of other molecules by donating electrons to them (and itself gets oxidized in the process)  Sodium thioglycollate media  Thioglycolic acid (TGA) is readily oxidized by air to disulfide  Oxidizing agent :  Causes the oxidation of other molecules by accepting their electrons (and itself gets reduced in the process)  Oxygen  Hydrogen peroxide

9. Copyright © 2011 Pearson Education Inc. Electron Carrier Molecules  Electron carrier molecules are used to carry electrons from one location in the cell to another.  Often they are carried in the form of hydrogen atoms (one proton and one electron)  Important electron carriers in cell metabolism – NAD + → NADH – Nicotinamide adenine dinucleotide – FAD → FADH2 – Flavine adenine dinucleotide https://highered.mcgraw- hill.com/sites/0072507470/student_view0/chapter25/animation__how_the_nad__works.ht mlhttps://highered.mcgraw- hill.com/sites/0072507470/student_view0/chapter25/animation__how_the_nad__works.ht ml

10. Copyright © 2011 Pearson Education Inc. ATP Production and Energy Storage  Energy produced by catabolism is stored as ATP  Is needed for many metabolic reactions  ATP is generated by the phosphorylation of ADP  (Addition of Phosphate)  Occurs ~ 3 million times/second

11. Copyright © 2011 Pearson Education Inc. ATP Structure When energy is needed the high energy bond that binds phosphate to ADP (ADP~P) is broken and energy is released

12. Copyright © 2011 Pearson Education Inc. ATP Production Substrate-level phosphorylation – high energy phosphate is transferred from a phosphorylated compound to ADP Oxidative phosphorylation – energy from redox reactions used to attach inorganic phosphate to ADP Photophosphorylation – light energy used to attach inorganic phosphate to ADP

13. Copyright © 2011 Pearson Education Inc. The Roles of Enzymes in Metabolism Enzymes Proteins that speed up a reaction by decreasing the ‘activation’ energy required to start a chemical reaction Biological catalysts- are NOT used up in reaction Necessary for chemical reactions to occur fast enough to maintain life Do not make reactions happen that would not ordinarily take place

14. Copyright © 2011 Pearson Education Inc. The effect of enzymes on chemical reactions Figure 5.4

15. Copyright © 2011 Pearson Education Inc. The Roles of Enzymes in Metabolism – Enzymes have active sites that bind to a very specific substrate Named according to type of catalyzed reaction or substrate Most end in “ –ase” Examples: – TransferaseTransfer functional groups – LigaseJoining of molecules – LipaseFat digestion – SucraseHydrolysis of sucrose

16. Copyright © 2011 Pearson Education Inc. Enzyme Components Many enzymes are made of protein alone Some enzymes are composed of: – Protein portions ( Apoenzymes ) and – Non-protein portions: – Cofactors -inorganic ions (Fe, Cu,Zn) or – Coenzymes –organic molecules – (NAD +, NADP +, FAD) Holoenzyme -Complete active enzyme Active site – Region that interacts with the substrate

17. Copyright © 2011 Pearson Education Inc. Enzyme Components

18. Copyright © 2011 Pearson Education Inc. Enzyme Activity

19. Copyright © 2011 Pearson Education Inc. Enzyme Activity  Many factors influence the rate of enzymatic reactions  Temperature  pH  Enzyme and substrate concentrations  Presence of inhibitors  Competitive  Non-competitive

20. Copyright © 2011 Pearson Education Inc. Temperature and pH Figure 5.8a Enzymes can be denatured by temperature and pH

21. Copyright © 2011 Pearson Education Inc. Substrate Concentration Figure 5.8c Saturation point Active sites on enzymes are filled

22. Copyright © 2011 Pearson Education Inc. Competitive Inhibitors Substances that block an enzyme’s active site Do not denature enzymes – Sulfa drugs compete with normal substrate for bacterial enzymes that make folic acid

23. Copyright © 2011 Pearson Education Inc. Noncompetitive Inhibitors Substances that alter an enzyme’s active site structure by binding to enzyme at allosteric site – Nevirapene (HIV) attaches to reverse transcriptase so it can’t make DNA

24. Copyright © 2011 Pearson Education Inc. Feedback Inhibition  Feedback inhibition stops the metabolic pathway when final end product accumulates  Conserves energy in organism

25. Copyright © 2011 Pearson Education Inc. Metabolic Pathway Sequence of chemical reactions that slowly releases energy from molecules. Usually each step catalyzed by a specific enzyme. Product of one reaction serves as the substrate for the next one. Occurs through oxidation-reduction reactions.

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27. Carbohydrate Catabolism The breakdown of carbohydrates to release energy (in the form of ATP) Carbohydrates are catabolized by: 1.Cellular respiration : – Utilizes glycolysis, Krebs cycle, and electron transport chain – Glucose is completely decomposed – Aerobic – Oxygen is final e - acceptor – Anaerobic – Inorganic substrate is final e - acceptor (ex. sulfur, iron, nitrogen) 2.Fermentation : – Utilizes glycolysis- then converts pyruvic acid into another compound (organic waste products)

28. Copyright © 2011 Pearson Education Inc. Aerobic Respiration  C 6 H 12 O 6 + 6O 2 + 38ADP+ 38P  6CO 2 + 6H 2 O +38ATP  Divided into “four” metabolic steps: 1. Glycolysis 2. Transition step 3. Krebs cycle 4. Electron Transport Chain Alternative pathways: Pentose phosphate pathway- breakdown of 5C sugar Entner-Doudoroff pathway- used by Pseudomonas aeruginosa & Enterococcus faecalis

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31. Carbohydrate Catabolism Glycolysis – Divided into three stages involving 10 total steps – Energy-investment stage – Lysis stage – Energy-conserving stage

32. Copyright © 2011 Pearson Education Inc. Glycolysis Oxidation of glucose to pyruvic acid 2 ATP are invested to begin the biochemical pathway. Four molecules of ATP are generated through substrate level phosphorylation. Result – – net 2 ATP and 2 molecules of NADH. Each molecule of NADH will be converted to 3 ATP in the Electron Transport Chain.

33. Copyright © 2011 Pearson Education Inc. Glycolysis Figure 5.13

34. Copyright © 2011 Pearson Education Inc. Carbohydrate Catabolism

35. Copyright © 2011 Pearson Education Inc. Substrate-level phosphorylation Figure 5.14

36. Copyright © 2011 Pearson Education Inc. Transition Step Pyruvic acid loses a carbon (released as CO 2 ) to become acetyl-coA which enters the Krebs Cycle. 1 NADH produced for each acetyl-coA formed from pyruvic acid- – 2 pyruvic acid molecules from one glucose molecule.

37. Copyright © 2011 Pearson Education Inc. Krebs Cycle 2 ATP’s produced by substrate-level phosphorylation 6 NADH (potential 18 ATP) 2 FADH2 (potential of 4 ATP) Each Acetyl Co-A goes through the Krebs Cycle

38. Copyright © 2011 Pearson Education Inc. Carbohydrate Catabolism Cellular Respiration – Electron transport – Most significant production of ATP occurs from series of redox reactions known as an electron transport chain (ETC) – Series of carrier molecules that pass electrons from one to another to final electron acceptor – Energy from electrons used to pump protons (H + ) across the membrane, establishing a proton gradient – Located in cristae of eukaryotes and in cytoplasmic membrane of prokaryotes

39. Copyright © 2011 Pearson Education Inc. Electron Transport Chain NADH and FADH2 carry protons and electrons in the form of hydrogen to the ETC. Sequence of carrier molecules where oxidation-reduction reactions occur. As electrons are passed along the carrier molecules in the ETC, some hydrogen ions are pushed across the membrane in which they are located

40. Copyright © 2011 Pearson Education Inc. An electron transport chain Figure 5.17

41. Copyright © 2011 Pearson Education Inc. Carbohydrate Catabolism

42. Copyright © 2011 Pearson Education Inc. Carbohydrate Catabolism Cellular Respiration – Electron transport – Four categories of carrier molecules – Flavoproteins – Ubiquinones – Metal-containing proteins – Cytochromes – Aerobic respiration: oxygen serves as final electron acceptor – Anaerobic respiration: molecule other than oxygen serves as final electron acceptor

43. Copyright © 2011 Pearson Education Inc. Figure 5.20 http://vcell.ndsu.nodak.edu/animations/etc/movie.htm Electron Transport Chain

44. Copyright © 2011 Pearson Education Inc. Chemiosmosis The process of releasing electrons from the carrier molecules in the ETC to produce ATP As hydrogen ions collect across the membrane, they produce a potential energy source (like a battery) called the Proton Motive Force. These hydrogen ions move from this area of high concentration back across the membrane through channels in the membrane that contain ATP synthase.

45. Copyright © 2011 Pearson Education Inc. Chemiosmosis ATP synthase converts the energy produced by the H+ flow into ATP. Electrons are passed to the final electron acceptor (oxygen or another inorganic compound). 34 ATP produced in ETC by oxidative phosphorylation.

46. Copyright © 2011 Pearson Education Inc. Chemiosmosis

47. Copyright © 2011 Pearson Education Inc. ATP From Aerobic Respiration Total Net ATP from 1 glucose molecule 4 ATP = 4 ATP 10 NADH = 30 ATP 2 FADH2 = 4 ATP Total 38 ATP for prokaryotes

48. Copyright © 2011 Pearson Education Inc. PathwayEukaryoteProkaryote GlycolysisCytoplasm Transition stepCytoplasm Krebs cycle Mitochondrial matrix Cytoplasm Electron Transport Chain Mitochondrial inner membrane Plasma membrane Aerobic Respiration

49. Copyright © 2011 Pearson Education Inc. Anaerobic respiration Electron acceptorProducts E. coli NO 3 – NO 2 –, N 2 + H 2 O Desulfovibrio SO 4 2- H 2 S + H 2 O Methanococcus CO 2 CH 4 + H 2 O The final electron acceptor in the ETC is not O 2. Yields less energy than aerobic respiration because only part of the Krebs cycles operates under anaerobic conditions.

50. Copyright © 2011 Pearson Education Inc. Fermentation Figure 5.13

51. Copyright © 2011 Pearson Education Inc. Fermentation Figure 5.22  Produces 2 ATP from glycolysis  Most of the potential energy remains in the bonds of fermentation products  Fermentation products are wastes to cells that make them, many are useful to humans

52. Copyright © 2011 Pearson Education Inc. Fermentation

53. Copyright © 2011 Pearson Education Inc. Catabolism of Organic Molecules

54. Copyright © 2011 Pearson Education Inc. Lipid Catabolism LIPID = 1 glycerol 3 fatty acids

55. Copyright © 2011 Pearson Education Inc. Protein Catabolism ProteinAmino acids Extracellular proteases and peptidases Krebs cycle Deamination, decarboxylation, dehydrogenation Organic acid Protein is too large to pass through plasma membrane Extracellular enzymes break down protein into amino acids that can pass through plasma membrane Amino acids are enzymatically converted to organic acids that can enter the Krebs cycle

56. Copyright © 2011 Pearson Education Inc. Protein Catabolism

57. Copyright © 2011 Pearson Education Inc. How Metabolism Is Used in Bacterial Identification  Test for presence of enzymes  Detect end product of metabolic pathways