Microbial Metabolism

1. Microbial Metabolism

2. What is metabolism? • Sum total of ALL chemical reactions in a living organism • Metabolism is about the energy balance in cells, production and use • CATABOLIC reactions release energy • ANABOLIC reactions require energy • These processes are COUPLED

3. Coupled Reactions

4. Enzymes • Enzymes are biological catalysts • Enzymes lower the activation energy required for biological reactions to occur • Enzymes are: • Proteins • Specific for the reactions they catalyze • Not used up in the reactions • Speed up reactions in living system • Generally end with -ase

5. Energy Requirements

6. Enzymes • Enzymes are proteins • Many enzymes are part protein and part something else • Protein part is called the APOENZYME • Non-protein part is called a COFACTOR • Cofactors are often metal ions • If the cofactor is an organic molecule it is called a COENZYME • Together the apoenzyme and cofactor form the HOLOENZYME

7. Holoenzyme

8. Coenzymes • Coenzymes are organic molecules • Often derived from vitamins • Some coenzymes act as electron carriers in metabolic reactions • Two important coenzymes in metabolism are: • NAD+ (nicotinamide adenine dinucleotide) • FAD (flavin adenine dinucleotide) • Both are electron carriers

9. Enzyme action • Enzymes have a specific region on the surface where reactions occur • ACTIVE SITE • Enzyme-substrate complex forms at the active site • Reaction occurs • Products are released

10. Enzyme action

11. Factors that influence enzyme activity • Temperature • pH • Substrate concentration • Inhibitors

12. Temperature, pH, Substrate

13. Enzyme Inhibition • Competitive inhibition • Inhibitor “looks like” the natural substrate and fills the active site preventing the enzymatic reaction • Noncompetitive inhibition • Inhibitor binds to another site on the enzyme • ALLOSTERIC SITE • Causes a change in the active site and loss of activity

14. Inhibition

15. Sildenifil citrate Cyclic GMP Competitive Inhibition

17. How to make ATP • First you need ADP • Next some phosphate • Add some energy → ATP • ADP + P +energy → ATP • But where does the required energy come from? • That’s the next slide…….

18. Where’s the energy? • Adding the phosphate to ADP to make ATP is called PHOSPHORYLATION • Cells do this in two ways: • 1. Substrate level phosphorylation - the phosphate is transferred directly to the ADP to make ATP • 2. Oxidative phosphorylation – electron carriers such as NAD+ transfer electrons to a series of carries in the electron transport chain where energy is released and transferred to ADP to make ATP

19. Oxidation and Reduction • Part of the energy production mechanism in cells • Oxidation = loss of electrons • Reduction = gain of electrons • These reactions are always coupled in cells • Oxidation reactions produce energy

20. Redox reactions

21. Electron Carriers of Oxidative Phosphorylation • NAD+ • NAD+ gains 1electron and 1H+ NADH • FAD • FAD gains two H atoms FADH2 • The electrons will eventually be passed through the electron transport system to generate energy

22. Carbohydrate catabolism • Cells break down glucose to make energy • Two processes are involved: • 1. Cellular respiration • 2. Fermentation • Glycolysis • Krebs cycle • Electron transport chain

23. Respiration and Fermentation

24. Glycolysis • Means glucose burning • Occurs in either the presence or absence of oxygen • Yields relatively little ATP • Most living cells use this process

25. Glycolysis

26. Glycolysis

27. Out come of glycolysis • Start with 1 molecule of glucose (6C) • The products are: • NET 2 molecules of ATP • 2 molecules of NADH • 2 molecules of pyruvic acid (3C) • Organisms that are aerobic or facultative proceed into the Krebs cycle and ETS • Anaerobes proceed to fermentation

28. Krebs Cycle

29. How are Glycolysis and the Krebs Cycle connected? • Pyruvic acid is the END PRODUCT of glycolysis • Pyruvic acid itself DOES NOT directly enter the cycle • It is first converted to acetyl CoA, and this compound enters the Krebs cycle • Pyruvic acid → acetyl CoA yeilds: • NADH x 2 = 2 NADH • CO2 x 2 = 2 CO2

30. Krebs Cycle • Aerobic process • Has the potential to generate the most energy for the cell • The products of this cycle are: • 3 molecules of NADH x 2 = 6 NADH→ ETS • 1 molecule of FADH2 x 2 = 2 FADH2 →ETS • 1 molecule of ATP x 2 = 2 ATP • Why x 2? • Because glycolysis generates 2 molecules of pyruvic acid as an end product so the cycle must “go around” twice to metabolize all the pyruvic acid!!

31. Electron Transport • Chain of carrier molecules that receive electrons from NADH. • The flow of electrons from carrier to carrier is exergonic. • The energy release is used to pump H+ from NADH across a membrane to form a concentration of H+ on one side of the membrane. • The flow of H+ back across the membrane is also exergonic and the energy released is used to convert ADP to ATP – PROTON MOTIVE FORCE! • ATP is made via CHEMIOSMOSIS by the enzyme ATP SYNTHASE • Final electron acceptor is oxygen • ETS occurs only in the presence of oxygen • The reaction to make ATP is called OXIDATIVE PHOSPHORYLATION. • The source of electrons are the NADH and FADH2 produced in earlier steps of glycolysis and the Krebs cycle

32. Electron Transport System

33. ATP Production by ETS • For every 1 NADH that goes into ETS you get 3 ATP • For every 1 FADH2 that goes into ETS you get 2 ATP • How many NADH and FADH2 are generated in ALL parts of AEROBIC respiration? • ANSWER 10 NADH and 2 FADH2

34. Total Energy Production • 1 molecule of glucose gives the following: • 10 NADH → ETS = 30 ATP • 2 FADH2 → ETS = 4 ATP • 2 ATP directly from glycolysis (substrate level phosphorylation) • 2 ATP directly from Krebs (substrate level phosphprylation) • TOTAL ATP from 1 glucose = 38

35. Summary of aerobic respiration

36. What about the anaerobes and energy? • If you are an anaerobe you can do only glycolysis and get how munch total ATP? • Right, just 2 ATP • Remember no ETS for these microbes! • But want about the end product pyruvic acid, what happens to it, it can’t just continue to build up? • Answer, these bacteria use the pyruvic acid in the process of FERMENTATION

37. Fermentation • Occurs in the absence of oxygen • Produces small amounts of ATP • Organic molecule, not oxygen, is the final electron acceptor • End products are alcohol or various acids • Anaerobes and facultative anaerobes

38. Let’s make wine! • What do you need? • Grape juice, that’s the source of sugar • Yeast, these cells will make the alcohol • Mix together and put in a bottle that can be sealed • What’s happening in the bottle? • The answer is fermentation – no oxygen, you sealed the bottle, the yeast break down the sugars in the grape juice, make pyruvic acid, then alcohol • Wait a few weeks, open the bottle, then PARTY!

39. What wine? • The party was no fun, there was no wine • What happened, yeast make alcohol don’t they? • Right, they do, but only if NO oxygen is present • You didn’t seal the bottle well, air got in and the yeast did this: • Grape juice→glycolysis→Krebs→ETS • NO alcohol!

40. Fermentation

41. Fermentation

42. End Products

43. Catabolism

44. Nutritional Classification

45. Energy comparisons • Aerobes and facultative anaerobes growing the presence of oxygen produce 38 ATP from one molecule of glucose • Anaerobes produce 2 ATP from one molecule of glucose • So the energy bottom line is that growth in the presence of oxygen is the way to go and grow!