Unit 12 1 UNIT 12 PART A: THE PENTOSE PHOSPHATE PATHWAY PART B: OXIDATIVE PHOSPHORYLATION PART A: THE PENTOSE PHOSPHATE PATHWAY The function of the pentose phosphate pathway is to generate pentose sugars and reducing power in the form of NADPH for use in biosynthetic pathways (for example, faty acid synthesis). The metabolic state of the cel determines the relative amounts of NADPH and pentose sugars that are generated. The pathway is also used to convert pentose sugars to glucose which can then be metabolized for energy. Asignment: Nelson & Cox, pp. 558 - 563 (stop at section entitled (Wernicke- Korsakoff Syndrome.."). Refer to the diagram that follows in addresing the following objectives. You are not required to memorize names or structures of intermediates. 1. Notice that the branch on the left is reversible and nonoxidative. It involves exchanges of 2- and 3-carbon fragments among intermediates. a. Considering the fact that no CO 2 is lost, how many moles of ribulose 5-P should be produced from 5 moles of glucose 6-P by these reactions? b. What types of enzymes catalyze the nonoxidative reactions (p. 560)? Unit 12 2 2. Notice that the branch on the right, labeled the oxidative or phosphogluconate branch, involves oxidation-reduction reactions and the loss of CO 2 . How many moles of ribulose 5-P are produced from 5 moles of glucose 6-P by these reactions? 3. Which pathway wil be favored under the following conditions. Explain your answers. a. A condition in which more ribose 5-P is needed (as for nucleic acid synthesis) than NADPH. b. A condition in which more NADPH is needed (as for faty acid synthesis) than ribose 5-phosphate. c. Which pathway (oxidative or nonoxidative) enables ribose 5- phosphate to be metabolized for energy (i.e . for ATP)? Unit 12 3 OH HC O CH OP 2 glucose 6-P CH OP 2 O C O 6-phosphogluconolactone CH OP 2 O C O 6-phosphogluconate - CH OP 2 ribulose 5-P CH OH 2 O NADP + NADPH H O 2 NADP + NADPH CO 2 O x i d a t i v e ( p h o s p h o g l u c o n a t e ) B r a n c h N o n o x i d a t i v e B r a n c h ( r e v e r s i b l e ) ribose 5-P Unit 12 4 PART B: OXIDATIVE PHOSPHORYLATION Glycolysis and the citric acid cycle catalyze the oxidation of carbohydrates and fats to CO 2, but the resulting electrons are left on the coenzymes NADH and FADH 2. Oxidative phosphorylation is the proces by which the energy of these electrons is used to make ATP. The electrons pas down a series of redox coenzymes and prosthetic groups or "cariers", caled the "respiratory chain", to the final aceptor, oxygen. The proces by which this pasage of electrons is coupled to ATP synthesis has been shown to involve the transport of protons across the mitochondrial inner membrane to form an electrochemical proton gradient which is the "high energy intermediate" of the energy coupling. Elucidation of this "chemiosmotic" coupling was a particularly dramatic chapter in the history of biochemistry because it involved such radicaly new concepts. Your first goal should be to understand the basic principles of electron transfer, proton gradients, and ATP synthesis before you tackle the details of each redox carier, the stoichiometries of proton transport reactions, and the mechanism of ATP synthesis Asignment: Nelson & Cox, pp. 707 ? 742. 1. The mitochondrion is the eukaryotic organele where the TCA cycle and oxidative phosphorylation take place. a. Study the diagram and description of a mitochondrion in Fig. 19-1 (p. 708), making special note of the presence of cristae, diferences betwen the inner and outer membranes, the matrix, and the intermembrane space (not labeled on the diagram). Note that mitochondria are found in almost al eukaryotic cels (animal and plant), but not in prokaryotic cels. b. Indicate the locations of the electron cariers of the respiratory chain and the reactions of the TCA cycle on the diagram of the mitochondrion (Fig. 19-1, p. 708). Unit 12 5 2. The prosthetic groups cary electrons (hemes, Cu + , and FeS) or hydrogen atoms (Q, FMN, FAD, NAD). The ones that cary only electrons are caled electron cariers. Those that cary protons as wel as electrons are caled hydrogen cariers. The half reduced forms of hydrogen cariers, caled semiquinones, alow the hydrogen cariers to be reduced or oxidized one electron at a time. The basic respiratory chain as it was viewed in the 1950?s is shown in Fig. 19-6 (p. 712). Follow the logic of the use of inhibitors to determine the order of the components. You should be familiar with the following inhibitors of oxidative phosphorylation from Table 19-4 (p. 714): Cyanide, Antimycin A, Rotenone, Oligomycin, and DNP. Note: Rotenone inhibits electron flow from complex I to ubiquinone as shown in Fig. 19-6 but has no afect when electrons enter the respiratory chain via succinate. 3. Electron transfer complexes Treatment of mitochondria with detergents alows purification of four complexes, sumarized in Table 19-3 (p. 713). Name the four dehydrogenases that gather electrons from various substrates (Fig. 19-8, p. 713). Note that the thre FAD-linked dehydrogenases are at a more positive E?° and lack the energy available from NADH. Also note that there is some evidence that complexes may asociate in the membrane (termed respirasome; p. 718), but it is not established. X-ray structures of the bc 1 complex and cytochrome oxidase have been done and it is clear that the prosthetic groups are held in globular domains for cyt c, c 1 , and FeS, or sandwiched betwen transmembrane alpha helices for cytochromes b, a, and a 3 (Figs. 19-11, p. 716, and 19-13, p. 717). Using Fig. 19-16 (p. 719), discuss electron flow from NADH and succinate to oxygen. What is diferent about cytochrome c and coenzyme Q, compared with the other cariers of the chain? The structure of the bc 1 complex shows that there is not a simple linear chain of electron flow but instead a loop caled the Q-cycle (p. 716) which functions to transport protons across the membrane. Follow the path of electrons and protons in the Q-cycle (Fig. 19-12, p. 717). 4. The Chemiosmotic Model Unit 12 6 a. Name the two components of the proton-motive force (Fig. 19-17, p. 720)? b. In the respiratory chain, which complexes contribute to the proton- motive force (Fig. 19-19, p. 723)? c. Discuss the evidence shown in Fig. 19-20 (p. 724) that demonstrates that electron transport is coupled to ATP synthesis. d. What is an uncoupler. How do they act (Figs. 19-20 and 19-21, p. 724)? Note: Uncouplers must be permeant in both the protonated and ionized forms, which is unusual. The DNP anion is permeant because the negative charge is delocalized by resonance structures. e. The structure of ATP synthase is very complex (e.g. Fig. 19-25f, p. 728). Note that Efraim Racker was a former Cornel Profesor (p. 725). To understand how ATP synthase works, consider the following questions (pp. 725 - 731): 1) Where do the protons go (se Fig. 19-25f, p. 728)? 2) How is the energy transfered from F o to F 1 ? Use Fig. 19-27 (p. 730) to describe the experimental evidence for this. 3) What is the "binding-change" mechanism for ATP synthesis on F 1 (Fig. 19-26, p. 729)? 4) One proton is used to drive the uptake by mitochondria of ADP and Pi in exchange for ATP (Fig. 19-28, p. 730). The proton crosses the membrane chemicaly with Pi and electricaly with ADP. What do we mean by that? Unit 12 7 5. Chemiosmotic Coupling a. Why is the P/O ratio of ATP synthesis from NADH and from succinate to oxygen 2.5 and 1.5 respectively (pp. 729 - 730)? Discuss these values in terms of the proton numbers transported by the various components. Note: On page 728 it is mentioned that yeast mitochondrial F o has ten F c subunits in a ring. Thus for each complete rotation of the c ring and the gama subunit, 10 protons enter F o and 3 ATPs are made by F 1 giving a H + /ATP ratio of 10/3 instead of the value of 9/3 based on the mechanism in Fig. 19-26 (p. 729). This would result in a P/O ratio that was slightly lower. Since the H + /ATP ratio of 10/3 is speculative, when calculating P/O ratios in this class, unles otherwise indicated, assume the ratio of 9H + /3 ATP. b. What is the yield of ATP per glucose (Table 19-5, p. 733)? c. Name the two main pathways by which mitochondria can oxidize external NADH (Figs. 19-29 and 19-30, pp. 731 - 732; you are not responsible for the details of these shuttle systems)? 6. Regulation of oxidative phosphorylation (pp. 732 - 735) Compared to other pathways ox phos stands out for its lack of regulation. As long as mitochondria have substrates available they are always ready to make ATP when ADP and Pi are added. 7. Uncoupling protein (Fig. 19-34, p. 736) is a hot topic because it is involved in heat generation and control of body weight. How does it work? 8. Bacteria cary out ox phos and have a variety of respiratory chains depending on the species and growth conditions. They also use the proton- motive force for some unusual functions such as rotation of their flagela (Fig. 19-39, p. 739). Note that the motor that drives flagelar rotation is completely diferent from ATP synthase structuraly - even though the rotation of both motors is driven by a proton-motive force. Jim Blankenship Microsoft Word - U12_F08.doc
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