MICROBIAL BIOCHEMISTRY BIOT 309, 2012 Kim and Gadd, Chapter 4

1. MICROBIAL BIOCHEMISTRYBIOT 309, 2012Kim and Gadd, Chapter 4

2. OVERVIEW OF BACTERIAL METABOLISM

3. BACTERIAL METABOLISM • Metabolism = all biochemical reactions taking place in organism Conversion (change, rearrangement) reactions • One molecule becomes another • Structure changes • Not use or generate energy Anabolism uses endergonic reactions • Uses energy • Forms bonds • builds larger molecules, ie, proteins, carbohydrates

4. BACTERIAL METABOLISM Catabolism is exergonic • Releases/produces energy, i.e., makes ATP • Breaks bonds • Hydrolyzes larger molecules into simpler molecules

6. BACTERIAL METABOLISM • Growth depends on metabolism • All 3 types of reactions happening at the same time but anabolism or catabolism dominate at different phases of growth • Carried out by wide variety of enzymes and co-factors • Involves single enzymes and enzyme complexes • Provides precursor metabolites to anabolic pathways • Occurs in three locations: • Cytosol • On or in cell membrane • In periplasmic space

7. ENZYMES • Characteristics • Reusable • Very specific – one reaction/enzyme • Minute amounts needed • Work fast (100-1000 reactions/minute) • Catalysts • Large proteins Cont’d

8. ENZYMES • Characteristics • 1 enzyme/reaction • Substrate specificity • “Lock and Key”

9. “LOCK AND KEY”

10. “LOCK AND KEY” with coenzyme* * See descriptions in White * *

11. ENZYMES • Active site aligns substrate(s) so reaction is highly favorable Free energy = G

12. ENZYMES • Primarily proteins • Some have co-factors; co-factor use based on needs of enzyme • Inorganic ions: Mg++, Fe++, Zn++ • Organic ions: important in redox reactions • NAD+ : EMP glycolysis, ED – Entner Duodorff pathway • NADP+: HMP – hexose monophosphate pathway, glycolysis • Both inorganic and organic used in reactions in TCA cycle and ETC (electron transport)

13. NAD+ (oxidized)NADH (reduced) As NAD+ is reduced, one electron is added at the Nitrogen atom (removing the + charge), (= becomes - ) and one (electron + proton = H atom) (= becomes -) is added at the upper position of the nicotinamide ring.

14. ENERGY STORAGE DURING CATABOLISM • Must be available as energy for anabolism • Forms of storage: • Held in high energy bonds, e.g., ATP • Reducing equivalents, such as NADH, NADPH & FADH2 • Proton gradient (formed by electron transport system) • Forms used depend on pathway/enzymes used by bacteria • ATP and NADH are most common

15. COUPLED REACTIONS OCCUR: BE ABLE TO IDENTIFY THEM

16. Also Important in Glycolysis and Kreb’s Cycle!!! • Substrate-level phosphorylation HOMEWORK: FIND EXAMPLES FROM SLIDES AND TEXTBOOK

17. ADDITIONAL REDOX MOLECULES • Used in Electron Transport – Ch 5 • Ubiquinone • Iron-sulfur • Will review then

18. ENZYMES OCCUR AS: • Single enzymes • Part of complexes with other proteins and cofactors * • Electron transport chain • Flagella • ATP synthase • Part of pathways • Glycolysis • Citric Acid Cycle • Etc. Slowest reaction is a rate limiting step

19. BACTERIA: FOCUS ON CATABOLISM Catabolism is exergonic • Releases/produces energy, i.e., makes ATP • Hydrolyzes larger molecules into simpler molecules • Breaks bonds 3 phases of catabolism: glycolysis, Kreb’s Cycle, Electron Transport Chain (ETC)

20. BIG PICTUREIntegrating3 Phases of Catabolism

21. Reaction Products have abbreviated names • Watch for their use and know to what they refer

22. GLYCOLYSIS • Occurs mainly in cytoplasm • 1st step in some bacteria occurs in membrane • Involves how many enzymes? TEN but two ways to make glucose-6-phosphate (See STEP 1 slide.) • Splits glucose • NOTE: Does not require O2, i.e., this stage is anaerobic

23. OVERALL GLYCOLYSIS* REACTION glucose (6C) + 2 NAD+ + 2 ADP +2 Pi 2 pyruvate (3C) + 2 NADH + 2 H++2 H2O + 2 net ATP Is NAD+ the oxidized or reduced form? *also called Embden-Myerhof-Parnas Pathway

24. GLYCOLYSIS AND ALTERNATIVES • Bacteria use 3 different pathways to convert glucose to PGA (3-phosphoglycerate) (see diagram) • Glycolysis/Embden-Myerhof-Parnas (shown in next slide) • Pentose phosphate shunt/hexose monophosphate shunt • Entner-Duodorff • Energy yields are different • Same pathway (transition or bridging reaction) takes PGA (3-phosphoglycerate) to pyruvate

25. GLYCOLYSIS 3 different glycolytic pathways operate: EMP, EDP, HMP  THIS IS EMP From PGA on same steps

26. GLYCOLYSIS PHASES Preparatory Phase Payoff Phase

27. HIGH ENERGY COMPOUNDS • ATP • Pyruvate • HOMEWORK: WHAT OTHER HIGH ENERGY COMPOUNDS ARE PART OF EMP PATHWAY?

28. GLYCOLYSIS, step 1 • Rapid reaction to keep glucose inside cell • Location 1 = cytoplasm; one enzyme = hexokinase, requires Mg2+ Glucose glucose-6-phosphate (G6P) • Location 2: membrane (Some bacteria) PEP pyruvate provides ~P to phosphorylate and transport glucose across the membrane • More proteins and enzymes are involved • Other sugars use similar mechanism phosoenolpyruvate: sugar phosphotransferase system (PTS) PEP + SUGAR PYRUVATE + sugar-phosphate In E.coli the PTS consists of two enzyme and a low molecular weight heat-stable protein (HPr) WHAT DOES PEP stand for?

29. GLYCOLYSIS, step 1Group Translocation – phosphotransferase system

31. ANIMATION: http://highered.mcgraw-hill.com/sites/9834092339/student_view0/chapter5/active_transport_by_group_translocation.html ANIMATIONS FOR GROUP TRANSLOCATION ANIMATION: http://www.microbelibrary.org/images/kaiser/grouptranslocat.html

32. GLYCOLYSIS, step 2 Rearrangement/change reaction, requires Mg2+

33. GLYCOLYSIS, step 3phosphorylation NOTICE use of ATP QUESTION: What type of reaction is this?

34. GLYCOLYSIS, step 4cleavage Aldose to ketoseisomerization Yield 2 G3P END OF PREPARATORY PHASE Aldol cleavage

35. GLYCOLYSIS, step 5coupled oxidation + phosphorylation ☐ QUESTION: Where is energy of NADH used? Where does it go? ~ = high energy bond Question: What is oxidized? What is reduced?

36. GLYCOLYSIS, step 6dephosphorylation Example of substrate level phosphorylation QUESTION: From what carbon atom is the Pi removed? Why is this Pi removed?

37. GLYCOLYSIS, step 7phosphate group shift

38. GLYCOLYSIS, step 8dehydration ~ = high energy bond QUESTION: why does this reaction create ~Pi?

39. GLYCOLYSIS, step 9dephosphorylation ~ = high energy bond

41. OVERALL GLYCOLYSIS* REACTION glucose (6C) + 2 NAD+ + 2 ADP +2 Pi 2 pyruvate (3C) + 2 NADH + 2 H+ + 2 net ATP + 2H2O WHICH is oxidized and which is reduced? NAD+ is __________ ; NADH is ___________ *also called Embden-Myerhof-Parnas Pathway

42. THE OTHER GLYCOLYTIC PATHWAYS

43. TRANSITION OR BRIDGING REACTIONConnects glycolysis to citric acid/Kreb’s Cycle OVERALL REACTION 2 pyruvate + 2 NAD+ + 2 CoA-SH (coenzyme A) 2 acetyl-CoA + 2 NADH + 2 H+ + 2 CO2 CONNECTION TO OTHER BIOLOGY: Where else is CO2 made?

44. NAD+ (oxidized)NADH (reduced) As NAD+ is reduced, one electron is added at the Nitrogen atom (removing the + charge), (= becomes - ) and one (electron + proton = H atom) (= becomes -) is added at the upper position of the nicotinamide ring.

45. ANOTHER COENZYMECoenzyme A • Energy generation • Molecule made from several component parts – complex • Highly polar • Key in glycolysis to Kreb’s cycle transition reaction • Key component in fatty acid reactions • Synthesis very similar pro- and eukaryotes

46. TRANSITION REACTION 3 carbon Co A 2 carbon

48. NEXT: MORE ON TCA CYCLE

49. Operate under different growth conditions Note energy yields

50. NOTE: MORE SLIDES WILL BE ADDED ON ED AND PPS PATHWAYS