Microbial Metabolism

1. Microbial Metabolism Chapter 5

2. Why microbial metabolism is important • How do cells gain energy to form cell structures? • How do pathogens acquire energy and nutrients at the expense of a patients health? • How does grape juice turn into wine?

3. Metabolism • Metabolism – the sum of all of the ________ ___________within a living organism • Biosynthetic • Energy harvesting processes • 1000s of chemical reactions and control mechanisms • The chemical reactions that occur in a cell are referred to _______________________

4. Metabolic Pathways • Metabolic pathway (Biochemical pathway): Series of chemical reactions required to breakdown or build a cellular component • ____________(“food”) • Intermediates (“partially digested food”) • ____________(“by-product”) • ____________are involved

5. Enzymes Role in Metabolic Pathways • Enzyme facilitate each step of a metabolic pathway • Made of _______________ • Act as a Biological _____________ • _________________________of a chemical reaction Enzyme 1 Enzyme 2 Enzyme 3 Enzyme + Substrate -----------------------------------------------> Enzyme + End Product

6. Activation energywithout enzyme Activation energywith enzyme Energy Products Progress of reaction

7. Enzymes Role in Metabolic Pathways • Naming Enzymes – many are named by adding “ase” • Enzymes are ________________ • Lipases Lipids • Proteases Proteins

8. Enzyme Specificity can be explained by a lock and key theory called the ____________________ Key (____________) _______________: substrate binding site Lock (______________: protein) Many protein enzymes are complete on their own, others have protein and non protein components.

9. Induced fit model ____________________ What happens to the enzyme once products have been made?

10. Apoenzyme Components Inorganic cofactor Active site Apoenzymes: • Enzyme - protein portion • Cofactor - non-protein • ______________- inorganic ions (iron, magnesium, or zinc) • ______________- organic vitamins which cannot be synthesized by certain organisms Coenzyme(organiccofactor) Apoenzyme (protein)

11. Coenzymes • E. coli can synthesize or make its own vitamins and convert them to coenzymes • Humans and other animals must consume vitamins from external sources • E. coli synthesizes vitamin K which we can absorb

12. Factors that Influence Enzymatic Activity • A cells ability to survive in extreme temperatures or pH is due to their enzymes • Enzymes are influenced by environmental factors • Temperature, pH, and substrate concentrations • Have optimal activity ranges

13. Temperature

14. Denaturation of an Active Protein

15. pH Many enzymes work best at neutral pH. Acetic acid pH 3.0 can act as a preservative

16. Substrate concentration When the substrate concentration (enzyme food) gets to high the enzymatic activity levels off, since all enzymes are working at their maximum rate.

17. Without enzymes… • Energy yielding reactions could occur but rates would be extremely slow

18. Allosteric Enzymes • Cells can rapidly regulate or control the activity of key enzymes • They have an _____________that is ________ from the active site • Molecules bind and _____________________ which prevents them from working

19. Feedback inhibition • When ______________of a biosynthetic pathway can act as an ______________of the ______________in that pathway. • Allows for controlling its own synthesis or building • Example: E. coli, the presence of the amino acid isoleucine allosterically inhibits the first enzyme in the pathway. Prevents the synthesis of isoleucine when available. Once depleted E. coli can resume production

20. Substrate Pathwayoperates Pathwayshuts down Enzyme 1 Boundend-product(allostericinhibitor) Allostericsite Feedbackinhibition Intermediate A Enzyme 2 Intermediate B End-product

21. Enzyme Inhibition Enzymes can be inhibited by a variety of compounds other than regulatory molecules such as allosteric regulators Inhibitors can effect enzymatic activity 1. Competitive Inhibitors 2. Noncompetitive Inhibitors Why is enzyme inhibition important?

22. Competitive Inhibitors • Generally this occurs since the inhibitor has a ________________________as the substrate

23. Example • Sulfanilamide (Sulfa Drugs) • Antibiotic • competes for the active site on bacterial enzymes that converts PABA into Folic Acid • Has a similar chemical structure to PABA • Prevents PABA from binding to active site • Folic Acid - required for the synthesis of DNA and RNA • No Folic acid= no DNA RNA synthesis= no cell replication • Selective toxicity: Doesn’t affect human cells since we can not synthesize Folic acid

24. Non-competitive Inhibitors

25. Substrate Pathwayoperates Pathwayshuts down Enzyme 1 Boundend-product(allostericinhibitor) Allostericsite Figure 5.11 Feedback inhibition Feedbackinhibition Intermediate A Enzyme 2 Intermediate B End-product Enzyme 3

26. WHO CARES? • What are the 3 methods we will use in this class to identify unknown bacteria?

27. WHO CARES????? • When laboratory personnel perform biochemical tests to identify unknown microorganisms we are testing for the production of an __________by that organism.

28. Scenario • You observe Gram positive coccus using the microscope. You are having a difficult time determining arrangement (which is common) • You see a mixture of chains: indicating Streptococcus • You also see clumps: indicating Staphylococcus • What biochemical test/tests can you perform to further differentiate between the two?

29. Biochemical Testing • Every bacterial species produce different enzymes which allow them to produce different metabolic by-products. • Identify and differentiate bacteria. Streptococcus pyogenes Staphylococcus aureus

30. Example of a biochemical test • Test used to demonstrate the ability of a bacterium to produce the enzyme catalase • Simplest test used to differentiate between Staphylococcus and Streptococcus • Streptococcus are catalase negative (obligate fermenter) • Staphylococcus are catalase positive Streptococcus pyogenes Staphylococcus aureus

31. Catalase test • The catalase test is simply performed by placing a few drops of H2O2 (hydrogen peroxide) on bacterial growth on an agar media. • A positive test is indicated by the formation of bubbles (H2O2 + catalase -> H2O + O2 (bubbles) Streptococcus pyogenes Staphylococcus aureus

32. Basics of Metabolism

33. Two components of metabolism • 1. __________________( Catabolic ) • _____________of complex organic molecules into simpler compounds • 2. _________________( Anabolic ) • the _____________of complex organic molecules from simpler ones

34. Energy • Energy: the capacity to do work • 2 forms • Potential: stored energy • Kinetic: energy of motion • Example: Rock on top of a hill? • Rock tumbling down a hill?

35. Role of ATP • Adenosine triphosphate (ATP) • Energy currency of a cell • Donor of free energy • 3 phosphates • Energy is stored in the phosphate bonds • Adenosine diphosphate (ADP) • Acceptor of free energy • 2 phosphates

36. ISM BOL CATA Energy lostas heat Energy lostas heat Energyused Energystored Metabolism ANABOLISM Larger buildingblocks Precursormolecules Macromolecules Energy storage(carbohydrates,lipids, etc.) Nutrients Cellular structures(membranes,ribosomes, etc.) Cellularprocesses(cell growth,cell division, etc.)

37. Role of ATP • Cells constantly generate and use ATP • Power biosynthetic reactions • 2 processes used by heterotrophic bacteria to form ATP • Substrate-level phosphorylation • Oxidative phosphorylation

38. How cells make ATP (part 1) • Substrate-level phosphorylation • Ex. Glycolosis • Only a small amount of ATP is made

39. How cells make ATP (part 2) • Oxidative phosphorylation

40. Chemical Energy Production • Energy Source or electron donor • Compound is broken down by a cell to release energy • Example: Glucose • 1. • refers to the ________of electrons • 2. • the _________of electrons

41. Chemical Energy Production Oxidation and Reduction Reactions Electron transfer from an electron _______to an electron ____________ Reactions always occur simultaneously Cells use ___________________to carry electrons (often in H atoms) Reduction Electrondonor Oxidizeddonor Electronacceptor Reduced acceptor Oxidation

42. Chemical Energy ProductionElectron carriers • Oxidized form Reduced form • NAD+ NADH • FAD FADH2

43. Chemical Energy ProductionTerminal Electron Acceptors • Electrons are transferred to a molecule such as oxygen which functions as a terminal e- acceptor. • Aerobic Respiration

44. Carbohydrate Catabolism • Microorganisms oxidize carbohydrates as their primary source of energy for anabolic reactions • Glucose - most common energy source • Energy obtained from Glucose by:

45. Key metabolic pathways • Gradually oxidize glucose completely to carbon dioxide • Glycolysis • Transition reaction • Krebs cycle • Electron Transport system

46. Oxidation of GlucoseChemical Equation • C6H12O6 + 6 O2 -------> 6 CO2 + 6 H2O • 38 ADP + 38 P 38 ATP • Oxygen is the terminal e- acceptor

47. Glycolysis • Oxidation of Glucose (_________) into ___ molecules of ____________ (___carbon) • Investment phase • Pay off phase • Generates __________ • Net gain ____________ • Overall End Products of Glycolysis: • ___ pyruvate • ___ NADH (reducing power used in electron transport system) • ___ ATP

49. Cellular Respiration • Includes • Synthesis of Acetyl-CoA (transition reaction) • Krebs cycle • ETS • This can be aerobic (with oxygen) • Or anaerobic (without oxygen) • Varying amounts of ATP are produced depending on e- acceptor

50. Cellular RespirationTransition Reaction (synthesis of Acetyl-CoA) • Connects Glycolysis to Krebs Cycle • Input from glycolysis • _____________ • End Products: