Paper : 04 Metabolism of carbohydrates Module : 15 Principal Investigator Paper Coordinator Content Reviewer Content Writer Dr.S.K.Khare,Professor IIT Delhi. Dr. Ramesh Kothari,Professor UGC-CAS Department of Biosciences Saurashtra University, Rajkot-5 Gujarat-INDIA Dr. S. P. SinghProfessor UGC-CAS Department of Biosciences Saurashtra University, Rajkot-5, Gujarat-INDIA Dr. Ramesh Kothari,Professor UGC-CAS Department of Biosciences Saurashtra University, Rajkot-5, Gujarat-INDIA 1
Description of Module Subject Name Paper Name Module Name/Title 04 Metabolism of carbohydrates 15 2
DESCRIPTION is the process whereby the free energy that is released when electrons are transferred along the electron transport (respiratory) chain is coupled to the formation of adenosine triphosphate (ATP) from adenosine diphosphate (ADP) and inorganicphosphate (P i ). In intact mitochondria and in specialpreparations of submitochondrial particles, the transportof electrons and the phosphorylation of ADP are tightly coupled reactions. In damaged mitochondria, respiration (i.e., electron transport) may occur unaccompaniedby oxidative phosphorylation. When this happens the mitochondria are said to be uncoupled. Oxidative phosphorylation is the main source of energy in aerobic cells. In uncoupled mitochondria, because electron transport may still occur, free energy may stillbe released as the electrons are transferred down the transport chain. However, this energy isnot trapped as ATP and appears instead as heat. MITOCHONDRIAL FRAGMENTATION (Fig. 17-1) A. Oxidative phosphorylation, being a mitochondrial process, is studied by isolating and thenfragmenting mitochondria. 1. In the first fragmentation step the outer membrane is removed by treatment with variousdetergents, e.g., phospholipase and digitonin. a. Two particulate fractions result; 1. The outer membrane, either in the form of vesicles or completely solubilized. 2. The inner membrane plus the mitochondrial matrix enzymes. This fraction containsthe enzymes of the electron transport chain(etc), those of oxidative phosphorylation,and those of the tricarboxylic acid (TCA) cycle. b. Asoluble fraction containing the enzymes from the intermembrane space is also obtained. 3
2. The inner membrane friction is then subjected to mild sonication. a. The enzymes present in the matrix are released into the medium, and the inner membraneforms vesicles. b. The vesiculated inner membrane can carry out coupled electron transport and oxidativephosphorylation. 3. If the phosphorylating particles are abraded, knoblike projections containing the so-calledcoupling factors are separated from the membrane, which now cannot carry out phosphorylation of ADP to ATP, but can still transport electrons through the transport chain. 4. Treatment of the transport-chain fraction of the inner membrane with bile salts, (NH4)2SO4,or potassium cholate (often used sequentially) breaks the fraction up into the four complexesconcerned with electron transport (see Chapter 16, IV). These isolated complexes can beused to study electron transport in reconstitution experiments. B. Orientation of the enzymes of the electron transport chain in relation to coupled oxidativephosphorylation is illustrated in Figure 1 7-2. Note that 1. Succinate dehydrogenase (SDH) faces the mitochondrial matrix, from which it acceptselectrons from succinyl CoA synthetase of the TCA cycle. 2. The cytochrome a-a 3 complex likewise faces the matrix, from it will obtain oxygen for the final step of the electron transport chain. 4
COUPLING OF PHOSPHORYLATION TO RESPIRATION A. Oxidative phosphorylation has been studied in isolated mitochiondria (or in phosphorylating submitochondrial particles) by incubating them in an oxygen electrode cell (called an oxygraph cell). 1. The oxygen cell measure the concentration of oxygen dissolved in the suspension medium and thus indicates the mitochondrial oxygen uptake. 2. Phosphorylation is assessed by measuring the rate of disappearance of ADP or P i. B. A typical oxygen electrode tracing is diagrammed in Figure 17-3. 1. At point A on the graph a substrate, Pi, and Mg+2 are all added to the suspension medium. The horizontal tracing indicates that there is no oxygen uptake from the mitochondria-free medium. 2. At point B an aliquot of a mitochondrial suspension is added. a. A slow rate of oxygen uptake ensues, as indicated by the rate of disappearance of oxygen from the medium. b. This oxygen uptake, or respiration, is called state IV respiration, and is probably related to the degree of uncoupling of the mitochondria (see III C, below) rather than to the utilization of endogenous substrate. 3. At point C a measured amount of ADP is added, producing a.more rapid rate of oxygen uptake. This rate, known as state III respiration, is limited only by the rate at which the transport chain can transfer electrons from the substrate to oxygen. 4. At point D, all of the added ADP has been phosphorylated to ATP, and the rate of oxygen uptake declines back to the state IV rate. State IV respiration is therefore limited by the concentration of ADP. (Points E and F will be discussed below.) 5
C. Acceptor control and the acceptor control ratio 1. The acceptor control ratio (the acceptor being ADP) is defined as Rate of respiration in state lll ------------------------------------------- Rate of respiration in state IV 2. The acceptor control ratio is a measure of the degree to which electron transport and phosphorylation are coupled. a. Tightly coupled mitochondria have acceptor control ratios of about 10. b. Uncoupled mitochondria may have ratios as low as 1, the value produced when state III and state IV respiration rates are equal. D. The ADP:O or the P:O ratio 1. The ADP:O (or P:O) ratio is a measure of how many moles of ATP are formed per gram atom of oxygen utilized. This is usually measured as the number of moles of ADP (or of P i ) which disappear per gram atom of oxygen utilized. 2. If at point A in Figure 17-3 the substrate added was malate, the ADP:O (or P:O) ratio would be 3, because the following steps take place: a. Malate, when it is oxidized, donates its electrons to oxidized nicotinamide adenine dinucleotide (NAD+). b. When the reduced nicotinamide adenine dinucleotide (NADH) that is formed enters the electron transport chain it does so at the level of the NADH-Q (coenzyme Q) reductase complex. c. As the electrons pass down the chain, three ATPs are formed per atom of oxygen utilized. 3. If α-glycerophosphate had been added as the substrate at point A in Figure 17-3, the ADP:O ratio would have been 2, because a. Some electrons are derived from the oxidized flavin adenine dlnucleotide (FAD)-linked dehydrogenase which oxidized the substrate. b. These electrons enter the transport chain at the level of coenzyme Q. c. Therefore, they miss the formation of ATP at the NADH-Q reductase complex. 6
E. Uncouplers of oxidative phosphorylation 1. Uncouplers of oxidative phosphorylation are compounds that allow mitochondria to utilize oxygen regardless of whether or not there is any phosphate acceptor (ADP) available. 2. At point E on the graph in Figure 17-3 an uncoupler has been added. This causes a marked increase in oxygen uptake, the new rate being greater than the state III rate. 3. Note that no ADP had to be added for the uncoupler to boost the oxygen uptake rate to the new high level. In fact, if at point F ADP is added, there will be no further change in the oxygen uptake rate. 4. Prototype uncouplers a. 2, 4-Dinitrophenol is a classic uncoupler of oxidative phosphorylation. It was once used as a weight-loss drug, but was discontinued because of its toxicity. b. Dicumarol and similar drugs are used clinically as anticoagulants. The closely related warfarin is also used as a rat poison, mainly because of its anticoagulant action, but also because it uncouples oxidative phosphorylation. c. Calcium transport into the mitochondria also changes the relationship between electron transport and oxidative phosphorylation. 1. The mitochondria of mammalian cells transport calcium against a concentration gradient, and this process is energetically coupled to electron transport. 2. The uptake of calcium by mitochondria is also obligatorily coupled to the uptake of a corresponding amount of Pi. 3. For every pair of electrons that pass from NADH to oxygen along the electron transport chain, approximately six Ca2+ ions accumulate in the mitochondria; that is, two Ca2+ ions per energy-conserving site (i.e., per site of ATP formation) are retained (Fig. 17-4). Note that in the diagram the possibility of a fourth Ca2+-accumulating site is indicated. 4. When Ca 2+ ions are taken up by the mitochondria, electron transport can proceed but energy is required to pump the Ca2+ ions into the mitochondria; therefore, there is no energy available to be stored as ATP. 5. if Ca 2+ ions are added to an oxygraph cell containing mitochondria, substrate, Pi, and Mg 2+, the respiration rate changes from state IV to the very fast rate typical of uncoupled respiration. If ADP is added there is no additional change in the oxygen uptake rate. 6. Ca 2+ ions do not increase the state IV rate unless Pi is present. 7. Ca 2+ and Pi are transported into the mitochondria, and apparently the calcium is stored as a calcium-phosphate complex. 7
F. Phosphorylation inhibitors Oligomycin prevents both the stimulation of oxygen uptake by ADP and the phosphorylation of ADP to ATP. 1. If oligomycin is added to a mitochondrial preparation in the presence of substrate, Pi, Mg 2+, and ADP, the state III respiration is immediately reduced to the state IV rate 2. Characteristically, the inhibition of oxygen uptake by oligomycin is relieved by the addition of dinitrophenol, which stimulates the usual fast uncoupled rate of oxygen uptake 3. Oligomycin appears to act by interfering with the ATP synthetase reaction, which causes the phosphorylation of ADP G. Inhibitors of the DDP-ATP carrier 1. Atractyloside is a toxic glycoside from a Mediterranean thistle; bongkrekicacid is derived from a mold that grows on coconut ''flesh.'' 2. Both of these compounds block the translocase that is responsible for the movement of ADP and ATP across the inner mitochondrial membrane. 3. The addition of either inhibitor to a mitochondrial preparation incubated in the presence of substrate, Pi, Mg 2+, and ADP reduces the state III respiration to state IV. MECHANISMS OF OXIDATIVE PHOSPHORYLATION. Three major proposals for the mechanism of oxidative phosphorylation have been considered: A. THE CHEMICAL COUPLING HYPOTHESIS developed from the concept of an intermediate common to both electron transport and phosphorylation of ADP. 1. Electron transport is postulated to generate a high-energy compound which is utilized in secondary reactions to form ATP from ADP and Pi. 2. A model for the reaction is the step in glycolysis at which glyceraldehyde 3-phosphate phosphorylated to 1,3-diphosphoglycerate (1,3-DPG), after which 1,3-DPG transfers one of its phosphates to ADP to form ATP. 8
3. It is postulated that as electrons move down the transport chain a phosphorylated intermediate is formed which in turn transfers its phosphate group to ADP. 4. Figure 1-7-5 is a simplified diagram of the chemical coupling hypothesis: The reduced form of A (AH 2 ) is oxidized by B, forming the reduced form of B (BH 2 ). BH 2 interacts with an intermediate, Y, to form Y. BH 2. C oxidized Y. BH 2 to form reduced C and a high-energy intermediate, Y-B. ln order to recover B, which is required if the chain is to recycle, Y-B interacts with another intermediate, X, to form the high-energy compound X-Y and free B. X-Y can interact with Pi to give yet another high-energy intermediate, X-P. X-P transfers its phosphate to ADP to form ATP and free X. 5. The complexities of the scheme outlined in Figure17-5 had to be introduced order to explain the actions of uncouplers like dinitrophehol (DNP) and of phosphorylation inhibitors such as oligomycin. a. It is postulated that DNP can substitute for X in the reaction that regenerates B from Y- B, so that DNP interacts with Y to form DNP. Y. However, DNP. Y is too unstable to interact with Pi and breaks down. Y is available to allow electron transport to continue, but no ATP is formed. b. Because oligomycin is postulated to act at the level of ATP synthatase, it allows no regeneration of the X intermediate from X-P. Therefore, X cannot enter back into the cycle, so that electron transport shuts down, an uncoupler, in contrast, by substituting for X, would allow electron transport, but not phosphorylation, to resume 6. The absence of data supporting the existence of the postulated intermediates is one of the major difficulties with this hypothesis. 9
B. THE CONFORMATIONAL COUPLING HYPOTHESIS 1. When mitochondria are carrying out electron transport associated with oxidative phosphorylation they change their architecture. a. In state IV respiration, the mitochondrial cristae have a relaxed appearance and are saidto be in the orthodox state. b. In the presence of ADP, when state 111 respiration is occurring, the cristae are very tightly condensed, and this condition is known as the condensed state. 2. It has been postulated that the changes in the architecture reflect changes in the relationship of different components of the electron transport chain to one another, and that this con- formational change represents the formation of the high-energy state C. THE CHEMIOSMOTIC COUPLING HYPOTHESIS is probably the most widely accepted of current theories of oxidative phosphorylation 1. It is proposed that an electrochemical gradient of protons (H ' ions) across the mitochondrial inner membrane serves as the means of coupling the energy flow of electron transport to the formation of ATP. 2. The electron carriers are hypothesized to act as pumps which cause vectorial (directional) pumping of H ' ions across the membrane (Fi8. 1 7-6) 3. Because the H ' ions are charged particles, the flow of free energy across the inner membranes due to the combination of a concentration gradient and a chafe gradient 4. In the electron transport chain, the H ' ions are separated from the electrons; th us, according to the chemiosmotic coupling hypothesis, as the electrons move down the chain the H+ ions are expelled, traveling from the matrix to the intermembrane space, as shown in Figure 1 7-6 5. The chemiosmotic coupling hypothesis then proposes that the protons in the intermembrane space pass through the inner membrane and back into the matrix at a special site, or ''pore. Where ATP synthetase resides. The dissipation of energy that occurs as the protons pass down the concentration gradient to the matrix allows the phosphorylation of ADP to ATP by the synthetase. 6. To account for the actions of dinitrophenol, oligomycin, and calcium, the chemiosmoticcoupling hypothesis proposes that. 10
a. Dinitrophenol, being a lipid-soluble molecule, creates holes in the inner membrane,rendering it permeable to H ' ions so that an H'.ion gradient cannot form. b. Oligomycin blocks the ATP synthetase reaction, and electron transport ceases as thegradients back up. c. Calcium transport acts to break down the proton gradient. 11