MCB100 Introductory Microbiology February 25, 2019 Chapter 5 - Microbial Metabolism

1. MCB100 Introductory Microbiology February 25, 2019 Chapter 5 - Microbial Metabolism

2. Conditions that can affect the activity of an enzyme - Protein denaturation See figure 5.8. - Temperature See figure 5.7 a on page 128. - pH See figure 5.7 b on page 128. - Substrate concentration See figure 5.7 c on page 128. - Presence of competitive inhibitors See figure 5.10. - Allosteric inhibitors or activators See figures 5.9 and 5.11. - Feedback inhibition See figure 5.12. (regulation of activity)

3. ENZYME INHIBITORS COMPETITIVE vs. ALLOSTERIC INHIBITORS

4. A competitive inhibitor has a chemical structure that is similar to one of the enzyme’s natural substrate. Competitive inhibitors bind to the active site of the enzyme. Competitive Inhibition

5. Increased substrate concentration can overcome inhibition

7. Enzyme Catalyzed Reactions The forward progress of an enzyme-catalyzed biochemical reaction can be slowed or stopped by all of the following things EXCEPT: A. the enzyme looses its’ shape due to an excessively high temperature B. a competitive inhibitor binds to the active site of the enzyme  C. the enzyme looses its’ shape due to an excessively high or low intracellular pH D. the enzyme looses it’s shape due to the binding of heavy metal ions that displace smaller ions like sodium E. the concentration of the substrate is very high

8. Enzyme Catalyzed Reactions The forward progress of an enzyme-catalyzed biochemical reaction can be slowed or stopped by all of the following things EXCEPT: A. the enzyme looses its’ shape due to an excessively high temperature B. a competitive inhibitor binds to the active site of the enzyme  C. the enzyme looses its’ shape due to an excessively high or low intracellular pH D. the enzyme looses it’s shape due to the binding of heavy metal ions that displace smaller ions like sodium E. the concentration of the substrate is very high

9. Conditions that can affect the activity of an enzyme - Allosteric inhibitors or activators - Feedback inhibition (end-product inhibition regulates enzymes)

11. Feedback Inhibition

12. Synthesis of ATP

13. ATP The Major Short-Term Energy Storage Molecule of Cells

14. What are some of the ways that a cell uses ATP? Substrate Activation - For glucose to be broken down it must be activated by the attachment of two phosphates. The cell converts glucose to glucose-1, 6-bisphosphate, which can then be broken down to yield energy and form ATP. - ATP and similar nucleotide triphosphates are the activated building blocks of nucleic acid biosynthesis. - Energy from ATP is used to attach amino acids to tRNA molecules. This is a key energy consuming step in protein biosynthesis. Power Cellular Motion - Energy from ATP is used to make flagella rotate and muscle fibers contract. - Energy from the hydrolysis of a similar compound, GTP, is used to make a ribosome move along a strand of mRNA in a specific direction. Energy from ATP can be used to make GTP. Pump ions or other molecules across a membrane - Active transport, to take in nutrients or excrete wastes

15. Cells make most of their ATP in two different ways: 1) Substrate-level phosphorylation The energy to form a high energy phospho-anhydride bond can come from the hydrolysis of a higher energy bond such as a mixed anhydride bond (carboxylic acid – phosphate) or a phospho-enol bond. 2) The H+-ATPase (proton-ATPase) This method uses the potential energy of the proton motive force (PMF). Also known as the chemoosmotic theory or oxidative phosphorylation. see figure 5.15 for an example of substrate level phosphorylation see figure 5.19 for a diagram showing the electron transport chain and synthesis of ATP by oxidative phosphorylation using the Proton Motive Force

16. Substrate Level Phosphorylation (SLP) Substrate level phosphorylation requires the sacrifice of a chemical bond that has a higher energy than the high energy bond of ATP. For example: The hydrolysis of the mixed anhydride bond in 1,3-bisphosphoglycerate releases about 40 kcal per mole so the breaking of this bond can drive the formation of a phosphoanhydride bond in ATP which requires about 30 k cal per mole.

17. In order for a cell to make ATP using a proton-ATPase there must be a membrane separating two compartments that have a difference in the concentration of protons. Protons flow through the portal from the side with the highest concentration to the side with the lowest. Energy from the flow of protons can be used to synthesize ATP. Proton gradients can be established using the chemical reactions of respiration via the electron transport chain, or through a photoactivated electron transport chain.

18. ATP synthesis by substrate level phosphorylation and oxidative phosphorylation In substrate level phosphorylation (SLP) an enzyme transfers a phosphate group from an organic compound to ADP to form ATP. The cell must form a metabolic intermediate that has a very high energy bond joining a phosphate group to an organic compound before the SLP occurs. General Mechanism of SLP: S~P + ADP S + ATP Enzymes that catalyze the transfer of the phosphate groups in SLP are called" kinases". SLP reactions take place in the cytoplasm and involve water-soluble enzymes. See figure 5.14 on page 132, steps 7 and 10 are SLP reactions of glycolysis. Enzymes and electron carriers of oxidative phosphorylation must be associated with an intact membrane. Redox reactions of the electron transport chain pump protons across the membrane, resulting in the formation of an electrical charge or voltage gradient across the membrane. Protons flow from the side with the highest concentration to the side with the lowest concentration by passing through a port in an enzyme called ATP synthase (AKA proton-ATPase). As the protons flow through ATP synthase the energy released by their movement is used to attach a phosphate group to ATP. See pages 134, 136 – 138 of your textbook.

19. Chemoheterotrophic Organisms Make Energy via Redox Reactions Redox Reactions = Oxidation – Reduction Reactions Redox reactions involve transfer of electrons from one atom to another. Redox reactions do not always require the involvement of oxygen. Electrons are not destroyed in a redox reaction, just transferred from one type of atom to another. When some compound gets oxidized (loss of electrons) some other compound has to be reduced (gain of electrons). The loss of electrons is called oxidation. The gain of electrons is called reduction. (A gain of electrons is called a reduction because the charge of the molecule becomes less. Remember, electrons are negatively charged.) In biochemistry an oxidation usually involves adding oxygen or removing a pair of hydrogens from a compound. A reduction is the addition of two hydrogen atoms or the removal of an oxygen atom from a compound.

20. The electron donor starts out as a reduced compound that getsoxidized during the course of the reaction. The electron donor is also known as the reducing reagent. The electron acceptor starts out as an oxidized compound thatgets reduced during the course of the reaction. The electron acceptor is also known as the oxidizing reagent.

21. In biochemistry (generally speaking): Oxidation is the addition of an Oxygen or the removal of 2 Hydrogens, Reduction is the addition of 2 Hydrogens of the removal of an Oxygen. Sugars are exactly mid-way between being completely oxidized (CO2) or completely reduced (a saturated alkane).

23. Lactic Acid Dehydrogenase is an NAD-dependent oxidoreductase When pyruvic acid gets reduced to lactic acid (gains 2 hydrogens), NADH gets oxidized to NAD+ (donates 2 hydrogens). In that reaction, pyruvic acid is the oxidizing reagent and NADH is the reducing reagent.

24. Biochemical Reaction Type Which one of the reactions (or half-reactions) shown would be an example of an oxidation? A. glucose is converted to fructose C6H12O6 C6H12O6 B. formaldehyde is converted to formic acid HCHO  HCOOH C. fumarate is converted to malate C4H4O4 C4H6O5 D. formaldehyde is converted to methanol HCHO  CH3OH

25. Biochemical Reaction Type Which one of the reactions (or half-reactions) shown would be an example of an oxidation? A. glucose is converted to fructose C6H12O6 C6H12O6isomerization B. formaldehyde is converted to formic acid HCHO  HCOOH oxidation C. fumarate is converted to malate C4H4O4 C4H6O5hydration D. formaldehyde is converted to methanol HCHO  CH3OH reduction

26. CHEMOHETEROTROPHY (both energy and carbon is derived from organic compounds) Type of Metabolismterminal electron acceptors - Aerobic Respiration: oxygen C6H12O6 + 6O26CO2 + 6H2O - - - - - - - - - - - - - - - - - Anaerobic Respirationan oxidized mineral ex: (NO3- reduction) sugar + nitrate CO2 + nitrite - - - - - - - - - - - - - - - - - - Fermentation an organic compound ex: (ethanol fermentation) sugar CO2 + ethanol The terminal e- acceptor is the oxidized compound that gets reduced in metabolic redox reactions.

27. COMPLEX METABOLIC PATHWAYS REQUIRED ARE FOR ENERGY PRODUCTION IN LIVING CELLS Redox reactions often release a lot of energy. This is good because living cells need energy, but if this energy is released in an uncontrolled fashion it could damage the cell. Redox reactions in cells occur in a series of gentle steps that often involve moving electrons or protons across a membrane to produce a charge gradient. Consider the oxidation of glucose: This reaction releases energy in a cell. C6H12O6 + 6 O26 CO2 + 6 H2O It is about the same as burning wood, which is mostly cellulose - a polymer of glucose. Combustion of wood requires a high ignition temperature and dry fuel. The energy of a fire is released in an uncontrolled fashion and serves only to ignite surrounding flammable material. Fire destroys things. Oxidation of glucose in a cell releases just as much energy as the oxidation of glucose in a fire. But in a cell the reaction takes place at 37o C, the fuel is dissolved in water and some of the energy released is used to make ATP.

28. A metabolic pathway is a series of enzyme catalyzed reactions that achieves the breakdown or building up of a complex molecule in small steps. The oxidation of glucose is achieved by about 20 discrete steps that allow that allow the energy to be released slowly so it can be efficiently utilized by the cell. The steps in the oxidation of glucose are divided into three metabolic pathways: - glycolysis, - the Kreb's cycle - and the electron transport chain (oxidative phosphorylation).

29. Glycolysis (ten steps) Glycolysis is a series of 10 reactions that results in a glucose molecule being partially oxidized to form two molecules of pyruvate. See figures 5.12 and 5.13 on pages 135 - 137 of the textbook For each glucose molecule converted to 2 molecules of pyruvate: 2 ATPs are required to start the process, 4 ATPs are produced by substrate level phosphorylation (for a net profit of 2 ATPs per glucose) also 2 molecules of NAD+ are reduced to NADH + H+.

31. Glycolysis

33. First steps of glycolysis

35. Glycolysis 1 Which ONE of the following statements about the reactions of the glycolysis pathway is TRUE? A. The reactions of the glycolysis pathway can’t occur in the absence of elemental oxygen (O2). B. The reactions of the glycolysis pathway produce carbon dioxide as a waste product. C. In the reactions of the glycolysis pathway one molecule of glucose is converted into two molecules of pyruvate. This is a partial oxidation of the glucose molecule. D. The reactions of the glycolysis pathway may occur in some types of bacteria but it doesn’t happen in humans.

36. Glycolysis 1 Which ONE of the following statements about the reactions of the glycolysis pathway is TRUE? • A. The reactions of the glycolysis pathway can’t occur in the absence of elemental oxygen (O2). • B. The reactions of the glycolysis pathway produce carbon dioxide as a waste product. • C. In the reactions of the glycolysis pathway one molecule of glucose is converted into two molecules of pyruvate. This is a partial oxidation of the glucose molecule. • D. The reactions of the glycolysis pathway may occur in some types of bacteria but it doesn’t happen in humans. • In the conversion of one molecule of glucose to two molecules of pyruvate by the reactions of the glycolysis pathway a cell can gain (net profit) 2 molecules of ATP. • - Before the 6-carbon sugar glucose can be broken down into 3-carbon compounds the cell must use 2 molecules of ATP.

40. The Kreb's cycle: 8 reactions that result in the oxidation of pyruvate to CO2. See pages 140-1431 of your textbook. When two molecules of pyruvate are oxidized to six molecules of carbon dioxide: 8 molecules of NAD+ are reduced to NADH + H+ 2 molecules of FAD are reduced to FADH2 2 ATPs made by substrate level phosphorylation At the end of glycolysis and the Kreb’s cycle the glucose has been oxidized to 6 molecules of CO2 and 10 molecules of NAD+ and 2 molecules of FAD have been reduced to NADH + H+ and FADH2.

41. Electron Transport Chain Oxidative phosphorylationis a series of redox reactions involving membrane bound enzymes and electron carriers that results in the reoxidation of NADH + H+ back to NAD+, the reduction of O2 to H2O and the production of about 34 ATPs per glucose. See pages 138 – 141 of the textbook. Oxidative phosphorylation yields a Proton Motive Force (PMF) that is sufficient to produce about 3 ATPs per NADH oxidized and 2 ATPs per FADH2 oxidized.

42. The Electron Transport Chain (ETC) is a series of enzymes and electron carriers embedded in a membrane. The ETC is located in the mitochondrial membrane in eukaryotic cells and in the cytoplasmic membrane in bacteria. 1) NADH + H+ is oxidized to NAD+ 2) Electrons are passed from carrier to carrier, protons are pumped across membrane. 3) Oxygen is reduced to water, in aerobic respiration oxygen is the terminal electron acceptor. Protons flow through ATP synthase and generate ATP from ADP + Pi. outside the cell or mitochondria membrane inside cell or mitochondria

43. Aerobic Respiration Oxidation of Glucose to Carbon Dioxide and Water Steps 1) Glycolysis 2) Krebs Cycle 3) Electron Transport Chain and Oxidative Phosphorylation Aerobic respiration yields about 38 ATPs per glucose molecule consumed. GlycolysisKrebs CycleETC and OP makes: 4 ATPs makes 2 GTPs 2FADH2 FAD uses: - 2 ATPs 10NADH  NAD+ profit = 2 ATPs 8NAD+ 8NADH 2FAD  2FADH2 makes PMF that 2NAD+ 2NADH can make 34 ATPs