Biochemistry Exam 2 Citric Acid Cycle

NAME: For GradeScope, please write your FIRST and LAST name in CAPITAL letters WITHIN the box: I. ________________/42 II. ________________/ 8 III. ________________/10 IV. ________________/12 V. ________________/28 Biochemistry II I. MULTIPLE CHOICE (42 points; 3 points each)) Choose the best answer as requested in each question and writing the appropriate letter in the box at the left.. 1. 2. A. the ratio of [ATP]/[ADP] is high. B. [AMP] is high. C. the ratio of [NAD+]/[NADH] is high. D. NADH is rapidly oxidized through the respiratory chain. Oxaloacetate uniformly labeled with 14C (i.e., with equal amounts of 14C in each of its carbon atoms) is condensed with unlabeled acetyl-CoA. After a single pass through the citric acid cycle back to oxaloacetate, what fraction of the original radioactivity will be found in the oxaloacetate? all B. 3/4 C. 1/2 D. 1/3 E. 1/4 Which of the following statements about the metabolic fate of pyruvate is incorrect ? A. B. C. D. 4. /100 Entry of acetyl-CoA into the citric acid cycle is decreased when: A. 3. TOTAL Under aerobic conditions, pyruvate is oxidatively decarboxylated and enters the citric acid cycle. In yeast growing anaerobically, pyruvate is oxidized to 3phosphoglycerate to use up excess NAD+ generated by the early steps of glycolysis. In anaerobic muscle, pyruvate is converted to lactate. The pyruvate dehydrogenase complex converts pyruvate to acetyl-CoA. Which of the following statements about the oxidation of pyruvate in aerobic conditions in animal cells is correct? A. B. C. D. E. The methyl (-CH3) group is eliminated as CO2. The process occurs in the cytosolic compartment of the cell. One of the products of the reactions of the pyruvate dehydrogenase complex is acetyl-CoA. The pyruvate dehydrogenase complex uses all of these as cofactors: NAD+, lipoic acid, pyridoxal phosphate (PLP), and FAD. The reaction is so important to energy production that pyruvate dehydrogenase operates at full speed under all conditions. Page 2 5. Which of the following statements is not true of the reaction catalyzed by the pyruvate dehydrogenase complex? A. B. C. D. 6. A new compound is isolated from mitochondria and a claim is made that it represents a previously unrecognized carrier in the electron transfer chain, and it is given the name coenzyme Z. Which line of evidence do you feel is the least conclusive in assigning this compound a position in the electron transfer chain? A. B. C. D. 7. E. ATP synthesis will continue, but the P/O ratio will drop to 1. electron transfer from succinate to O2 will continue unabated. all ATP synthesis from the mitochondria will stop. electron transfer from NADH will cease, but O2 uptake will continue. energy diverted from the cytochromes will be used to make ATP, and the P/ O ratio will rise. Which of the following enzymatic activities would you expect to be decreased by thiamin deficiency? A. B. C. D. E. 9. When added to a mitochondrial suspension, coenzyme Z very rapidly and specifically is taken up by the mitochondria. Removal of coenzyme Z from the mitochondria results in a decreased rate of oxygen consumption. Alternate oxidation and reduction of the mitochondrion-bound coenzyme Z can be readily demonstrated. The rate of oxidation and reduction of mitochondrion-bound coenzyme is of the same order of magnitude as the overall rate of electron transfer in mitochondria as measured by oxygen consumption. If electron transfer in tightly coupled mitochondria is blocked (with antimycin A) between cytochrome b and cytochrome c 1, then: A. B. C. D. 8. The reaction occurs in the mitochondrial matrix. Both NAD+ and a flavin nucleotide act as electron carriers. Biotin participates in the decarboxylation. Two different cofactors containing -SH groups participate. pyruvate carboxylase isocitrate dehydrogenase a-ketoglutarate dehydrogenase complex fumarase malate dehydrogenase In 1935, Szent-Gyorgi showed that the production of CO2 in an extract of muscle increased when succinate was added. For every mole of succinate added, many extra moles of CO2 were produced. Explain this effect in terms of the known catabolic pathways. Succinate is an intermediate in a cycle; it is not consumed but is A. regenerated by the operation of the cycle during which CO2 is produced. B. Succinate is an intermediate in the production of oxaloacetate, which generates citrate, which has three moles of carboxylates. C. Succinate is an activator of isocitrate decarboxylase, which generates multiple moles of CO2. D. Succinate is actually an enzyme and thus acts catalytically to generate multiple moles of CO2. Page 3 10. If the mitochondrial electron carriers are artificially fully oxidized and NADH is then added to the system, the last carrier to become reduced is: A. B. C. D. E. 11. In eukaryotic cells, the enzymes of glycolysis are in the __________, and the enzymes of the citric acid cycle are in the _____________. A. B. C. D. E. 12. cytochrome a 3. coenzyme Q. cytochrome b . flavoprotein. ubiquinone. inner mitochondrial membrane; mitochondrial matrix cytosol; mitochondrial matrix mitochondrial matrix; cytosol cytosol; inner mitochondrial membrane inner mitochondrial membrane; cytosol When the F1 portion of the ATP synthase complex is removed from the mitochondrial membrane and studied in solution, it functions as an ATPase. Why does it not function as an ATP synthase? A. B. C. D. E. 13. 14. The free-energy change favors ATP hydrolysis (-30.5 kJ/mol). The free-energy change favors ATP synthesis (-30.5 kJ/mol), There is a proton motive force driving the hydrolysis reaction. The free-energy change favors ATP hydrolysis (30.5 kJ/mol). There is a proton motive force driving the synthesis reaction. How do the P/O ratios for NADH differ in a species where the mitochondrial ATP Synthases contain either 9 and 17 c subunits of the F0 complex? A. The F0 with 9 has a P/O ratio of 2.5 and that with 17 has a P/O ratio of 4.7 B. The F0 with 9 has a P/O ratio of 4.7 and that with 17 has a P/O ratio of 2.5 C. The F0 with 9 has a P/O ratio of 9 and that with 17 has a P/O ratio of 17 D. The F0 with 9 has a P/O ratio of 5.7 and that with 17 has a P/O ratio of 3.0 E. The F0 with 9 has a P/O ratio of 3.0 and that with 17 has a P/O ratio of 5.7 F. The F0 with 9 has a P/O ratio of 2.5 and that with 17 has a P/O ratio of 1.5 G. The F0 with 9 has a P/O ratio of 1.5 and that with 17 has a P/O ratio of 2.5 The citric acid cycle is frequently described as the major pathway of aerobic catabolism, which means that it is an oxygen-dependent degradative process.. However, none of the reactions of the cycle directly involves oxygen as a reactant. Why is the pathway oxygen-dependent? A. Oxygen is needed to keep aconticase reduced. B. Oxygen is needed to keep aconitase oxidized. C. Oxygen is needed to absorb electrons from the reduced cofactors the citric acid cycle produces. D. Oxygen is needed to produce the needed water for the fumarase reaction. E. Oxygen is needed to oxidize the NAD+ and FAD cofactors back to NADH and FADH2, which are normally needed in the citric acid cycle. II. TRUE/FALSE Put a T or F in the box to the left. (8 points) Page 4 15. The condensation to make ATP from ADP + Pi in the active site on ATP Synthase has a DG°' of +7.3 kcal/mole. 16. The mechanism of ATP Synthase is termed the Binding-Change Model. 17. Substitution of Asn for the Asp in the c-subunit of the F0 portion of the ATP synthase would have no effect on the function of the intact ATP synthase. 18. The catalytic step requiring the most energy input in the mechanism of ATP synthesis by the ATP synthase is the condensation reaction. 19. Consider a liver cell carrying out the oxidation of glucose under aerobic conditions. Suppose that we added a very potent and specific inhibitor of the mitochondrial ATP synthase, completely inhibiting this enzyme. Tell whether each of the following statements about the effect of this inhibitor is true or false; if false, explain in a sentence why it is false. (4 pt) (a) ATP production in the cell will quickly drop to zero. (b) The rate of glucose consumption by this cell will decrease sharply. (c) The rate of oxygen consumption will increase. (d) The citric acid cycle will speed up to compensate. III. MATCHING (10 points) 20. Match the attribute, strategy, or intermediate used in enzyme-catalyzed reaction mechanisms listed on the right with an enzyme listed on the left. There is one best answer for each. (10 pts) A. B. C. D. 1. Isocitrate dehydrogenase 2. Citrate synthase 3. Fumarase 4. Aconitase 5. Succinyl-CoA synthetase 6. F. a-ketoglutarate dehydrogenase (E1) 7. cytochrome oxidase 8. E3 of the PDH complex 9. 10. E. enolic intermediate mixed anhydride with phosphate 4Fe-4S cluster for dehydration/rehydration manganese cofactor to polarize bond at beta carbon Acid/base catalysis to generate a carbanion for nucleophic attack after induced fit Schiff-base to polarize bond at beta carbon G. use of TPP carbanion for decarboxylation alpha to a carbonyl H. Tyr radical intermediate Malate dehydrogenase I. disulfide exchange with lipoic acid Acetoacetate decarboxylase J. stereo-specific hydride abstraction IV. SHORT ANSWER & PROBLEMS. provided. (12 points) 21. Write answers as directed in the boxes Page 5 A recently discovered bacterium carries out ATP synthesis coupled to the flow of electrons through a chain of carriers to some electron acceptor. The components of its electron transfer chain differ from those found in mitochondria; they are listed below with their standard reduction potentials. (6 pt) Electron carriers in the newly discovered bacterium: Electrons E o' Oxidant Reductant transferred (V) NADH 2 -0.32 + NAD flavoprotein b 2 -0.62 flavoprotein b (FP-b)(reduced) (oxidized) cytochrome c (Fe3+) cytochrome c (Fe2+)(Cty-c) 1 +0.22 Fe-S protein (oxidized) flavoprotein a (oxidized) Fe-S protein (reduced)(Fe-S) flavoprotein a 2 +0.88 2 +0.77 (reduced) (FPa) (a) Place the electron carriers in the order in which they are most likely to act in carrying electrons. Use abbreviations under reductant noted by underlines. (b) Is it likely that O2 for which E o' = 0.82 V) is the final electron acceptor in this organism? Y or N? Why or why not? (c) How would you calculate the maximum number of ATP molecules that could theoretically be synthesized, under standard conditions, per pair of electrons transferred through this chain of carriers? (Faraday constant, ¡, is about 100 kJ/V•mol or 25 kcal/V•mol) ∆Eo'= – = = ∆Go'= Number of ATP molecules = Page 6 Cyanide ion (CN-) blocks electron transfer in mitochondria at the level of cytochrome a + a3. 2,4-Dinitrophenol (DNP) is a potent uncoupler of mitochondrial oxidative phosphorylation. Add labeled curves to the graphs below to show the effects of adding each of these compounds separately to a suspension of mitochondria supplied with O2 succinate, ADP, and Pi. Arrow indicates time of addition of drug. (You need to draw and label 2 curves on each graph; Use ● for +CN– and use ○ for +DPN) (4 pt) No addition No addition Drug Drug Time 23. O2 consumed ATP synthesized 22. Time The complete oxidation of glucose to CO2 uses water at certain steps and produces water at other steps. How many moles of water are produced and how many are required per mole of glucose? moles of water produced moles of water required Page 7 IV. PATHWAYS ((28 points) Write out the pathway as requested. For each step give the name of the enzyme that catalyzes the reaction (NO abbreviations), any cofactors required (abbreviations OK), the name of each intermediate and their structures (including structures of starting and ending compounds; you can abbreviate phosphate as Pi, and a phosphoryl group by a circled "P"). 24. Draw the pathway from succinate to citrate. (18 pts) structures & names cofactors/ cosubstrates enzyme names Page 8 25. Draw second phase of the Q-cycle by filling in the boxes appropriately. The arrows indicate just the flow of electrons. You do NOT need to show the redox states of each carrier, only the order, the substrates, products, and where they end up as requested in the blank boxes. (10 pts) released in the Intermembrane Space 1 e– 1 e– 1 e– Qp site CoQH2 1 e– + released in the: released in the Inner Mitochondrial membrane 1 e– 1 e– QN site CoQ•– released in the Inner Mitochondrial membrane Page 1 No. on Test Correct Answer 1 2 3 4 5 6 7 8 9 10 11 12 13 14 A C B C C A C C A A B A F C 15 16 17 18 F T F F 19 (a) False. Mitochondrial ATP synthesis will cease, but to compensate, cells will accelerate the production of ATP by glycolysis, preventing ATP levels from dropping to zero. (b) False. The acceleration of glycolysis noted above will actually increase the rate of glucose consumption. (c) False. Because electron transfer through the respiratory chain is tightly coupled to ATP synthesis, blocking ATP synthase blocks electron flow and oxygen consumption. (d) False. The citric acid cycle is an oxidative pathway, producing NADH. When electron flow from NADH to O2 is blocked, NADH accumulates, NAD+ is depleted, and the citric acid cycle slows for lack of an electron acceptor (NAD+). 20 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. D E A C B G H I J F Page 2 No. on Test Correct Answer 21 (a) FPb Æ NAD+ Æ cyt c Æ FPa Æ Fe-S (b) No; Fe-S has a larger E o', so will probably be the terminal acceptor (c) First calculate ∆G °' for e- flow from FPb to Fe-S: ∆E o' = E o'(oxidant) - E o'(reductant) = +0.88 - (-0.62) = +1.5 V ∆G °'¸ = -n ¡∆E o'= (-2)(100 kJ/V•mol)(1.5 V) = -300 kJ/2e∆G °' for ATP synthesis is +30.5 kJ/mol, so theoretically the flow of 2 electrons from FPb to Fe-S could drive the synthesis of (300 kJ)/(30.5 kJ/mol) = 10 mol ATP. No addition No addition Drug Drug +DNP and/or CN - O2 consumed ATP synthesized 22 +CN - Time 23 24 Time moles of water produced: moles of water required: 2 (from enolase) 4 (to fumarase and citrate synthase) 4 (from enolase and aconitase) 6 (to fumarase, CS, and aconitase) +12 (if including ET) 25 15 50 26 +DNP