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BiochemBIGA0:CentralMetabolism-APreparatoryReview ! The Plan: 1. Form teams of four. In this exercise, you may select your own team members and determine your own roles. If there is a role you have not played yet, you are encouraged to volunteer for that role. I’m keeping track. a. Manager: this person manages the group and ensures that the assigned tasks are being accomplished on time and that everyone in the group is participating. The manager will also be the only one to relay any questions the group may have to the instructor. b. Recorder: the recorder records the names and roles of the group members and the final responses for the group. c. Presenter/Spokesperson: presents oral reports to the class. Because of the number of groups, not all presenters will present in every small-group session. d. Skeptic: presents opposing viewpoints in order to generate discussion. 2. Answer the questions and go through the exercises as indicated. 3. Prepare the spokesperson to articulate responses for the team if the group is called upon. Group No:_________ This page was left blank intentionally 2 SECTION 1: Warm-Up Part I: The Citric Acid Cycle-Overview 1. What is the purpose or are the purposes of the citric acid cycle in metabolism, in general? 2. Discuss with your group the ways that the citric acid cycle differs from glycolysis and list the ones you all agree on. (Consider reaction types, locations and directionality of the pathways when constructing your answer.) Part II: Electron Transport 3. Considering only glycolysis and electron transport: a. Through what molecule(s), enzyme(s) or intermediate(s) are glycolysis and the electron transport chain directly linked to one another? (i.e., Which do they have in common?) b. Given the way(s) that the two pathways are directly interconnected, what could one say the purpose of electron transport is as it relates to glycolysis only? 3 4. Through what molecules, enzymes or intermediates are the citric acid cycle and the electron transport chain directly linked to one another? 5. During the process of electron transport, protons are pumped from the matrix into the intermembrane space. a. How do the pH values differ in the matrix and the intermembrane space? b. What does this suggest about the sign of ∆G for proton pumping into the intermembrane space? Explain. SECTION 2: Making Connections For this part of the exercise, provide fairly thorough answers. 6. Oxygen is not a metabolite, reactant or product of glycolysis, but in cells, the flux through glycolysis is increased in the presence of oxygen relative to the flux in the absence of oxygen. Why? 4 7. Which reaction in glycolysis is likely to become the rate limiting one at low oxygen concentrations and why? (i.e., Which reaction in glycolysis is most likely to be directly affected by low oxygen concentrations? You may want to revisit question 3) Use the following information to answer questions 8 and 9. In cells, glycolysis can still continue in the absence of oxygen. In humans, this is possible because under anaerobic conditions an additional reaction, catalyzed by lactate dehydrogenase (LDH), is used to oxidize NADH in the cytosol. The reaction is shown below: Anaerobic glycolysis is known as fermentation. In this case, it is homolactic fermentation because the product is lactate (aka lactic acid). In yeast cells, the fermentation process works a little differently as illustrated below. This is alcoholic fermentation and has been exploited for centuries in making bread and alcoholic beverages. (Humans do not have the enzyme pyruvate decarboxylase.) 5 8. Why are the lactate dehydrogenase reaction in humans and the pyruvate decarboxylate and alcohol dehydrogenase reactions in yeast needed under anaerobic conditions and not under aerobic conditions? 9. What do you think would happen to the cell if it was under anaerobic conditions and its lactate or alcohol dehydrogenase was not functional? 10. Explain the biochemical logic of ONE of the following regulatory mechanisms of the citric acid cycle I. The pyruvate dehydrogenase complex catalyzes the reaction pyruvate → acetyl-CoA and it is activated by AMP, CoA and NAD+, but it is inhibited by ATP, acetyl-CoA and NADH. II. Citrate syntase catalyzes the reaction oxaloacetate → citrate and it is activated by ADP but it is inhibited by succinyl-CoA, NADH, citrate and ATP. III. Isocitrate dehydrogenase catalyzes the reaction isocitrate → α-ketoglutarate and it is activated by ADP and inhibited by ATP. 6 Choose ONE of the following to answer 11. The pyruvate dehydrogenase complex catalyzes the reaction pyruvate→acetyl-CoA (Reaction 0 in the TCA cycle). Individuals who express a defective form of the pyruvate dehydrogenase complex constantly feel tired. What is the biochemical basis for this? 12. One reason that arsenic is poisonous is that arsenate is able to substitute for Pi in the glyceraldehyde 3-phosphate dehydrogenase reaction of glycolysis to produce 1-arseno, 3-phosphoglycerate (see figure below). This compound is very unstable and hydrolyzes rapidly and spontaneously to produce 3-phosphoglycerate, which is the next intermediate in glycolysis, and is further metabolized to ultimately yield pyruvate. Why, then, is arsenate toxic? Answer to Question Number ____ is below. 7 SECTION 3: If there is time... Oxidative Phosphorylation (10 minutes) 13. Oxidative phosphorylation is a mechanism of ATP synthesis that is distinct from substrate-level phosphorylation. a. What is substrate level phosphorylation? b. Why is the term “oxidative phosphorylation” appropriate for ATP synthesis by ATP synthase? 14. The synthesis of ATP is catalyzed by ATP synthase, which is sometimes called complex V. ATP synthesis is driven by changes in the structure of complex V at what structural level(s)? (Remember, the levels of protein structure are primary, secondary, tertiary and quaternary.) 8 Continue your answer on this page if Necessary 9 Table 1: Oxidation of Pyruvate and the Reactions of The TCA Cycle Reaction ∆G’° (kJ/mol) pyruvate + CoA + NAD+ → NADH + H+ + CO2 + acetyl CoA -33.4 acetyl CoA+ Oxaloacetate → citrate -32.2 citrate → isocitrate 13.3 isocitrate → α-ketoglutarate -8.4 α-ketoglutarate + NAD+ + CoA→ succinyl-CoA + CO2 + NADH + H+ -33.5 succinyl-CoA + GDP + Pi →CoA + GTP + succinate -2.9 succinate + FAD →FADH2 + fumarate 0 fumarate + H2O → malate -3.8 malate + NAD+ → NADH + H+ + oxaloacetate 29.7 10 This page was left blank intentionally 11