Carbohydrate Synthesis

Transcription

1 arbohydrate Synthesis 4-1 Lec # 11 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: ( ) G' o = -868 kj/mol (energy released) bviously, the material to be degraded must have originated somewhere and the starting point for all organic carbon is the fixation of into carbohydrate via photosynthesis. 1. Photosynthesis - Introduction To reverse the above reaction such that is reduced or converted into glucose, energy must be supplied and in photosynthetic cells light energy is used ( ) G' o = +868 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 respiration photosynthesis energy released (ATP) energy used (hν or light energy)

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 ( ) is the usual representation but if 6 are added to both sides we get: ( ) Green sulfur bacteria S ( ) S Purple non-sulfur bacteria () 3 ( ) (isopropanol) (acetone) omparison of the three overall reactions produces the generalized overall reaction: ( ) electron electron acceptor donor 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 while the molecular oxygen ( ) must have originated from the input electron donor ( ). In short, there may be two stages to the process as follows: Stage e Stage e ( ) Two key experiments addressed and confirmed this idea. 1. The first experiment identified the electron acceptors that became reduced electron carriers (ill reagents after the scientist) generated when electrons are removed from (clearly electrons don't float around loose in solution), and demonstrated that they were generated independent of.

3 4-3 ill found that isolated chlorplasts were capable of generating molecular oxygen ( ) in the absence of if they were provided with an electron acceptor. A variety of electron acceptors were found to work in vitro and these became known as "ill reagents". hν + Fe 3+ 1/ + Fe donor acceptor Eventually, it was determined that the actual in vivo electron acceptor in plants was ADP +. + ADP + 1/ + ADP + +. In order to prove that molecular oxygen was derived from the and not from, heavy water ( 18 ) was used with the result that only 18 was found - no 16 (from the 16 ) or ( 18 ) 6. Then if the overall reaction is written normally, it is not balanced correctly ( 16 ) owever, it can be balanced simply by adding 6 18 to the left and 6 16 to the right side ( 16 ) 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 Stage ADP ADP Stage ADP ( 16 ) ADP + Because stage 1 requires light energy, it is referred to as the light stage or light reactions. n the other hand, stage 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 4-4 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 ( reduction to carbohydrate) occur in the stroma.. 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. 3 3 Mg This is the structure of a chlorophyll but you are T responsible for it. The point here is to illustrate the conjugated double bond system. 3 3 phytol 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 10 3 ) of photons increases with decreasing wavelength.

5 λ kj/mol red purple 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. hν (red) E 3 E E 1 hν (purple) E 0 hν must equal the energy required for excitation from one level to another. 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. 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 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) E' o = +1.0 v E' o = -0.6 v hν P700 P700* (in PSI) E' o = +0.4 v E' o = -1.0 v

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 ADP + of -0.3 v, it means that the photoexcitation of the reaction center can lead to reduction of ADP v E o ' AD ADP + Effectively, electrons are pumped up hill or against an energy barrier using light energy. +1.0v energy out as ATP light energy in Two key photoexcitations in PS II and PS I are used to bring about the electron transfer from to ADP v P700* P680* ATP hν PSI ADP + E o ' 0.0v hν PSII P v E P680 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 ADP +, () that is evolved and (3) that ATP is generated as a result of electron flow (the mechanism will come shortly).

7 v PS II P680* see p 4-9 Fe-S protein ferridoxin PS I Lec #1 P700* Fe-S protein ferridoxin E o ' pheophytin plastoquinone hν ADP + 0.0v cytochrome bf hν plastocyanin cytochrome bfquinone complex P700 E 1/ +1.0v + P680 The E 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 hain in the membranes of mitochondria and bacteria. The similarity extends further to there being protons pumped across the thylakoid membrane to generate a p and electrical gradient. This gradient or energized state is then used by ATPase to generate ATP. Getting a little bit ahead of ourselves, ADP and 3 ATP are required to fix 1 into organic form and the quantum yield refers to the number of photons required to fix 1 or the number of photons required to generate ADP and 3 ATP. From what we have seen so far, determining the number of photons required to generate ADP is clear. 8 hν electrons are involved and each electron must be photoexcited times: x 4 = 8 or ADP + ADP it takes 8 photons to generate ADP.

8 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 IT the thylakoid vesicle. This creates a region of positively charged electrical character and low p inside the vesicle and a region of negatively charged electrical character and high p 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 E and another 8 protons are pumped at the cytochrome bf / quinone complex for a total of 1 + 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 + yielding 1 ATP. In response to 8 photons: 1. + ADP + + ADP + + and,. 1 + are pumped across the membrane and, 3. 3 ADP + 3 Pi 3 ATP + 3 as protons flow back through the ATPase. This gives rise to a quantum yield of 8 photons per ADP and 3 ATP or 8 photons per 1 fixed into organic form. Stroma (outside) ATP + 3 4hν 4hν 3 ADP + 3 Pi ADP ADP (high p) E PS II yt bf PS I ATPase (1 + per e - ) ( + per e - ) Thylakoid vesicle (inside) (low p)

9 4-9 The Z-diagram as just presented depicts a process of non-cyclic electron flow during which electrons are pumped from to form ADP. 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 ADP 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 p gradient to be used by the ATPase to generate ATP. P700* hν Fe-S protein plastocyanin ferridoxin plastoquinone cytochrome bf cytochrome bf quinone complex 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 ADP + 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 PS I ATPase PS II PS II yt bf yt bf PS I

10 3. Dark Reactions 4-10 The dark stage or dark ractions are responsible for the fixation of into organic form and are collectively known as the alvin ycle [ 3 6 ] (profit) As presented at this lowest common denominator of intermediates, the process requires 6 ADP and 9 ATP (3 are fixed each requiring ADP and 3ATP). To generate one hexose (glucose), this process would occur twice generating 3 which would be combined into a 6, and the energy requirement would be 1 ADP and 18 ATP. The dark reactons can be sub-divided into two stages; 1. the fixation stage and. the rearrangement stage. In neither stage is light energy used, and only in the fixation stage are the ADP and ATP used. fixation stage ADP + 9ATP 6 ADP ADP rearrangement stage A. Fixation stage 1 P 3 ATP ADP Ribulose phosphate kinase P 3 Ribulose-5-phosphate P 3 Ribulose-1,5-bisphosphate

11 5 + [ 6 ] P 3 P 3 Ribulose-1,5- bisphosphate P 3 P 3 P 3 P 3 P 3 P 3 reaction intermediates 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 ATP ADP P 3 P 3 3-Phosphoglycerate kinase P 3 3- phosphoglycerate 1,3-bisphosphoglycerate 4 P 3 ADP + + ADP + P 3 1,3-bisphosphoglycerate Pi Glyceraldehyde-3-phosphate dehydrogenase P 3 glyceraldehyde- 3-phosphate Triose phosphate isomerase P 3 1 dihydroxy acetone phosphate

12 In summary, for the fixation stage reaction 4-1 Lec # , 3 ATP and ADP are required. R B. Rearrangement stage (profit) The overall reaction is 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 R Steps 1 and 3 utilize almost the same enzymes, and steps and 4 also utilize a second enzyme P 3 P 3 glyceraldehyde- 3-phosphate + P 3 dihydroxy acetone phosphate Aldolase P 3 fructose-1,6- bisphosphate Pi Fructose, 1,6- bisphosphatase P 3 fructose-6- phosphate

13 P 3 fructose-6- phosphate + P 3 glyceraldehyde- 3-phosphate Transketolase TPP P 3 erythrose-4- phosphate + P 3 xylulose- 5-phosphate P 3 P 3 erythrose-4- phosphate 4 + P 3 dihydroxy acetone phosphate Aldolase P 3 sedoheptulose- 1,7-bisphosphate Pi Sedoheptulose-1,7- bisphosphatase P 3 sedoheptulose- 7-phosphate + Transketolase + P 3 glyceraldehyde- 3-phosphate P 3 sedoheptulose-7-phosphate TPP P 3 ribose- 5-phosphate P 3 xylulose- 5-phosphate

14 4-14 To finish up reactions and 4, it is necessary to convert the xylulose-5-p and 1 ribose-5-p to ribulose-5-p and this is accomplished as follows: Ribose phosphate isomerase Ribulose phosphate 3-epimerase P 3 1 ribose- 5-phosphate P 3 ribulose- 5-phosphate P 3 xylulose- 5-phosphate In summary the overall process of fixation and rearrangement is: 6 ADP + 9ATP fixation rearrangement (profit) 5. 4 plants The process just described occurs in all plants and produces a 3 carbohydrate (3- phosphoglycerate) as the immediate product of fixation. Some plants have evolved an accessory system that results in a 4 carbohydrate being the immediate product of fixation. Such plants are referred to as 4 -plants and the primary reason for the auxiliary pathway is to allow the plants to grow more efficiently at lower concentrations. That is, 3 plants express only the alvin ycle process while 4 plants express both the alvin ycle enzymes and the enzymes of the 4 process. The reason that the 4 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 - phosphoglycolate and 3-phosphoglycerate. In other words, the enzyme uses up both oxygen and carbohydrate.

15 4-15 P 3 P 3 - phosphoglycolate (salavageable but expensive) P 3 + Ribulose bisphosphate carboxylase/oxygenase 3 - phosphoglycerate Ribulose-1,5-bisphosphate P 3 The problem for the enzyme lies in its relative affinities for and in comparison to the aqueous concentrations of the two compounds. for K M = 350 M compared to the aqueous [ ] of 50 M This means the enzyme will work as an oxidase at less than 1/ V max. for K M = 9 M compared to the aqueous [ ] of 10 M This means the enzyme will work as a carboxylase at about 1/ V max. r 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 ADP and ATP generated in the light reactions during day time creates a favorable environment for the other reactions of the alvin ycle and pushes the enzyme to be a carboxylase. owever, at night, in the absence of ADP 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. 4 plants have evolved a system that circumvents this problem by creating an effective " pump" that increases the intracellular [ ] 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 P 3 ( 3 - ) Pi PEP PEP carboxylase G' o =-8.6 kj/mol AA

16 ADP + + ADP Malate dehydrogenase G' o =-9.7 kj/mol AA Malate 3 ADP + ADP + + Malate Malic enzyme G' o =+1.7 kj/mol 3 Pyruvate to ribulose bisphosphate carboxylase IPPase 4 3 Pyruvate + Pi ATP Pyruvate orthophosphate dikinase AMP + PPi P 3 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. Pi ATP equivalents (atmosphere) The process occurs in two different cell types and results in the being pumped into the bundle sheath cell creating an elevated intracellular concentration of for use by ribulose bisphosphate carboxylase. AA malate malate PEP pyruvate pyruvate alvin ycle Mesophyll cell Bundle sheath cell

17 4-17 This system provides better reaction conditions for ribulose bisphosphate carboxylase in the form of a higher [ ] making possible a more efficient fixation of and a more rapid accumulation of organic carbon with less oxidation reaction. Generally, 3 plants are found in temperate regions and 4 plants are found in the tropics, but there are obvious exceptions. Rapidly growing plants such as crab grass and corn are 4 plants and the growth advantage provided by the 4 process is obvious. The 4 process requires more energy but with the benefit of faster growth. 3 plants 1 ADP ADP + 6 ( ) ATP ADP + 18 Pi 4 plants 1ADP ADP + 6 ( ) ATP ADP + 30 Pi In other words, an additional ATP per are required for the 4 process to proceed. 6. arbohydrate from acetate (AcoA) From section 1, and 3 involving the degradation of various substrates to form acetyl oa, one take home lesson should have been that the only thing that can happen to acetate (a carbon molecule) is that it can be fed into the TA cycle to generate. In other words, there can be no net gain in organic carbon atoms using acetate as a carbon source for growth acetyl oa ( ) (AA) (citrate)

18 4-18 owever, 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 TA cycle and this is achieved by the glyoxalate shunt. This involves two additional enzymes superimposed on the TA cycle. TA cycle Isocitrate Isocitrate lyase glyoxalate succinate acetyl-oa oas Malate synthase Malate Ac-oA 1 citrate AA isocitrate malate glyoxalate α-kg fumarate succinyl-oa bypassed reactions sucinate The difference between the TA cycle and the glyoxalate shunt is as follows: AA + AcoA AA + AcoA AA (glyoxalate shunt) AA + 4 (TA cycle)

19 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. Glyoxalate enzymes seed germination plant Time Another way of looking at the process to reinforce its role is as follows: AcoA AA (net gain of 4 via glyoxalate) ( ) ( 4 ) AcoA + AA Isocitrate ( ) ( 4 ) ( 6 ) AA (no net gain of in TA cycle) (1 4 ) 7. Synthesis and storage of glucose nce photosynthesis produces the glyceraldehyde-3-p and the glyoxalate shunt produces AA, 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 AA PEP -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.

20 4-0 3 P Phosphoglucose mutase P 3 glucose-6-phosphate glucose-1-phosphate glucose-1-phosphate P 3 UDPG pyrophosphorylase UTP PPi IPPase P P Pi uridine diphosphoglucose (UDPG) Glycogen synthase (GS) glycogen n+1 n glc added to end of chain last removed from chain first UDP last in - first out glycogen n+ n glc

21 Pi Glycogen phosphorylase (GP) glycogen n+1 glycogen n+ n glc n glc P 3 glucose-1-phosphate 4-1 verall gluconeogenesis GP Pi glc-6-p glc-1-p glycogen n+1 glycogen n+ glycolysis UTP UDPG GS UDP PPi 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.

22 8. Regulation of glycogen metabolism 4- 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. losely 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. Pi IPPase - P - P - P - ATP PPi Adenylate cyclase P - camp 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 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. 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 () which is activated as a result. camp R camp R - P - protein 5'-AMP active protein-p Any protein that is phosphorylated has to be dephosphorylated and this is accomplished by protein phosphatase (PP). ATP ADP Pi Protein phosphatase

23 4-3 nce activated the protein kinase phosphorylates three proteins that affect glycogen metabolism. 1. Glycogen synthase (GS) is inactivated. Protein kinase GS GS-P (active) () ATP ADP This results in the turning off of glycogen synthesis.. Glycogen phosphorylase kinase (GPK) is activated to phosphorylate and activate glycogen phosphorylase (GP). Protein kinase GPK GPK-P () (active) ATP ADP GP () ATP This results in the turning on of glycogen degradation. GPK-P GP-P (active) ADP 3. Protein phosphatase inhibitor (PPI) is activated so that it can bind to and inactivate protein phosphatase (PP). Protein kinase PPI PPI-P () (active) ATP ADP PP PP-PPI-P (active) () 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.

24 Adrenalin energy storage 4-4 -adrenergic receptor adenylate cyclase camp PK PK active GS active GS-P PPI glycogen n+ glycogen n+1 energy release GPK-P active GPK GP-P active GP PP active PPI-P active PP - PPI-P Adrenalin energy storage -adrenergic receptor adenylate cyclase camp 5'AMP PK PK active GS active GS-P glycogen n+ glycogen n+1 energy release GPK-P active GP-P active GP PPI GPK PP active PPI-P active PP - PPI-P

25 9. Summary of carbohydrate synthesis 4-5 Lec #15 1. Photosynthesis a) overall reactions b) light reactions - Z-diagram, ADP and ATP synthesis c) dark reactions - fixation d) 4 reactions. Growth on acetate - glyoxalate shunt 3. Glycogen metabolism a) review of gluconeogenesis b) synthesis and degradation c) regulation