Microbiology

1. Microbiology Microbial Metabolism

2. The Metabolism of Microbes metabolism – all chemical reactions and physical workings of a cell Two types of chemical reactions: anabolism – biosynthesis; process that forms larger macromolecules from smaller molecules; requires energy input catabolism – degradative; breaks the bonds of larger molecules forming smaller molecules; releases energy

4. Cell Energetics • Cells manage energy in the form of chemical reactions that make or break bonds and transfer electrons. • Endergonic reactions – consume energy • Exergonic reactions – release energy • Energy present in chemical bonds of nutrients are trapped by specialized systems as the bonds of the nutrients are broken. • Energy released is temporarily stored in high energy phosphate molecules (ATP in particular). The energy of these molecules is used in endergonic cell reactions.

5. Enzymes • Enzymes are biological catalysts that increase the rate of a chemical reaction • The enzyme is not permanently altered in the reaction. • Enzyme promotes a reaction by serving as a physical site for specific substrate molecules to position. Substrate(s) Product(s)

6. Enzyme Structure • Simple enzymes – consist of protein alone • Some enzymes contain protein and nonprotein portions • apoenzyme –protein portion • cofactors – nonprotein portion • metallic cofactors – iron, copper, magnesium • coenzymes -organic molecules - vitamins

7. Enzyme Specificity and the Active Site • Site for substrate binding is active site, or catalytic site • A temporary enzyme-substrate union occurs when substrate moves into active site – induced fit • Appropriate reaction occurs; product is formed and released

8. Induced Fit Model

9. Location of Enzyme Action • Exoenzymes – transported extracellularly, where they break down large food molecules or harmful chemicals • cellulase, amylase, penicillinase • endoenzymes – retained intracellularly and function there

10. Metabolic Pathways • Sequence of metabolic reactions that proceed in a systematic, highly regulated manner – metabolic pathways • Each reaction requires a different enzyme to make it go

11. Controls on Enzyme Activity • Competitive Inhibitors • Bind at the active site and have a shape similar to the substrate • Non-competitive Inhibitors (allosteric inhibitors • Bind to an area other than the active site (an allosteric site) and do not resemble the substrate

13. Real WorldAntifreeze (ethylene glycol) poisoning • Dogs and cats are attracted to ethylene glycol by its sweet taste. Animals will voluntarily drink ethylene glycol if antifreeze is spilled or leaks onto garage floors or driveways. • Perhaps as many as 10,000 dogs and cats are victims of accidental poisoning by automobile antifreeze every year

14. Symptoms of ethylene glycol poisoning Depression, vomiting, incoordination, excessive urination, excessive thirst, and muscle twitching. Within 30 minutes of ingestion. In as little as 12 to 36 hours, severe kidney dysfunction characterized by swollen, painful kidneys and the production of minimal to no urine, may occur. The dog or cat may be seizuring, be comatose. Or dead! WHY?

15. The Problem Ethylene glycol itself is not the problem per se. The PROBLEM is the toxic products that are created by liver enzymes (alcohol dehydrogenase) acting upon the ethylene glycol. These metabolites cause acidosis (the blood becomes dangerously acidic) and destruction of kidney function.

16. The Solution…Let’s get Rover hammered!!!! Hooray for Competitive inhibition! Ethanol can be used as a competitive inhibitor. It can outcompete ethylene glycol for the active site of the alcohol dehydrogenase enzyme. If the enzyme molecules are busy with ethanol they won’t be available to turn ethylene glycol into toxic metabolites. Fomepizole, an inhibitor of alcohol dehydrogenase is replacing the use of ethanol.

17. Feedback Inhibition

18. Oxidation-Reduction (Redox reactions) • Lose electrons, oxidize (“LEO”) • Gain electrons, reduce (“GER”) • Coupled reactions…as one molecule is oxidized something else is always simultaneously reduced. • A great number of the catabolic reactions we will discuss are step-by-step oxidations of organic nutrient molecules. • The process salvages electrons and their energy • Released energy can be captured to phosphorylate ADP to ATP

19. Methane combustion as an energy-yielding redox reaction

20. Electron Carriers NAD+ • The transfer of electrons in redox reactions from donor to acceptor involves intermediates (carriers) • NAD+ is reduced to NADH • NADH must pass the electrons eventually so that it can be reoxidized to NAD+ and become available once again as a carrier

21. Adenosine Triphosphate: ATP • Three part molecule consisting of: • adenine – a nitrogenous base • ribose – a 5-carbon sugar • 3 phosphate groups • ATP utilization and replenishment is a constant cycle in active cells. • Removal of the terminal phosphate releases energy.

22. Formation of ATP ATP can be formed by three different mechanisms: • Substrate-level phosphorylation – transfer of phosphate group from a phosphorylated compound (substrate) directly to ADP • Oxidative phosphorylation – series of redox reactions occurring during respiratory pathway • Photophosphorylation – ATP is formed utilizing the energy of sunlight

23. Pathways of Bioenergetics Primary catabolism of fuels (glucose) proceeds through a series of three coupled pathways: • glycolysis • tricarboxylic acid cycle, Kreb’s cycle • respiratory chain, electron transport

24. Pathways Figure 5.13

25. Metabolic Strategies • Nutrient processing is varied, yet in many cases is based on the three catabolic pathways that convert glucose to CO2 and gives off energy. • Aerobic respiration – glycolysis, the TCA cycle, respiratory chain • Anaerobic respiration - glycolysis, the TCA cycle, respiratory chain; molecular oxygen is not final electron acceptor

26. Aerobic Respiration • Series or enzyme-catalyzed reactions in which electrons are transferred from fuel molecules (glucose) to oxygen as a final electron acceptor • Glycolysis – glucose (6C) is oxidized and split into 2 molecules of pyruvic acid (3C) • TCA – processes pyruvic acid and generates 3 CO2 molecules • Electron transport chain – accepts electrons NADH and FADH; generates energy through sequential redox reactions (oxidative phosphorylation)

27. Glycolysis

28. The energy input and output of glycolysis

29. The Krebs Cycle Figure 5.19

30. A summary of the Krebs cycle

31. Electron Transport and Oxidative Phosphorylation • Final processing of electrons and hydrogen and the major generator of ATP • Chain of redox carriers that receive electrons from reduced NADH and FADH2 • ETS shuttles electrons down the chain, energy is released and subsequently captured and used by ATP synthase complexes to produce ATP. – oxidative phosphorylation

32. Electron Transport Figure 5.21

33. The Formation of ATP and Chemiosmosis • Chemiosmosis – as the electron transport carriers shuttle electrons, they actively pump hydrogen ions (protons) across the membranesetting up agradient of hydrogen ions - proton motive force. • Hydrogen ions diffuse back through the ATP synthase complex causing it to rotate, causing a 3-dimensional change which results in the production of ATP.

35. The Terminal Step • Oxygen accepts 2 electrons from the ETS and then picks up 2 hydrogen ions from the solution to form a molecule of water. Oxygen is the final electron acceptor. 2H+ + 2e- + ½O2→ H2O

36. Final Totals for Aerobic respiration • For Each Molecule of Glucose you get: • 34 ATPs from Electron Transport Chain • 2 ATPs from Krebs Cycle • 2 ATPs from Glycolysis

37. Anaerobic Respiration • Functions like aerobic respiration except it utilizes oxygen containing ions, rather than free oxygen, as the final electron acceptor • Nitrate (NO3-) and nitrite (NO2-)

38. Fermentation • Incomplete oxidation of glucose or other carbohydrates in the absence of oxygen • Uses organic compounds as terminal electron acceptors • Yields a small amount of ATP • Examples: • Production of ethyl alcohol by yeasts acting on glucose (pyruvic acid) • Formation of acid, gas and other products by the action of various bacteria on pyruvic acid

39. Fermentation Figure 5.22

41. Non-Carbohydrate Energy/Carbon Sources • Lipids Lipases split lipids into: Glycerol (enters cycle at 3PGAL step of glycolysis) Fatty acids (disassembled 2 carbons at a time to enter the Krebs cycle at Acetyl-CoA step) • Proteins Proteases release amino acids which are converted to glycolytic and Krebs Cycle intermediates.

42. The catabolism of various food molecules

43. Biosynthesis and the Crossing Pathways of Metabolism • Many pathways of metabolism are bi-directional or amphibolic. • Catabolic pathways contain molecular intermediates (metabolites) that can be diverted into anabolic pathways. • pyruvic acid can be converted into amino acids • amino acids can be converted into energy sources • glyceraldehyde-3-phosphate can be converted into precursors for amino acids, carbohydrates and fats

45. Photosynthesis • Every food chain begins with anabolic pathways in organisms that synthesize their own organic molecules from inorganic carbon dioxide • Most of these organisms capture light energy from the sun and use it to drive the synthesis of carbohydrates from CO2 and H2O by a process called photosynthesis 6 CO2 + 6 H2O  C6 H12O6 + 6 O2