Biochemistry Study Guide Exam 4
Biochemistry Study Guide Exam 4 BC 351
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This 31 page Study Guide was uploaded by Grace Gerhards on Monday August 1, 2016. The Study Guide belongs to BC 351 at Colorado State University taught by Paul Laybourn PH.D. in Summer 2016. Since its upload, it has received 33 views. For similar materials see Biochemistry in Biochemistry & Molecular Biology at Colorado State University.
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Biochemistry Study Guide Exam 4 Module 13: Metabolic Regulation Study questions: 1. Briefly describe the intrinsic and system properties that give hydrolysis of ATP to ADP and Pi a very large negative change in free energy. 2. Why is the actual free energy (G) of hydrolysis of ATP in the cell different from the standard free energy (G'°)? 3. Explain the difference between homeostasis and equilibrium. 4. What are the regulatory implications for the cell with regard to ATP and AMP, given that the former are generally high, and the latter are low? 5. Describe four major principles of metabolic regulation that have selectively evolved throughout evolution. 6. Compare the amount of ATP and NADH yield in the pathway of glycolysis to the amounts of ATP and NADH required to convert two pyruvates to glucose in gluconeogenesis. Explain the significance of the difference between the two sets of numbers. 7. What are the three key regulatory steps in glycolysis/gluconeogenesis? Indicate the substrates and products, the enzymes catalyzing each step, and how these enzymes are regulated. 8. In the glycolytic path from glucose to phosphoenolpyruvate, two steps are practically irreversible. What are these steps, and how is each bypassed in gluconeogenesis? What advantages does an organism gain from having separate pathways for anabolic and catabolic metabolism? What are the disadvantages? 9. Why is citrate, in addition to being a metabolic intermediate in aerobic oxidation of fuels, an important control molecule for a variety of enzymes? 10. Diagram and describe the function of fructose 2,6 bis phosphate in overriding the control of glycolysis/gluconeogenesis in liver cells in response to lowered blood glucose levels. 11. Diagram or describe how the TCA cycle is catalytic, is the hub of metabolism, and is amphibolic. What are the anaplerotic reactions of the TCA cycle and what are their functions? 12. There are few, if any, humans with defects in the enzymes of the citric acid cycle. Explain this observation in terms of the role of the citric acid cycle. 13. The citric acid cycle begins with the condensation of acetyl-CoA with oxaloacetate. Describe three possible sources for the acetyl-CoA. 14. Suppose you found an overly high level of pyruvate in a patient’s blood and urine. One possible cause is a genetic defect in the enzyme pyruvate dehydrogenase, but another plausible cause is a specific vitamin deficiency. Explain what vitamin might be deficient in the diet, and why that would account for high levels of pyruvate to be excreted in the urine. How would you determine which explanation is correct? Page 1 of 2 BC 351: Principles of Biochemistry 15. Write the three reactions in the citric acid cycle in which NADH is produced. None of these reactions involves molecular oxygen (O 2), but all three reactions are strongly inhibited by anaerobic conditions; explain why. 16. In which reaction of the citric acid cycle does substrate-level phosphorylation occur? 17. Explain in quantitative terms the circumstances under which the following reaction can proceed. L-Malate + NAD oxaloacetate + NADH + H G'° = + +29.7 kJ/mol 18. Preparation of an extract of muscle results in a dramatic decrease in the concentration of citric acid cycle intermediates compared to their concentrations in the tissue. However, in 1935, Szent-Gyorgi showed that the production of CO 2by the extract increased when succinate was added. In fact, for every mole of succinate added, many extra moles of CO 2were produced. Explain this effect in terms of the known catabolic pathways. 19. Germinating plant seeds can convert stored fatty acids into oxaloacetate and a variety of carbohydrates. Animals cannot synthesize significant quantities of oxaloacetate or glucose from fatty acids. What accounts for this difference? 20. You are in charge of genetically engineering a new bacterium that will derive all of its ATP from sunlight by photosynthesis. Will you put the enzymes of the citric acid cycle in this organism? Briefly explain why or why not. Answers: 1. The intrinsic property of ATP hydrolysis is the biochemical standard free energy change that is quite negative primarily due to the neutral pH in cells. The system property is the [ATP]/[ADP] ratio in cells being at least 10/1, causing K = to be at lease 0.1. The two properties combine to give ATP hydrolysis a very negative change in free energy in cells. This negative free energy change can then be coupled to unfavorable reactions in the cell, like biosynthesis, to make the overall reaction still have a negative free energy change. The other two “energy currency” molecules in the cell are NADH and Acetyl Co Enzyme A. 2. The concentrations of the reactant (ATP) and the products (ADP and P ) are not all equal to 1 M. The pH is not exactly equal to 7.0 (the standard pH) and the temperature is usually higher than the standard temperature of 25 °C. 3. Homeostasis is constant conditions, typically far from equilibrium – such that there is significant net reaction in the desire direction. At equilibrium, there is no net reaction; for a cell, this would be lethal. 4. Normally, [ATP] is 5-10 mM, while [AMP] is < 0.1 mM, thus AMP is a much more sensitive indicator of a cell’s energetic state. Small changes in ATP concentration are amplified into large changes in AMP concentration (see Table 15-1), hence many regulatory processes hinge on changes in the concentration of AMP. 5. a. Maximize the efficiency of fuel utilization by preventing the simultaneous operation of opposing pathways (i.e., futile cycles). b. Partition metabolites appropriately between alternative pathways.c. Draw on the fuel best suited for the immediate needs of the organism. d. Shut down biosynthetic pathways when their products accumulate. 6. The conversion of glucose to pyruvate via glycolysis yields 2 ATP (net) and 2 NADH. In contrast, the conversion of 2 pyruvates to glucose via gluconeogenesis requires the input of 6 ATP (4 ATP & 2 GTP) and 2 NADH. The 6 ATP are required to overcome the positive G rxn(negative G rxin glycolysis and therefore positive Grxn in gluconeogenesis) associated with reversible steps (steps 6 & 7) of glycolysis/gluconeogenesis and to drive the two bypass steps converting pyruvate back to phosphoenol pyruvate (via oxaloacetate, see PowerPoint slides). In addition, phosphate rather than ATP is produced in steps 1 and 3 (the “investment” step of glycolysis) of gluconeogenesis. The significance, of course, is that running both glycolysis and gluconeogenesis simultaneously would result in the net use of ATP forming a “futile cycle”. 7. The three key regulatory steps in glycolysis/gluconeogenesis are steps 1, 3, and 10 (see PowerPoint slides). Substrates/products are for step 1 – glucose and glucose 6-P, for step 3 – fructose 6-P and fructose 1,6 bisP, and for step 10 – phosphoenol pyruvate and pyruvate (via Page 1 of 4 BC 351: Principles of Biochemistry oxaloacetate in gluconeogenesis). The enzymes for each step and their regulation are for step 1 - hexokinase/glucokinase and glucose 6 phosphatase, step 3 – phosphofructokinase-1 and fructose 1,6 bisphosphatase, and step 10 - pyruvate kinase and puruvate carboxylase (phosphoenol pyruvate carboxykinase is not highly regulated). In general, glycolysis is upregulated by a lower [ATP]/[ADP] ratio (energy charge) and down regulated by a higher ratio while gluconeogenesis is regulated inversely. More specifically, hexokinases and glucokinase are repressed by glucose 6P and fructose 6P, while glucose 6 phosphatase is activated. Phosphofructokinase-1 is repressed by ATP and activated by AMP, while fructose 1,6 bis phosphatase is represses by AMP. Pyruvate kinase is inhibited by ATP and acetyl CoA and activated by fructose 1,6 bisP, while pyruvate carboxylase is activated by acetyl CoA. 8. The two irreversible steps in glycolysis are conversion of glucose to glucose 6-phosphate, catalyzed by hexokinase, and conversion of fructose 6-phosphate to fructose 1,6- bisphosphate, catalyzed by phosphofructokinase-1 (Table 15-3, p. 593 or 575). The first reaction is bypassed during gluconeogenesis by the reaction catalyzed by glucose 6- phosphatase, an enzyme unique to the liver. The second is bypassed by fructose 1,6- bisphosphatase-1 (FBPase-1). By having separate pathways that employ different enzymes, an organism is able to control anabolic and catabolic processes separately, thus avoiding futile cycles. A potential disadvantage is the need to produce separate sets of enzymes for catabolism and anabolism. 9. As the key biochemical intermediate in the citric acid cycle resulting from the condensation of oxaloacetate and acetyl-CoA, citrate is at a junction of amino acid, fatty acid, and pyruvate oxidation, serving as an intracellular signal that the cell’s current energy needs are being met. In particular, it is an allosteric regulator of PFK-1, increasing the inhibitory effect of ATP, and further reducing the flow of glucose through glycolysis. 10. Fructose 2,6 bis P(F2,6bisP) overrides glycolysis/gluconeogenesis regulation by cellularenergy charge in livercells by throughregulation of step 3 enzymesphosphofructokinase-1and fructose 1,6 bisphosphatase (see PowerPoint slides). F 2,6bisP activatesglycolysis (phosphofructokinase-1) and inhibits gluconeogenesis (fructose1,6 bisphosphatase). The level of F2,6bisP is determined by the bifunctional enzyme phosphofructokinase-2/fructose 2,6 bis phosphase (PFK- 2/F2,6BPtse). When blood glucose is low pancreatic islet cells produce glucagon. Glucagon binds its receptor on liver cell membranes signaling through G protein complexes for increased cAMP production by adenylyl cyclase. PKA is bound and activated by cAMP, resulting in PFK-2/F2,6BPtse phosphorylation and greater phosphatase over kinase Page 2 of 4 BC 351: Principles of Biochemistry activity. This in turn, decreases the F2,6bisP levels favoring gluconeogenesis over glycolysis. The glucose produced is released into the blood restoring normal blood glucose levels. 11. The anaplerotic reactions function to build up the level of TCA cycle intermediate, for example glutamate to alpha-ketoglutarate. Any TCA cycle intermediate can be interconverted to any other. Building up the levels of TCA cycle intermediates allows more acetyl CoA oxidation and energy currency production. 12. The citric acid cycle is central to all aerobic energy- yielding metabolisms and also plays a critical role in biosynthetic reactions by providing precursors. Mutations in the enzymes of the citric acid cycle are likely to be lethal during fetal development. 13. Acetyl-CoA is produced by (1) the pyruvate dehydrogenase complex, (2) oxidation of fatty acids, or (3) degradation of certain amino acids. 14. The most likely explanation is that the patient has a deficiency of thiamine, without which the cell cannot make thiamine pyrophosphate, the cofactor for pyruvate dehydrogenase. The inability to oxidize pyruvate produced by glycolysis to acetyl-CoA would lead to accumulation of pyruvate in blood and urine. The most direct test for this deficiency is to feed a diet supplemented with thiamine and determine whether urinary pyruvate levels fall. 15. NADH is produced in the reactions catalyzed by isocitrate dehydrogenase, the - ketoglutarate dehydrogenase complex, and malate dehydrogenase. These reactions are indirectly dependent on the presence of O 2because the NADH produced in the reactions is normally recycled to NAD by passage of electrons from NADH through the respiratory chain to O 2. See also Fig. 16-7, p. 621. 16. Substrate-level phosphorylation of GDP to GTP occurs in the succinyl-CoA synthetase reaction in which succinyl-CoA is converted to succinate during the citric acid cycle. (See p. 626.) 17. A reaction for which G'° is positive can proceed under conditions in which the actual G is negative. From the relationship: G = G'° + RT ln [product], [reactant] it is clear that if the concentration of product is kept very low (e.g., by its removal in a subsequent metabolic step), the logarithmic term becomes negative and the actual G can then have a negative value. (See also Chapter 13.) Page 3 of 4 BC 351: Principles of Biochemistry 18. Succinate is an intermediate in the citric acid cycle that is not consumed but is regenerated by the operation of the cycle. By adding succinate to an extract that is depleted in citric acid cycle intermediates, these intermediates are replenished and the cycle can resume operating, oxidizing acetyl-CoA to CO 2. 19. Plants use the glyoxylate cycle to convert two molecules of acetyl-CoA into one four-carbon compound (such as oxaloacetate), then use this compound to make glucose (gluconeogenesis). In animals that lack the glyoxylate cycle, each acetyl group that enters the citric acid cycle yields two CO , allowing no net conversion of acetyl groups into oxaloacetate. 20. Yes; even though the citric acid cycle is not needed for catabolic reactions in this organism, the enzymes of the cycle are still essential. They produce precursors of amino acids (such as - keto-glutarate and oxaloacetate), of heme (succinyl-CoA), and of a variety of other essential products. Module 13 sample questions: The hydrolysis of ATP has a large negativeG'°; nevertheless it is stable in solution due to The hydrolysis reaction having a large activation A reaction at equilibrium in a metabolic pathway is A rapid and reversible step Regarding ATP: The cycling between ATP and ADP+Pi provides an energy coupling between catabolic and anabolic pathways All of the following enzymes involved in the flow of carbon from glucose to lactate (glycolysis) are also involved in the reversal of this flow (gluconeogenesis) 3-phosphoglycerate kinase aldolase enolase phosphoglucoisomerase Gluconeogenesis must use “bypass reactions” to circumvent three reactions in the glycolytic pathway that are highly exergonic and essentially irreversible. Reactions carried out by which three of the enzymes listed must be bypassed in the gluconeogenic pathway? Hexokinase, phosphofructokinase-1, pyruvate kinase Which of the following statements about gluconeogenesis in animal cells is true? The conversion of fructose 1,6 biphosphate to fructose 6- phosphate is not catalyzed by phosphofructokinase-1, the enzyme involved in glycolysis The regulated steps in glycolysis and gluconeogenesis Are paired, but involve different enzymes Phosphofructokinase-2 and fructose 2,6 bisphosphatase Are regulated in their relative activities through phosphorylation by PKA Allosteric enzymes Usually have more than one polypeptide chain Which of the following statements about allosteric control of enzymatic activity is false? Heterotropic allosteric effectors compete with substrate for binding sites Which of the following is not true of the reaction catalyzed by the pyruvate dehydrogenase complex? Module 14: Metabolism Module 14 Study questions: 1. Using an energy diagram (free energy or G vs. reaction plot), (A) describe electron flow from NADH and FADH 2to O 2in the electron transport chain. (B) In this reaction plot indicate the two intermediate carriers and the large complexes that transfer the electrons between the carriers and indicate the complexes and intermediate carrier molecules that function in proton translocation across the mitochondrial inner membrane. 2. Diagram the mitochondrial structure and indicate where the electron transport chain and ATP synthase are located in this structure. 3. Why does FADH 2generate fewer ATP than NADH by way of the electron transport chain and oxidative phosphorylation? 4. What are the four main chemical components that form the business ends of four ETC complexes? How many electrons and protons can each of them carry? 5. Describe or diagram the flow of electrons from NADH to O2and the three sites of proton pumping in the context of structure of the electron transport chain complexes in inner mitochondrial membrane. Include also the nonpolar electron and proton carrier molecule and the peripheral membrane protein electron carrier. Indicate the main product produced by coupling to the electron flow and the three sites of production. Finally, include the coenzyme Q cycle in your description. 6. Diagram the path of electron flow from NADH to the final electron acceptor during electron transport in mitochondria. For each electron carrier, indicate whether only electrons, or both electrons and protons, are accepted/donated by that carrier. Also, indicate where electrons from succinate oxidation enter the chain of carriers. 7. 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. Electron carriers in the newly discovered bacterium: ———————————————————————————— ————————— Electrons E'° Oxidant Reductant transferred (V) ———————————————————————————— ————————— NAD flavoprotein b (FPb) (oxidized) NADH 2 –0.32 flavoprotein b 2 –0.62 (reduced) cytochrome c (Fe ) Fe-S protein (oxidized) flavoprotein a (FPa) cytochrome c (Fe )1 Fe-S protein 2 (reduced) flavoprotein a 2 (reduced) +0.22 +0.89 +0.77 (oxidized) ———————————————————————————— ————————— (a) Place the electron carriers in the order in which they are most likely to act in carrying electrons. (b) Is it likely that O2(for which E'° = 0.82 V) is the final electron acceptor in this organism? 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 transfered through this chain of carriers? (The Faraday constant, , is 96.48 kJ/V·mol.) G'° for ATP synthesis is +30.5 kJ/mol. 8. Although molecular oxygen (O ) does not participate directly in any of the reactions of the citric acid cycle, the cycle operates only when O2is present. Explain this observation. 9. What are the two main functions of the “bimetallic center” in complex IV of the ETC? 10. 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. Indicate whether each of the following statements about the effect of this inhibitor is true or false; if false, explain in a sentence or two why it is false. ____ (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. ____ (e) The cell will switch to fatty acid oxidation as an alternative to glucose oxidation, and the inhibitor will therefore have no effect on ATP production. 11. Describe, in simple diagrams and a few words, the chemiosmotic theory for coupling oxidation to phosphorylation in mitochondria. Study Questions Answers: 1. A. &B. 2. 3. FADH 2is a part of complex II (succinate dehydrogenase) and the G between FADH 2 and CoQ is insufficient to drive the transport of protons out of the matrix. This, as diagrammed above, there are only two sites of proton pumping for electrons coming in from FADH 2as compared to three for electrons from NADH through complex I. 4. The four main chemical components are FAD/FMN, FeS complexes, cytochromes and Cu complexes. The flavin in FAD and FMN can carry 1 or 2 electrons and protons. The iron (Fe3+) in iron-sulfur (FeS) complexes and in the cytochromes can only carry 1 electron and no protons. The same is true of the Copper (Cu2+) complexes (can carry only 1 electron no protons). The other chemical components important to electron transport chain function are NAD+ (carries only 2 electrons and one proton) and Coenzyme Q (carries 1 or 2 electron and proton). 5. Electrons flow from NADH to O , forming H 2O (see figure on next page). They enter the ETC at Complex I or (NADH dehydrogenase and are passed to Coenzyme Q (ubiquinone, non polar electron carrier), which picks up two protons and transports the electrons to Complex III (cytochrome c reductase). Complex III passes the Page 1 of 3 BC 351: Principles of Biochemistry electrons to cytochrome c (peripheral membrane electron carrier), which carries the electrons to Complex IV (cytochrome c oxidase), the site of O 2reduction. Electrons also enter the ETC from FADH 2in Complex II, which also feeds its electrons to Coenzyme Q. The three sites of proton pumping out of the mitochondrial matrix are Complex I, III and IV. The proton electrochemical gradient is the main product produced by coupling the flow of electrons from a higher G (lower redox potential) of NADH to a lower G (higher redox potential) of H 2O (reduced O ). The Coenzyme Q cycle occurs between two CoQ binding sites on Complex III (see figure to the right). Electrons fed in on one site are first recycled to CoQ molecules in the CoQ pool at a second binding site, reducing them to CoQH . These reduced CoQH 2bind the first site, but the now lower energy electrons are passed through Complex III onto cytochrome c. 6. NADH (both)FP (both)Q (both)cyt b (e– only)cyt c 1 (e– only)cyt c (e– only) cyt (a + a3) (e– only)O2(both) Electrons from succinate enter at Q. 7.(a)FPbNAD + cytc FPaFe-S(b) No; Fe-S has a larger E'°, so will probably be the terminal acceptor. (c) First, calculate G'° for e flow from FP to Fe-S: b E'° = E'°(oxidant) – E'°(reductant) = +0.89 – (–0.62) = +1.51 V G'° = –nE'° = (–2)(96.48 kJ/V·mol)(1.51 V) = – –291 kJ/2e Theoretically, the flow of two electrons from FP bto Fe-S could drive the synthesis of 291 kJ/30.5 kJ/mol = 9.5 mol ATP. Because only whole numbers of molecules can be made, the correct answer is 9 mol ATP per electron pair. 8. The citric acid cycle produces NADH, which is normally reoxidized to NAD + by the passage of electrons through the respiratory chain to O 2. With no O2to accept electrons, NADH + accumulates, NAD is de+leted, and the citric acid cycle slows for lack of NAD . 9. The bimetallic center of Complex IV functions:a. to sequentially feed four electrons to an O 2 molecule to fully reduce it to H 2O, whileb. holding onto O 2 and the highly reactive oxygen intermediate species tightly until all four electrons have been fed in. Of course, they accept electrons from the upstream cytochrome a and Cu A complexes to be regenerated. 10. (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 fr+m NADH to O 2is blocked, NADH accumulates, NAD is depleted, and the citric acid + cycle slows for lack of an electron acceptor (NAD ).(e) False. Oxidation of fats produces NADH, FADH 2, and acetyl-CoA, which is further oxidized via the citric acid cycle. For the reasons noted above, blocking electron flow through the respiratory chain prevents ATP synthesis with energy from fatty acid oxidation. 11. There are two central elements in the chemiosmotic model:(1) Energetically favorable electron flow through asymmetrically arranged membrane- bound carriers causes transmembrane flow of H , creating a proton gradient (a proton motive force).(2) The energy released by“downhill” movement ofprotons is captured when ADP and P iare condensed by ATPsynthase (F oF1). (See Fig. 19- 19.) Module 14 Sample Questions In mammals, each of the following occurs during the citric acid cycle except: Net synthesis of oxaloacetate Conversion of 1 mol of acetyl-CoA to 2 mol of CO2 and CoA via the citric acid cycle results in the net production of: 1 mole of FADH2 which of the following electron carriers is not able to transfer one electron at a time? NADH In the reoxidation of QH2 by purified ubiquinone- cytochrome c reductase (Complex III) from heart muscle, the overall stoichiometry of the reaction requires 2 mol of cytochrome c per mole of QH2 because Cytochrome c is a one- electron acceptor, whereas QH2 is a two- electron donor When a glucose molecule is broken down to CO2 in glycolysis and the TCA cycle, little ATP is directly produced, most of the energy is Stored as NADH Almost all of the oxygen (O2) one consumes in breathing is converted to: Water Antimycin A blocks electron transfer between cytochromes b and c . If 1 intact mitochondria were incubated with antimycin A, excess NADH, and an adequate supply of O , w2ich of the following would be found in the oxidized state? Cytochrome a3 Reduced QH2 is NOT formed by which of the following? Complex III and Cytochrome c In the reoxidation of QH b2 purified ubiquinone-cytochrome c reductase (Complex III) from heart muscle, the overall stoichiometry of the reaction requires 2 mol of cytochrome c per mole of QH becau2e: Cytochrome c is a one-electron acceptor, whereas QH2 is a two- electron donor What is are the features of complex IV? Cytochrome c is a one-electron donor Oxygen is a substrate Copper is an essential metal for the reaction For every electron passed through coplex IV, two protons are consumed form the matrix (N) side Module 15: The Electron Transport Chain Study Guide Questions: 1. Describe two experiments using mitochondrial inner membrane (MIM) vesicles out of the three described in class that provide evidence that electron flow through the electron transport chain is coupled directly to the synthesis of ATP from ADP and Pi by ATP synthase in oxidative phosphophorylation by showing (A) that a H gradient is sufficient and (B) that running of the ETC and the function of the ATP synthase are directly coupled. 2. Describe or diagram an experiment using MIM vesicles that indicates the function of the F 0and F 1components of the ATP synthase and describe their functions (a structurally detailed description of the mechanism is not necessary). 3. Diagram or describe the effect of an uncoupler on the ETC and ATP synthesis. What is the remaining product of the ETC in the presence of an uncoupler? (hint: brown adipose tissue) 4. When the F 1portion of the ATP synthetase complex is removed from the mitochondrial membrane and studied in solution, it functions as an ATPase. Why does it not function as an ATP synthetase? 5. The F F ATP synthase can be used to catalyze the reverse of its normal physiological reaction. Explain under what conditions the F oF1ATP synthase could be used to create a proton gradient. 6. Describe the general structure and function of ATP synthase including the location of the five F 1subunits and the three F 0 subunits and how they participate in ATP synthesis using the energy of protons flowing down the electrochemical gradient into the mitochondrial matrix. 7. When the G'° of the ATP synthesis reaction is measured on the surface of the ATP synthase enzyme, it was found to be close to zero. Describe briefly why this is so. 8. Explain briefly the current model for how the proton motive force that is generated by electron transport is used to drive the ATP synthesis reaction. Answers: 4. Like all enzymes, the F1subunit of the ATP synthase catalyzes a reaction in both directions: ADP+P iATP+H 2O The standard free-energy change (G'°) for ATP hydrolysis is –30.5 kJ/mol. With no proton motive force to drive the reaction toward ATP synthesis, the hydrolysis (ATPase activity) occurs spontaneously. 5. If no proton gradient or membrane potential existed across the mitochondrial inner membrane, then ATP in the matrix could be used to pump protons out of the matrix space. For example, if there were no NADH or O 2 to run the ETC. Some bacteria do use ATP synthase to maintain a proton gradient across their cytoplasmic membrane under anaerobic conditions at the expense of ATP generated through glycolysis. Bacteria use a proton gradient in secondary active transport of nutrients, etc. into the cell. 6. For the structure of the F 0F 1ATPsynthase complex see the figure to theright. The three subunits of F 0are a, b, and c and form an integral membraneprotein complex. The five subunits of F are , , , , and form a peripheralmembrane protein complex. Ten tofourteen copies of subunit c form a“wheel” that binds protons and turns.Subunit a forms a pair of incompleteproton channels that carry protons to andfrom the c subunits producing a path forprotons through the membrane that must include binding a c subunit on one side and traveling around with the c wheel as it turns to be able to escape on the other side. The subunit forms a crank or camshaft that is fixed into the c wheel by , such that when the c wheel turns turns. Three copies each of and form a complex over the other end of that is the knob seen in electron micrographs. In addition, has ATPase activity. Subunits b and form a “stator” or arm that binds the 3 3head to the a subunit proton channel keeping the 3 3head from turning with . Protons flowing in the intermembrane face side partial ion channel of subunit a bind a c subunit, the c wheel turns bringing this c subunit binding site to the matrix side partial ion channel of the a subunit. Protons flowing down the electrochemical gradient drive the turning of the c wheel. The turning of the c wheel turns in the 3 3head. The turning of causes conformation changes in the three subunits. There are three subunit conformations, the first binds ADP and Pi loosely, the second binds ADP and Pi tightly, and the third binds ATP tightly. The third conformation catalyzes ATP synthesis. The subunit then cycles back to the first conformation releasing ATP and allowing the loose binding of new ADP and Pi. 7. The enzyme binds ATP more tightly than ADP thus stabilizing the former (i.e., the product of the synthesis reaction) relative to the latter (i.e., the reactant in the synthesis reaction). 8. The enzyme binds ATP more tightly than ADP thus stabilizing the former, making the G'° of the synthetic reaction more favorable. Once the reaction has occurred, the ATP product must be released from the enzyme (which is unfavorable). The proton motive force causes protons to move across the inner mitochondrial membrane through the pore in the F o complex. This movement leads to conformational changes that decrease the affinity of the F 1portion of the synthase for ATP, resulting in its release from the enzyme. Module 15 Sample questions: 1. when a gluecose molecule is broken down to CO2 in glycolysis and the TCA cycle, little ATP is directly produced most of the energy is: a. Captured as NADH 2. In normal mitochondira, the rate of NADH consumption (oxidation) will: a. Be increased in active muscle, decreased in inactive muscle b. Be very low if the ATP synthase is inhibited byt increase when an uncoupler is added c. Decrease if mitochondrial ATP is depleted d. Decrease when cyanide is used to prevent electron transfer through the cytochrome a +a3 complex 3. Which of the following statements about the chemiosmotic theory is correct? a. Electron transfer in mitochondria is accompanied by an asymmetric release of protons on one side of the inner mitochondrial membrane 4. During oxidative phosphorylation, the proton motive force that is generated by electron transport is used to: a. Induce a conformational change in the ATP synthesis 5. Cyanide, oligomycin and 2,4-dintriophenol (DNP) are inhibitors of mitochondrial aerobic phosphorylation. What is true about describing the mode of action of the three inhibitors a. Cyanide inhibits the respiratory chain whereas, oligomycin and 2,4=dinitrophenol inhibit the synthesis of ATP 6. 2,4-dinitrophenol and oligomycin inhibit mitochondrial oxidative phosphorylation. 2,4- dintirophenol is an uncoupling agent: oligomycin blocks the ATP synthesis reaction itself. Therefore 2,4 dintrophenol wil: a. allow electron transfer in the presence of oligomycin 7. ATP synthase is often described as a. A molecular motor
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