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Transcript
4-1 Lec # 11 Carbohydrate Synthesis Up to this point in the course, the main focus has been the breakdown of metabolites, including carbohydrates, lipids and amino acids. The primary purpose of these pathways is to extract energy in useable form with the common end product being ATP, the "energy currency" of the cell. In the case of glucose, the break down can be expressed by: (CH2O)6 + 6 O2 6 CO2 + 6 H2O ∆G'o = -2868 kJ/mol (energy released) Obviously, the material to be degraded must have originated somewhere and the starting point for all organic carbon is the fixation of CO2 into carbohydrate via photosynthesis. 1. Photosynthesis - Introduction To reverse the above reaction such that CO2 is reduced or converted into glucose, energy must be supplied and in photosynthetic cells light energy is used. 6 CO2 + 6 H2O (CH2O)6 + 6 O2 ∆G'o = +2868 kJ/mol (energy used) In this one process, not only is reduced carbon (ie. carbohydrate) produced but molecular oxygen, required for respiration, is produced. When the two processes of photosynthesis and respiration are combined, the carbon cycle is generated. carbohydrates lipids amino acids nucleotides organic carbon O2 respiration energy released (ATP) photosynthesis CO2 energy used (hν or light energy) 4-2 Both procaryotes and eucaryotes are capable of carrying out photosynthesis. While the overall reactions are similar, there are differences apparent and the following section compares three cases to highlight the commonalities. 1. Green plants and algae 6 CO2 + 6 H2O (CH2O)6 + 6 O2 is the usual representation but if 6 H2O are added to both sides we get: 6 CO2 + 12 H2O (CH2O)6 + 6 O2 + 6 H2O 2. Green sulfur bacteria 6 CO2 + 12 H2S (CH2O)6 + 12 S + 6 H2O 3. Purple non-sulfur bacteria 6 CO2 + 12 CH3CH(OH)CH3 (isopropanol) (CH2O)6 + 12 CH3COCH3 + 6 H2O (acetone) Comparison of the three overall reactions produces the generalized overall reaction: 6 CO2 + 12 H2X electron electron acceptor donor (CH2O)6 + 12 X + 6 H2O Reflection on this generalized reaction in relation to the most common reaction from plants and algae leads to the realization that the oxygen atoms in the product waters must have originated in the input CO2 while the molecular oxygen (O2) must have originated from the input electron donor (H2O). In short, there may be two stages to the process as follows: 24 H+ + 24 e- + 12 X Stage 1 12 H2X Stage 2 24 H+ + 24 e- + 6 CO2 (CH2O)6 + 6 H2O Two key experiments addressed and confirmed this idea. 1. The first experiment identified the electron acceptors that became reduced electron carriers (Hill reagents after the scientist) generated when electrons are removed from H2X (clearly electrons don't float around loose in solution), and demonstrated that they were generated independent of CO2. 4-3 Hill found that isolated chlorplasts were capable of generating molecular oxygen (O2) in the absence of CO2 if they were provided with an electron acceptor. A variety of electron acceptors were found to work in vitro and these became known as "Hill reagents". hν 3+ H2O + 2 Fe 1/2 O2 + 2 Fe2+ + 2 H+ donor acceptor Eventually, it was determined that the actual in vivo electron acceptor in plants was NADP+. H2O + NADP+ 1/2 O2 + NADPH + H+ 2. In order to prove that molecular oxygen was derived from the H2O and not from CO2, heavy water (H218O) was used with the result that only 18O2 was found - no 16O2 (from the C16O2) or (CH218O)6. Then if the overall reaction is written normally, it is not balanced correctly. 6 C16O2 + 6 H218O (CH216O)6 + 618O2 However, it can be balanced simply by adding 6 H218O to the left and 6 H216O to the right side. 6 C16O2 + 12 H218O (CH216O)6 + 618O2 + 6 H216O This makes it very clear that the input and output waters are treated separately and this is easily explained by there being two stages to the process as deduced above. hν 18 + Stage 1 12 H2 O + 12 NADP 6 18O2 + 12 NADPH + 12 H+ Stage 2 6 C16O2 + 12 NADPH + 12 H+ (CH216O)6 + 6 H216O + 12 NADP+ Because stage 1 requires light energy, it is referred to as the light stage or light reactions. On the other hand, stage 2 does not require light energy and is referred to as the dark stage or dark reactions. Within plants, the whole process occurs within the intracellular organelle, the chloroplast which is believed to have bacterial origins. stroma or cytoplasm thylakoid vesicles 4-4 All of the reactions to do with the light stage (absorption of light energy and oxidation of water) occur in the membrane of the thylakoid vesicles and all of the reactions to do with the dark stage (CO2 reduction to carbohydrate) occur in the stroma. 2. Light Reactions The principal light absorbing molecule is the chlorphyll of which there are several different types varying in substituents on the porphyrin ring. There are light harvesting complexes composed of many proteins, chlorophylls and other pigments that absorb light energy and transfer it to one of two complex apparatuses called photosystems I and II (PS I and PS II). The complexity of the photosystems is evident in photosystem I which contains 16 proteins, 168 chlorphylls, as well as carotenoids, Fe-S clusters and phylloquinones. CH2CH3 H3C N N This is the structure of a chlorophyll but you are NOT responsible for it. The point here is to illustrate the conjugated double bond system. Mg N N CH3 H3C C20H39OOCH2CH2C phytol H3COOC O To understand how light energy is absorbed, we must first briefly review what light energy is. Light energy has the properties of both a particle or photon and a wave. The speed of light is expressed by: c = λν (where λ is the wavelength and ν is the frequency.) The energy of light is expressed by: E = hν or E = hc/λ (where h is Planck's constant) It is more common to use wavelength than frequency as a measure and the energy of an Einstein (6 x 1023) of photons increases with decreasing wavelength. 4-5 red purple λ 650 590 490 395 kJ/mol 181.8 201.0 242.0 300.1 The important point here is that the energy of a photon of light increases as you move to shorter wavelength. This is an important consideration for exposure to sunlight. UV All electrons in a molecule have the capability to exist in a number of different energy levels or energy states. The most stable is the ground state, but absorption of energy can result in an electron being excited to a higher energy level. Light energy can be used for this photoexcitation. E3 E2 hν must equal the energy required for excitation from one level to another. E1 The energies of the transitions vary with the type of electron and π electrons in an extended aromatic system are not as tightly bound and can be excited at lower energy levels whereas those more tightly bound (n electrons) require higher energy. hν (purple) hν (red) E0 Absorbance The absorbance spectrum of a chlorophyll exhibits two main peaks of absorbance, one in the red region (650 to 700 nm) and the second in the blue region (400 nm) of the spectrum. The trough between the two peaks where light is not absorbed falls in the green region of the spectrum where light is reflected giving chlorophyll and plants their green color. 300 400 500 600 700 Wavelength (nm) The light reactions actually involve two key photoexcitations involving photosystems I and II once. In fact, some of the light energy is transferred in from the light harvesting complexes but the photosystems themselves have a very complex array of chlorophylls and pigments that can absorb light. The light energy is eventually transferred to a key chlorophyll or reaction center in each of the photosystems, P700 in PS I and P680 in PS II (denoted by the wavelength of maximum absorbance). hν P680 P680* (in PSII) o o E' = -0.6 v E' = +1.0 v hν P700 P700* (in PSI) o o E' = +0.4 v E' = -1.0 v 4-6 The significance of an electron photoexcited to a higher energy level is that it is less tightly bound to the molecule and can therefore be donated or given up to another molecule more easily. In other words, the molecule becomes a better reducing agent or is more easily oxidized. This is evident in the change in reduction potentials from +1.0 v to -0.6 v and +0.4 v to -1.0v. When compared to the standard reduction potential of NADP+ of -0.32 v, it means that the photoexcitation of the reaction center can lead to reduction of NADP+. -1.0v Effectively, electrons are pumped up hill or against an energy barrier using light energy. NADP+ NADH Eo' energy out as ATP O2 light energy in H2O O2 +1.0v -1.0v P700* P680* Eo' Two key photoexcitations in PS II and PS I are used to bring about the electron transfer from H2O to NADP+. NADP+ ATP hν PSI 0.0v hν PSII P700 O2 H2O OEC P680 +1.0v This diagram is often referred to as the "Z" diagram of the light reactions of photosynthesis and its basic tenet is that it delineates (1) how electrons are photoexcited from water to NADP+, (2) that O2 is evolved and (3) that ATP is generated as a result of electron flow (the mechanism will come shortly). PS I see p 4-9 -1.0v P700* PS II Fe-S protein P680* Fe-S protein ferridoxin ferridoxin pheophytin hν Eo' 4-7 Lec #12 NADP+ plastoquinone cytochrome bf 0.0v hν plastocyanin cytochrome bfquinone complex P700 OEC H2O +1.0v 2H+ 1/2O2 P680 The OEC or oxygen evolving complex is a manganese containing complex that is responsible for pulling electrons out of water and generating molecular oxygen. The series of electron transfer reactions linking P680* and P700 are similar in concept to the series of oxidation-reduction reactions that make up the Electron Transport Chain in the membranes of mitochondria and bacteria. The similarity extends further to there being protons pumped across the thylakoid membrane to generate a pH and electrical gradient. This gradient or energized state is then used by ATPase to generate ATP. Getting a little bit ahead of ourselves, 2 NADPH and 3 ATP are required to fix 1 CO2 into organic form and the quantum yield refers to the number of photons required to fix 1 CO2 or the number of photons required to generate 2 NADPH and 3 ATP. From what we have seen so far, determining the number of photons required to generate 2 NADPH is clear. 8 hν 2 H2O O2 + 2 H+ 4 electrons are involved and each electron must be photoexcited 2 times: 2 x 4 = 8 or + 2 NADP 2 NADPH it takes 8 photons to generate 2 NADPH. 4-8 The next question is how is ATP generated and how many photons are required to generate 3 ATP? The answer lies in the chemiosmotic theory, first introduced for oxidative phosphorylation in the mitochondria. As the electrons flow through the system, protons are pumped across the membrane INTO the thylakoid vesicle. This creates a region of positively charged electrical character and low pH inside the vesicle and a region of negatively charged electrical character and high pH in the stroma. In other words, an energized state is created which can be dissipated by the flow of protons across the membrane. 4 protons are generated at the OEC and another 8 protons are pumped at the cytochrome bf / quinone complex for a total of 12 H+ per 4 electrons. As in the mitochondria, there is an ATPase in the thylakoid membrane through which protons can flow and the released energy is coupled to the phosphorylation of ADP to form ATP. The yield is also similar with 4 H+ yielding 1 ATP. In response to 8 photons: 1. 2 H2O + 2 NADP+ O2 + 2 NADPH + 2 H+ and, 2. 12 H+ are pumped across the membrane and, 3. 3 ADP + 3 Pi 3 ATP + 3 H2O as protons flow back through the ATPase. This gives rise to a quantum yield of 8 photons per 2 NADPH and 3 ATP or 8 photons per 1 CO2 fixed into organic form. Stroma (outside) 12 H+ 4hν 3 ADP + 3 Pi 3 ATP + 3 H2O 4hν 2 NADP+ + 2 H+ 2 NADPH OEC 2 H2O + - (1 H per e ) PS II O2 + 4 H+ Cyt bf PS I (high pH) ----------- ATPase ++++++++++++++ (low pH) 8 H+ (2 H+ per e-) Thylakoid vesicle (inside) 4-9 The Z-diagram as just presented depicts a process of non-cyclic electron flow during which electrons are pumped from H2O to form NADPH. There also exists a modification to this system that allows a process of cyclic electron flow that seems to have evolved as a means to generate additional ATP. The system is a hybrid of PS I and the plastoquinone/cytochrome bf complex that bypasses NADPH formation. PS I Electrons are photoexcited in PS I and as they flow through the plastoquinone and cytochrome bf complexes, protons are pumped across the thylakoid membrane. This generates the pH gradient to be used by the ATPase to generate ATP. P700* Fe-S protein ferridoxin hν plastoquinone cytochrome bf cytochrome bf quinone complex plastocyanin P700 The locations or distribution of ATPase, PS I, PS II and the cytochrome bf - quinone complex differ in the thylokoid vesicles. The ATPase and PS I are found mainly in the unstacked regions giving them access to NADP+ and ADP in the stroma. PS II is found mainly in the regions of stacked lamellae while the cytochrom bf - quinone complex is spread evenly throughout the membrane. ATPase ATPase PS I PS II PS II Cyt bf Cyt bf PS I 4-10 3. Dark Reactions The dark stage or dark ractions are responsible for the fixation of CO2 into organic form and are collectively known as the Calvin Cycle. 3 C5 + 3CO2 [ 3 C6] 6 C3 5 C3 1 C3 (profit) As presented at this lowest common denominator of intermediates, the process requires 6 NADPH and 9 ATP (3 CO2 are fixed each requiring 2 NADPH and 3ATP). To generate one hexose (glucose), this process would occur twice generating 2 C3 which would be combined into a C6, and the energy requirement would be 12 NADPH and 18 ATP. The dark reactons can be sub-divided into two stages; 1. the fixation stage and 2. the rearrangement stage. In neither stage is light energy used, and only in the fixation stage are the NADPH and ATP used. fixation stage 3 C5 + 3CO2 6 C3 6 NADP+ + 9 ADP 6 NADPH + 9ATP rearrangement stage 3 C5 5 C3 A. Fixation stage 1 2 H 2C C HC HC H 2C OH ATP O ADP OH OH 2 CH2OPO3 Ribulose-5-phosphate Ribulose phosphate kinase C OPO3 O HC OH HC OH 2 CH2OPO3 Ribulose-1,5-bisphosphate 4-11 2 C5 + CO2 [C6] 2 C3 2 2 H 2C 2 OPO3 C H 2C O HC OH HC OH 2 OPO3 C OH C OH HC OH 2 H 2C OOC OPO3 C OH C O CO2 HC CH OH O C H2O OH HC OH 2 CH2OPO3 Ribulose-1,5bisphosphate OOC OPO3 O 2 CH2OPO3 H 2C 2 CH2OPO3 CH2OPO3 reaction intermediates 2X 3- phosphoglycerate Ribulose bisphosphate carboxylase Ribulose bisphosphate carboxylase is a multimer of 8 large and 8 small subunits, L8S8 and is probably the most abundant protein in nature. 3 2 O O O C 2 ATP OH HC 2 CH2OPO3 OPO3 C 2 ADP HC 3-Phosphoglycerate kinase OH 2 CH2OPO3 2X 1,3-bisphosphoglycerate 2X 3- phosphoglycerate 4 2 O OPO3 2 NADPH + 2 H C HC OH 2 O + 2 NADP + H CH2OH C HC 2 2 Pi CH2OPO3 Glyceraldehyde-3-phosphate dehydrogenase 2X 2X glyceraldehyde1,3-bisphosphoglycerate 3-phosphate CH2OPO3 C OH Triose phosphate isomerase O 2 CH2OPO3 1X dihydroxy acetone phosphate 4-12 In summary, for the fixation stage reaction OR 1 CO2 + 1 C5 2 C3, 3 CO2 + 3 C5 6 C3 Lec #13 3 ATP and 2 NADPH are required. 5 C3 1 C3 (profit) B. Rearrangement stage The overall reaction is 5 C3 3 C5 and much of the process should be considered along side the pentose phosphate pathway. Like the pentose phosphate pathway, the easiest way to keep things straight is to have an overall scheme that illustrates duplicated processes and then fit the details into the overall template. C3 1 Steps 1 and 3 utilize almost the same enzymes, and steps 2 and 4 also utilize a second enzyme. C6 C3 2 C5 + C4 C3 3 C7 C3 4 C5 + C5 C3 OR 5 C3 3 C5 1 C3 + C3 O H 2 CH2OH C HC C6 CH2OPO3 CH2OH C C O H2O OH + C O HO 2 2 CH2OPO3 glyceraldehyde3-phosphate CH2OPO3 dihydroxy acetone phosphate Aldolase Pi HO CH HC OH HC OH 2 CH2OPO3 fructose-1,6bisphosphate O Fructose, 1,6bisphosphatase CH HC OH HC OH 2 CH2OPO3 fructose-6phosphate 4-13 2 C6 + C3 C4 + C5 CH2OH CH2OH C HO O O HC H C C CH + HC Transketolase OH HC TPP 2 HC HC HO O CH OH CH2OPO3 OH + OH HC OH O HC 2 OH CH2OPO3 glyceraldehyde3-phosphate 2 CH2OPO3 2 CH2OPO3 erythrose-4phosphate fructose-6phosphate xylulose5-phosphate 2 3 C4 + C3 HC O HC OH HC C7 CH2OPO3 CH2OH C C O CH2OH HO C dihydroxy acetone phosphate erythrose-4phosphate HC OH HC OH Sedoheptulose-1,7bisphosphatase HC OH HC OH HC OH Aldolase CH2OPO3 CH2OPO3 OH HC 2 2 CH O + OH Pi HO H2O CH 2 2 CH2OPO3 CH2OPO3 4 O C7 + C3 sedoheptulose1,7-bisphosphate C5 + C5 sedoheptulose7-phosphate CH2OH C HO HC O CH2OH HC OH C Transketolase HC OH HC OH O O CH H C HC OH HC OH HC OH 2 + HC OH 2 CH2OPO3 glyceraldehyde3-phosphate CH2OPO3 sedoheptulose-7-phosphate TPP + 2 CH2OPO3 ribose5-phosphate HO O CH HC OH 2 CH2OPO3 xylulose5-phosphate 4-14 To finish up reactions 2 and 4, it is necessary to convert the 2 xylulose-5-P and 1 ribose-5-P to ribulose-5-P and this is accomplished as follows: HC O CH2OH CH2OH HC OH C C HC OH HC OH HC OH HC OH Ribose phosphate isomerase 2 O HO Ribulose phosphate 3-epimerase O CH HC OH 2 2 CH2OPO3 CH2OPO3 CH2OPO3 1X ribose5-phosphate ribulose5-phosphate 2X xylulose5-phosphate In summary the overall process of fixation and rearrangement is: 6 NADPH + 9ATP fixation 3 C5 + 3 CO2 6 C3 rearrangement 5 C3 1 C3 (profit) 5. C4 plants The process just described occurs in all plants and produces a C3 carbohydrate (3phosphoglycerate) as the immediate product of CO2 fixation. Some plants have evolved an accessory system that results in a C4 carbohydrate being the immediate product of CO2 fixation. Such plants are referred to as C4-plants and the primary reason for the auxiliary pathway is to allow the plants to grow more efficiently at lower CO2 concentrations. That is, C3 plants express only the Calvin Cycle process while C4 plants express both the Calvin Cycle enzymes and the enzymes of the C4 process. The reason that the C4 process may have evolved is that there is an inherent inefficiency in ribulose bisphosphate carboxylase in the form of a side reaction which leads to the oxidation of ribulose bisphosphate, rather than its carboxylation, and subsequent cleavage of the product to 2phosphoglycolate and 3-phosphoglycerate. In other words, the enzyme uses up both oxygen and carbohydrate. 4-15 2 CH2OPO3 2 H 2C OPO3 C O O HC OH HC + 2 - phosphoglycolate (salavageable but expensive) O O2 Ribulose bisphosphate carboxylase/oxygenase OH 2 O O C 3 - phosphoglycerate HC CH2OPO3 OH 2 Ribulose-1,5-bisphosphate CH2OPO3 The problem for the enzyme lies in its relative affinities for O2 and CO2 in comparison to the aqueous concentrations of the two compounds. for O2 KM = 350 M compared to the aqueous [O2] of 250 M This means the enzyme will work as an oxidase at less than 1/2 Vmax. for CO2 KM = 9 M compared to the aqueous [CO2] of 10 M This means the enzyme will work as a carboxylase at about 1/2 Vmax. Or in other words, under normal conditions, the enzyme is a reasonably efficient oxidase as well as a carboxylase. "Product pull" caused by the presence of NADPH and ATP generated in the light reactions during day time creates a favorable environment for the other reactions of the Calvin Cycle and pushes the enzyme to be a carboxylase. However, at night, in the absence of NADPH and ATP, the enzyme works effectively as an oxidase and some estimates have has much as 50% of fixed carbon actually being metabolized as a result. C4 plants have evolved a system that circumvents this problem by creating an effective "CO2 pump" that increases the intracellular [CO2] available for ribulose bisphosphate carboxylase. The system involves four additional enzymes and an extra cell type. We will look at the enzymes individually and then consider the overall picture. 1 (HCO3-) CO2 CO2 CO2 Pi 2 C CH2 PEP C OPO3 PEP carboxylase G'o=-28.6 kJ/mol H 2C CO2 OAA O 4-16 2 CO2 O C H OH C Malate dehydrogenase ∆G'o=-29.7 kJ/mol CH2 3 NADP+ NADPH + H+ CO2 CH2 CO2 CO2 OAA Malate CO2 HC NADP+ NADPH + H+ CO2 OH C H 2C O Malic enzyme CH3 ∆G'o=+1.7 kJ/mol CO2 Malate H2O 4 ATP CO2 to ribulose bisphosphate carboxylase Pyruvate CO2 AMP + PPi IPPase 2 Pi CO2 2 ATP equivalents 2 C O + Pi CH3 C Pyruvate orthophosphate dikinase Pyruvate OPO3 CH2 PEP In this case, it is the inorganic pyrophosphatase that pulls the reaction towards PEP generating an overall ∆G'o for both reactions of ~0. CO2 (atmosphere) The process occurs in two different cell types and results in the CO2 being pumped into the bundle sheath cell creating an elevated intracellular concentration of CO2 for use by ribulose bisphosphate carboxylase. OAA PEP malate pyruvate malate pyruvate Mesophyll cell Bundle sheath cell CO2 Calvin Cycle 4-17 This system provides better reaction conditions for ribulose bisphosphate carboxylase in the form of a higher [CO2] making possible a more efficient fixation of CO2 and a more rapid accumulation of organic carbon with less oxidation reaction. Generally, C3 plants are found in temperate regions and C4 plants are found in the tropics, but there are obvious exceptions. Rapidly growing plants such as crab grass and corn are C4 plants and the growth advantage provided by the C4 process is obvious. The C4 process requires more energy but with the benefit of faster growth. C3 plants 12 NADPH + 12 H+ 12 NADP+ 6 CO2 (CH2O)6 + 6 H20 18 ATP + 18 H2O C4 plants 12NADPH + 12 H+ 18 ADP + 18 Pi 12 NADP+ 6 CO2 (CH2O)6 + 6 H2O 30 ATP + 30 H2O 30 ADP + 30 Pi In other words, an additional 2 ATP per CO2 are required for the C4 process to proceed. 6. Carbohydrate from acetate (AcCoA) From section 1, 2 and 3 involving the degradation of various substrates to form acetyl CoA, one take home lesson should have been that the only thing that can happen to acetate (a 2 carbon molecule) is that it can be fed into the TCA cycle to generate 2 CO2. In other words, there can be no net gain in organic carbon atoms using acetate as a carbon source for growth acetyl CoA (C2) (citrate) (OAA) CO2 CO2 4-18 However, there are many obvious examples in nature where acetate can be used as a carbon source from which larger organic molecules are generated. The most obvious lies in plants during seed germination when lipids in the seeds are broken down to acetate (β-oxidation) and used to generate rootlets and stems. Also, bacteria can grow using acetate as the sole carbon source. In order to do this, it is necessary to bypass the two decarboxylation steps in the TCA cycle and this is achieved by the glyoxalate shunt. This involves two additional enzymes superimposed on the TCA cycle. TCA cycle CO2 CO2 CH2 CH2 H 2C H C HC CO2 CO2 succinate CO2 Isocitrate lyase OH acetyl-CoA HC CH2 CoASH O HO CO2 CO2 Malate synthase glyoxalate Isocitrate C CO2 Malate Ac-CoA 1 citrate isocitrate OAA CO2 2 α-KG glyoxalate malate succinyl-CoA CO2 fumarate sucinate The difference between the TCA cycle and the glyoxalate shunt is as follows: OAA + 2 AcCoA OAA + 2 AcCoA 2 OAA (glyoxalate shunt) OAA + 4 CO2 (TCA cycle) bypassed reactions H Glyoxalate enzymes 4-19 Lec #14 As noted earlier, plants utilize the glyoxalate shunt during seed germination which is a very specific growth stage. As a result the glyoxalate enzymes are synthesized for only a short length of time as they are needed and then synthesis is turned off. This is illustrated in the graph. seed plant germination Time Another way of looking at the process to reinforce its role is as follows: AcCoA (C2) Isocitrate (C6) AcCoA + OAA (C2) (C4) 2 OAA (net gain of 4 C via glyoxalate) (2C4) 2 CO2 OAA (no net gain of C in TCA cycle) (1C4) 7. Synthesis and storage of glucose Once photosynthesis produces the glyceraldehyde-3-P and the glyoxalate shunt produces OAA, the products can be converted into glucose via the gluconeogenesis pathway which is basically the reversal of glycolysis with a couple extra enzymes added. This was covered in detail in section 1 of the notes. Pyr OAA PEP 2-PGA 3-PGA 1,3-bisPGA Ga-3P Frc-1,6-bisP Frc-6-P Glc-6-P Excess glucose produced in this way can then be stored as glycogen: glc-6-P glc-1-P glycogen The following section considers the reactions involved in glycogen synthesis and breakdown and then looks at the more interesting (and complicated) regulatory system that controls the process. 4-20 2 O3POH2C HOH2C O HO O HO HO HO Phosphoglucose mutase OH OH 2 OH OPO3 glucose-6-phosphate glucose-1-phosphate O HOH2C NH O HO HO N OH O OPO2OPO2O HOH2C O HO UDPG pyrophosphorylase O HO OH PPi UTP 2 IPPase H2O H OH H OH H OPO3 glucose-1-phosphate H uridine diphosphoglucose (UDPG) 2 Pi HOH2C O HO HO HOH2C O O OH Glycogen synthase (GS) HO OH n glc glycogenn+1 UDP HOH2C O HO HO HOH2C OH O O HO HOH2C added to end of chain last removed from chain first last in - first out OH glycogenn+2 O O HO OH n glc 4-21 HOH2C O HO HO HOH2C OH O O HO HOH2C OH Pi O O HO OH glycogenn+2 Glycogen phosphorylase (GP) n glc HOH2C HOH2C O HO O HO HO HO HOH2C OH OH O O 2 OPO3 HO glucose-1-phosphate OH glycogenn+1 n glc Overall GP Pi gluconeogenesis glc-6-P glycolysis glc-1-P glycogenn+1 glycogenn+2 GS UTP UDP UDPG PPi 2 Pi In the absence of a system to control these reactions, there is the potential for a "futile cycle" which would use up UTP (equivalent to ATP in energy terms). Therefore, glycogen metabolism is highly regulated with hormones affecting the activity and synthesis of the enzymes. 4-22 8. Regulation of glycogen metabolism While there are several levels of control of glycogen metabolism, the best studied and understood is the one activated by adrenalin (epinephrin or norepinephrin) secreted in the adrenal cortex in response to some external stimulus. The hormone binds to a β-adrenergic cell surface receptor present on certain tissue types. Closely associated with the receptor is the enzyme adenylate cyclase and whatever change in the receptor is induced by adrenalin binding results in activation of the cyclase and an increase in cAMP levels. NH2 N O -O P O O O- P N O O P O- O N 2 Pi H2O H N IPPase PPi N N O O- NH2 O H H OH H OH Adenylate cyclase O P H H O H OH H O- cAMP H2O The phosphodiesterase is active at a low level at all times (except when inhibited by caffeine) and the levels of cAMP are determined by the activity of the cyclase and whether it is turned on or not. Phosphodiesterase NH2 Therefore, levels of cAMP rise when it is turned on and drop when it is turned off as a result of the slow action of the phosphodiesterase. N N O cAMP is an intracellular messenger with roles in many different processes. In the case of glycogen metabolism regulation, it interacts with the regulatory subunit (R) of protein kinase (PK) causing its dissociation from the catalytic subunit (C) which is activated as a result. cAMP -O P O N N O OH H H OH H OH 5'-AMP protein R Pi cAMP R N O ATP C N ATP C ADP Protein phosphatase H2O inactive protein-P active Any protein that is phosphorylated has to be dephosphorylated and this is accomplished by protein phosphatase (PP). 4-23 Once activated the protein kinase phosphorylates three proteins that affect glycogen metabolism. 1. Glycogen synthase (GS) is inactivated. Protein kinase GS GS-P (active) (inactive) ATP ADP This results in the turning off of glycogen synthesis. 2. Glycogen phosphorylase kinase (GPK) is activated to phosphorylate and activate glycogen phosphorylase (GP). Protein kinase GPK GPK-P (inactive) (active) ATP ADP GPK-P GP (inactive) ATP GP-P (active) ADP This results in the turning on of glycogen degradation. 3. Protein phosphatase inhibitor (PPI) is activated so that it can bind to and inactivate protein phosphatase (PP). Protein kinase PPI PPI-P (inactive) (active) ATP ADP PP PP-PPI-P (active) (inactive) This results in the phosphorylation reactions not being reversed. The net effect of these three reactions is that glycogen synthesis is stopped, glycogen breakdown is turned on and the enzyme that reverses the effect of protein kinase, protein phosphatase, is turned off. This is summarized in the two diagrams on the following page. The top diagram shows how the presence of adrenalin activates energy release and the bottom diagram shows how in the absence of adrenalin, the system is shifted to carry out energy storage. 4-24 energy storage Adrenalin X glycogenn+2 -adrenergic receptor adenylate cyclase X GS active GP-P active glycogenn+1 X X PK inactive cAMP GS-P inactive X GP inactive energy release GPK-P active PK active X PPI inactive PP active GPK inactive X PP - PPI-P inactive PPI-P active Adrenalin energy storage -adrenergic receptor glycogenn+2 X GS active adenylate cyclase cAMP 5'AMP PK inactive glycogenn+1 X X X GS-P inactive energy release PK active GP-P active X GP inactive GPK-P active PPI inactive X GPK inactive X PPI-P active PP active PP - PPI-P inactive 4-25 Lec #15 9. Summary of carbohydrate synthesis 1. Photosynthesis a) overall reactions b) light reactions - Z-diagram, NADPH and ATP synthesis c) dark reactions - CO2 fixation d) C4 reactions 2. Growth on acetate - glyoxalate shunt 3. Glycogen metabolism a) review of gluconeogenesis b) synthesis and degradation c) regulation