Bioc 3021 Notes - Glycolysis and Gluconeogenesis PDF

BioC 3021 Notes Robert Roon Lecture 14: Glycolysis and Gluconeogenesis Slide 1. Glycolysis and Gluconeogenesis The monosaccharide glucose is one of the most common metabolic intermediates. Glycolysis is a metabolic pathway in which glucose is converted to...

BioC 3021 Notes Robert Roon Lecture 14: Glycolysis and Gluconeogenesis Slide 1. Glycolysis and Gluconeogenesis The monosaccharide glucose is one of the most common metabolic intermediates. Glycolysis is a metabolic pathway in which glucose is converted to pyruvate or lactate. The Gluconeogenesis pathway achieves the reversal of glycolysis—that is, in the gluconeogenesis pathway, lactate or pyruvate are converted back to glucose. These two pathways are at the core of human metabolism. Slide 2. The Major Catabolic Pathways The combined pathways of glycolysis and the citric acid cycle serve as the basic energy yielding mechanisms in most organisms. A number of carbohydrates, amino acids, and glycerol feed into the glycolysis pathway. Under aerobic conditions, the product of glycolysis is pyruvate. The pyruvate can be converted to acetylCoA and carbon dioxide by the pyruvate dehydrogenase complex. The acetylCoA is then oxidized to two molecules of carbon dioxide in the citric acid cycle. Slide 3. Glycolysis Pathway Summary Glucose is one of the main metabolic fuels for humans. Glucose has a high percentage of reduced carbon, giving it a lot of potential energy as a substrate for oxidation. The glycolysis pathway converts glucose to pyruvate, which is the first step in oxidizing glucose to carbon dioxide. Glycolysis occurs in two phases: -In phase 1, one molecule of glucose (a 6 carbon compound) is converted into two molecules of glyceraldehyde-3-phosphate (a 3 carbon compound). Phase 1 utilizes two ATP molecules. -In phase 2, two glyceraldehyde-3-phosphate molecules are converted into two pyruvate molecules. Phase 2 generates four ATP molecules. Slide 4. Key Intermediates and Products of Glycolysis An overview of glycolysis shows the following: 1 BioC 3021 Notes Robert Roon -The six carbon hexose, glycose, is transformed into two molecules of the three carbon triose, pyruvate. -At the hexose level, a single intermediate is phosphorylated at two sites, and two ATP's (2 sites X 1 ATP per site) are cleaved to two ADP's -At the triose level, two compounds transfer phosphate to ADP at two sites, so four ADP's (2 sites X 2 ADP's per site) are phosphorylated to four ATP's -If glycolysis proceeds through to pyruvate (aerobic metabolism), two NADH’s are produced The net balance sheet for aerobic glycolysis is: the metabolism of one glucose molecule results in the production of two pyruvates, two ATP’s and two NADH’s. Slide 5. Glycolysis Part I. This slide shows the top half of the glycolysis pathway. (The division of glycolysis into two parts is arbitrary, serving only to improve visualization of the overall process.) There are five steps in this part of the pathway, catalyzed by five different enzymes. (The names of the enzymes are shown in blue, and the nature of the reactions catalyzed is indicated in green.) In this part of glycolysis, one molecule of the six carbon compound glucose is converted into two molecules of the three carbon intermediate glyceraldehyde 3-phosphate. There are two kinase reactions in which intermediates are phosphorylated. These reactions utilize ATP and produce ADP. This means that the initial stages of glycolysis costs the cell two ATP equivalents. This cost must be taken into account when we calculate the overall energy balance of glycolysis. The last reaction interconverts dihydroxyacetone phosphate and glyceraldehyde 3-phosphate, effectively producing two molecules of glyceraldehyde 3-phosphate. This intermediate continues on down the second half of the pathway. So from here on down, there are two three-carbon molecules in the pathway. This is an important consideration when we calculate the energy 2 BioC 3021 Notes Robert Roon balance for glycolysis. Slide 6. Glycolysis Part II. Part two of glycolysis also has five steps and five different enzymes, making a delightful symmetry with part one. This second part starts and ends with two three-carbon intermediates. The first reaction shown, the conversion of glyceraldehyde 3- phosphate to 1-3-bisphosphoglycerate, is the key to energy production by the overall pathway. This oxidation-reduction reaction not only introduces additional phosphate residues into the pathway, but also results in the production of two molecules of NADH. Under oxidative conditions, those two molecules of NADH can enter the oxidative phosphorylation scheme, which ultimately results in the net production of four molecules of ATP. The addition of an extra phosphate residue produces a bis- phosphate product, 1-3-bisphosphoglycerate. The two 1-3- bisphosphoglycerate molecules are immediately converted to two 3-phosphoglycerates with the concomitant production of two molecules of ATP. This is an example of coupling the production of ATP to an intermediate with a high phosphate hydrolysis potential. At this point, the two ATP’s used in part I of glycolysis have been regenerated, so that initial energy price has been repaid. Two more ATP’s are produced in the last reaction of the pathway, which is catalyzed by pyruvate kinase. This means that glycolysis gives a net yield of two ATP molecules directly, with the potential for four more ATP’s from the oxidation of the two NADH’s. Two molecules of pyruvate are the other end products of glycolysis. Under aerobic conditions, these two pyruvate molecules are converted into acetylCoA, which can then enter the TCA cycle. Under anaerobic conditions, where oxygen is rate limiting, pyruvate is converted into lactate. Slide 7. Standard Free Energies of Hydrolysis 3 BioC 3021 Notes Robert Roon A glance at the standard energies of hydrolysis reveals that the phosphorylation of hexoses, such as glucose and fructose, requires the input of about 3 -5 kcal/mol, whereas the dephosphorylation of the trioses phosphoenolpyruvate and bis-phosphoglycerate releases 15.8 and 11.8 kcal/mol, respectively. This means that ATP, which has a standard energy of hydrolysis of 7.3 kcal/mol, can donate a phosphate to the hexoses in an energy releasing reaction, and ADP can accept phosphate from the trioses also in an energy releasing reaction. That means that all of the reactions in glycolysis that involve the transfer of phosphate to or from ATP have a favorable standard free energy, and thus tend to proceed in the direction of product formation. Slide 8. Change in Free Energy in Glycolysis and the TCA Cycle This figure shows the free energy changes for the reactions of glycolysis and the TCA cycle. There are a number of key points that we should make about this figure. -The overall free energy change for the conversion of glucose to carbon dioxide is highly negative. It is this loss of free energy that drives the reaction pathways in the direction of product formation. -The free energy changes for all of the individual reactions should be at least slightly negative. Some of the reactions shown on this figure seem to be slightly uphill with positive free energy changes. Theoretically, that has to be wrong. If true, it would mean that these reactions were going backwards, and the overall pathway would not be working. My conclusion is that the data (for the reactions that seem to have a positive free energy change) must be incorrect. It seems necessary that each and every reaction must release at least a little bit of energy. -The reactions that produce ATP directly (substrate level phosphorylation) seem to have small free energy changes, and the reactions that produce reduced nucleotide products have large negative free energy changes. Again, every reaction must have at 4 BioC 3021 Notes Robert Roon least a small negative free energy change, or the reactions would be going backward. Slide 9. Standard Free Energy for Hexokinase Reaction Here, we revisit the first reaction in glycolysis—the hexokinase reaction. In this reaction, a phosphate is transferred from a higher energy donor, ATP, to a lower energy product, glucose 6-P. The reaction proceeds in the direction of product because the phosphoanhydride bond of ATP releases more energy than it takes to forge the phosphoester bond in glucose 6-P. Slide 10. The Importance of Phosphorylating Glucose. The phosphorylation of glucose in the hexokinase reaction is an important process that keeps glucose and its products moving through the glycolysis pathway. Because it is phosphorylated, glucose 6-P cannot diffuse out of the cell directly through the cell membrane. Furthermore, glucose 6-P is not recognized by glucose transport proteins in the plasma membrane, and thus it cannot diffuse out of the cell through those systems either. Finally, the chemical transformation of glucose into glucose 6-P keeps the intracellular glucose concentration low, so diffusion of glucose into the cell is favored. It turns out that most cells use hexokinase to phosphorylate glucose. This enzyme has a Km for glucose of 0.1 mM, and so it is very efficient at transforming glucose into glucose 6-P. In contrast, the liver uses an enzyme called glucokinase, which has a Km = 10.0 mM. This higher Km assures that glucokinase only works when glucose is high in the cell. This allows glucose to diffuse out of liver cells into the blood. The glucose is transported from the liver and serves as an energy source for other tissues. It is interesting that glucokinase is induced by insulin, and insulin resistant diabetics thus have difficulty processing high glucose loads. 5 BioC 3021 Notes Robert Roon Slide 11. Phosphoglucose Isomerase The next step in glycolysis is the isomerization of glucose 6- phosphate to fructose 6-phosphate catalyzed by phosphoglucoisomerase. The overall reaction scheme as shown on this slide looks rather complicated, but a step-by-step analysis reveals that it involves a series of relatively straightforward transformations. The glucose 6-phosphate begins in the pyranose ring form, but in order for the overall reaction to proceed, mutarotation to the open chain form must first occur. The open chain structure then undergoes a rearrangement that converts the C-1 carbonyl oxygen to a hydroxyl group, and the C-2 hydroxyl group to a carbonyl oxygen atom. Translated, that means that the aldose, glucose 6-phosphate, is converted into the ketose, fructose 6-phosphate. The open chain form of fructose 6-phosphate then undergoes mutarotation to form a cyclic furanose ring structure. Slide 12. Phosphofructokinase In the next step, fructose 6-phosphate is phosphorylated in an ATP-dependent kinase reaction, yielding fructose 1-6-bisphosphate. This is the second ATP coupled reaction in the pathway, and again the net result is a reaction that is favorable in the direction of product formation. In the gluconeogenesis pathway, the reversal of this step is accomplished by an alternate reaction catalyzed by a different enzyme. Directly reversing this step using phosphofructokinase would be thermodynamically unfavorable. We will come back to this reaction again when we look at the regulation of glycolysis, because phosphofructokinase is subject to a number of interesting regulatory processes. Slide 13. Aldolase and Triose Phosphate Isomerase The aldolase reaction is a key step in glycolysis, in which one six- carbon compound is converted into two three-carbon compounds. Aldolase catalyzes an aldol cleavage reaction in which the substrate is split between adjacent alcohol groups to produce two 6 BioC 3021 Notes Robert Roon products, one an aldehyde and the other an alcohol. The new aldehyde group is found in glyceraldehyde 3-phosphate, and the alcohol group occurs in dihydroxyacetone phosphate. The triose phosphate isomerase reaction converts dihydroxyacetone phosphate into a second molecule of glyceraldehyde 3-phosphate. The mechanism of this isomerization is the same as that of phosphoglucoisomerase. At this point in glycolysis, one molecule of glucose has been converted into two molecules of glyceraldehyde 3-phosphate. From here on, each step in the glycolysis pathway involves two identical, three carbon molecules. Slide 14. One Six Carbon Intermediate Converted into Two Three Carbon Intermediates The combined action of the aldolase and triose phosphate isomerase constitutes an important watershed in glycolysis. For the remainder of the pathway, there are a series of two identical, three carbon intermediates. This means that for each energy rich intermediate, there will be two molecules formed. As a result, the energy yield of the pathway is doubled. This doubling is what allows the glycolysis pathway to have a net yield of ATP. Slide 15. Energy Producing Reactions of Glycolysis. In the remaining reactions of glycolysis, there are two sites at which ATP is produced, and one site where NADH is the product. Since two intermediates participate in each of these steps, the net result in this stage is the production of 4 molecules of ATP and two molecules of NADH. Slide 16. Glyceraldehyde 3-Phosphate Dehydrogenase and Phosphoglycerate Kinase. The next two reactions are also critical in making the glycolysis pathway an energy yielding process. Glyceraldehyde 3-phosphate dehydrogenase introduces an extra phosphate residue into the 7 BioC 3021 Notes Robert Roon pathway and also produces a molecule of NADH (which will ultimately yield energy). The phosphoglycerate kinase reaction then transfers that newly added phosphate to ADP to produce the first ATP product in the pathway. Slide 17. Glyceraldehyde 3-Phosphate Dehydrogenase. There are two elements in the glyceraldehyde 3-phosphate dehydrogenase reaction that ultimately enable glycolysis to sequester energy in the form of ATP. Glyceraldehyde 3-phosphate dehydrogenase catalyzes the incorporation of an additional phosphate residue into its product, and it simultaneously carries out an oxidation-reduction reaction. Lets deal with the phosphorylation first. The starting material, glyceraldehyde 3- phosphate, has a single phosphate residue, but the product of the reaction, 1,3-bisphosphoglycerate, has two phosphates. The 1,3- bisphosphoglycerate is a mixed acid anhydride, which has a very high negative ΔG of hydrolysis. In the further steps of glycolysis, this extra phosphate group will yield ATP. So the addition of an extra phosphate at this point allows glycolysis to gain two additional ATP’s. (Remember there are two molecules of each three carbon intermediate at this point in the pathway.) The oxidation-reduction reaction uses NAD+ as the oxidant. In the reaction, an aldehyde is oxidized to an acid, and NAD+ is reduced to NADH. Under aerobic conditions, the two molecules of NADH can enter the oxidative phosphorylation pathway, resulting in the net production of four molecules of ATP. Slide 18. The Overall Mechanism of Glyceraldehyde 3- Phosphate Dehydrogenase. The catalytic mechanism of glyceraldehyde 3-phosphate dehydrogenase involves two reactions. In one reaction, the aldehyde group at C-1 is oxidized to a carboxylic acid coupled to the reduction on NAD+ to NADH. In the other reaction, the newly 8 BioC 3021 Notes Robert Roon formed carboxyl group is joined to a phosphate group to yield an acyl-phosphate product. Slide 19. Structure of NAD+. For your review, here is another look at the structure of NAD+. Remember that the oxidation-reduction process involves the nicotinamide ring system, which can accept two electrons and one proton. Slide 20. Reduction of NAD+ to NADH. Here, we see the reduction of NAD+ to NADH. The two electrons enter the nicotinamide ring. One proton sits on the top carbon atom, and the other proton is released into the solvent. Slide 21. Energy from NADH Drives ATP Synthesis Under aerobic conditions, the NADH from glycolysis is used in the oxidative phosphorylation system to provide high energy electrons that are used to create a proton gradient across the inner membrane of the mitochondria. That gradient is then used to drive the synthesis of ATP by the ATP synthase. Slide 22. Phosphoglycerate Kinase There is an immediate payoff from the glyceraldehyde 3-phosphate dehydrogenase reaction in the next step of glycolysis. The enzyme phosphoglycerate kinase catalyzes the transfer of a phosphoryl group from the mixed acid anhydride of 1,3-bisphosphoglycerate to ADP. This is the first direct energy payback in glycolysis, effectively replacing the two ATP’s that were used in the earlier steps. The other product of the reaction is 3-phosphoglycerate. Slide 23. Substrate Level Phosphorylation The phosphoglycerate kinase reaction is our first example of substrate level phosphorylation. The term substrate level phosphorylation refers to a process in which a phosphate residue is directly transferred from an organic phosphorylated intermediate to 9 BioC 3021 Notes Robert Roon ADP to make ATP. In the phosphoglycerate kinase reaction, the phosphate donor is 1,3-bisphosphoglycerate. In contrast, oxidative phosphorylation uses the downhill transport of high energy electrons to form a proton gradient, which in turn is used to drive the synthesis of ATP from ADP and inorganic phosphate. (see Slide 21 above) Slide 24. Reactions Converting 3-Phosphoglycerate to Pyruvate. The next step in glycolysis converts 3-phosphoglycerate into 2- phosphoglycerate. This reaction, which is catalyzed by phosphoglyceratemutase, involves the transfer of a phosphoryl group from the number 3 carbon to the number 2 carbon of glycerate. The next step is a dehydration reaction, catalyzed by enolase, in which water is removed from 2-phosphoglycerate to produce a double bond. The product of this reaction is phosphoenolpyruvate (PEP). This compound is a phosphoenol derivative, meaning the phosphate group is adjacent to a carbon-carbon double bond. The proximity of the phosphate to the double bond gives phosphoenolpyruvate a very high negative ΔG of hydrolysis (in fact, it has the highest negative ΔG value of any compound on our hydrolysis table). The final reaction in glycolysis is catalyzed by pyruvate kinase. This is the second reaction in glycolysis that accomplishes substrate level phosphorylation—a process in which ATP is formed by the direct transfer of a phosphoryl group from an organic phosphate molecule to ADP. In this reaction, the substrate phosphoenolpyruvate is converted to pyruvate with the transfer of a phosphate residue to ADP to form ATP. The production of two molecules of ATP by pyruvate kinase is 10 BioC 3021 Notes Robert Roon what finally gives glycolysis a net positive ATP yield. The other two molecules of ATP produced by the phosphoglycerate kinase reaction replaced the ATP’s used in the early stages of glycolysis. The two ATP’s from the pyruvate kinase reaction constitute the actual net ATP gain in the pathway. I would like to make one final point about the energy balance in glycolysis. In addition to the ATP’s formed directly by substrate level phosphorylation, four extra ATP’s can be formed from the NADH produced by glyceraldehyde 3-phosphate dehydrogenase. However, this only occurs under aerobic conditions. Under anaerobic conditions, NADH does not participate in oxidative phosphorylation and does not yield ATP. Thus, the only net payoff of anaerobic glycolysis is the two ATP’s formed directly in the pathway. The next few slides focus on the difference between aerobic and anaerobic glycolysis. Slide 25. Summary of Aerobic Glycolysis To summarize the energetics of aerobic glycolysis: -One glucose molecule yields two molecules of pyruvate. -The ATP Investment is one for the hexokinase reaction and two for the phosphofructokinase reaction. -The ATP return is one for the phosphoglycerate kinase reaction and two for the pyruvate kinase reaction. -The NADH Return is two for the glyceraldehyde 3-P dehydrogenase reaction. Under aerobic conditions, these two NADH molecules can ultimately yield four molecules of ATP. Slide 26. Aerobic and Anaerobic Glycolysis This figure highlights the divergence in glucose metabolism that occurs after the formation of pyruvate. At rest or during relatively long and slow exercise, aerobic glycolysis is most prevalent. Glucose is converted into pyruvate, which in turn is oxidized to acetylCoA. The acetylCoA is then oxidized in the TCA cycle. 11 BioC 3021 Notes Robert Roon Under more stringent conditions, such as those that would prevail in a sprint (or semi-sprint) like a 400m race, the rate of acetylCoA oxidation is limited by the availability of oxygen. Pyruvate is diverted into a side reaction, employing lactate dehydrogenase. Under these anaerobic conditions, the product of glycolysis is lactate. In some fermentative microorganisms such as yeast, another reaction scheme is employed under anaerobic conditions. The pyruvate is diverted into a two step pathway that produces ethanol as an end product. This is the basis for the commercial production of alcoholic beverages. Slide 27. Lactate Dehydrogenase Mechanism. During vigorous exercise, pyruvate is reduced to lactate by lactate dehydrogenase. This is an oxidation-reduction reaction in which the reduction of pyruvate is coupled to the oxidation of NADH. The ketone carbonyl group of pyruvate is reduced to the secondary alcohol group of lactate. Slide 28. Conversion of Glucose to Lactate. The conversion of pyruvate to lactate serves an important function under anaerobic conditions. When oxygen is rate limiting, the production of pyruvate by glycolysis exceeds its rate of utilization by the TCA cycle. Even more critical is the buildup of NADH, which is produced by glycolysis faster than it can be used in oxidative phosphorylation. The reduction of pyruvate to lactate is coupled to the oxidation of NADH back to NAD+. This renewal of the NAD+ supply allows glycolysis to proceed past the glyceraldehyde 3-phosphate dehydrogenase reaction. So, glycolysis can continue unabated until the muscles start to scream from the accumulation of lactate (lactic acid). Eventually (after exercise is terminated) much of the accumulated lactate is released from muscle cells into the blood and is carried to the liver where it is converted back into glucose. 12 BioC 3021 Notes Robert Roon Slide 29. Net Energy Yield for Lactate Fermentation. The anaerobic fermentation of glucose to lactate has the same direct ATP yield as the aerobic conversion of glucose to pyruvate. There are ATP’s used in the hexokinase and phosphofructokinase reactions for a total of two ATP’s used. The phosphoglycerate kinase and pyruvate kinase reactions each produce two ATP’s, so the net return of anaerobic glycolysis is two molecules of ATP. Unlike aerobic metabolism, there is no net yield of NADH in anaerobic fermentation because NADH is recycled to NAD+. Slide 30. Conversion of Pyruvate to Ethanol. Under anaerobic conditions, many microorganisms convert pyruvate to ethanol. This reaction sequence accomplishes the same function as the conversion of pyruvate to lactate. That is, it serves to regenerate NAD+ and to get rid of excess pyruvate when oxygen is not available. This process starts with the decarboxylation of pyruvate to acetaldehyde, a reaction that is accomplished by pyruvate decarboxylase. That reaction is followed by the reduction of acetaldehyde to ethanol with the concomitant conversion of NADH to NAD+. This reaction is catalyzed by alcohol dehydrogenase. The NAD+ is used in further rounds of glycolysis, and the ethanol is excreted from the cell. In a closed system such as yeast fermentation, the ethanol rises to levels where it can eventually kill the cells. For yeast, I believe that lethal alcohol level is somewhere around 7-10% w/v. Slide 31. Conversion of Glucose to Ethanol. Ethanol fermentation has the same net energy balance as lactate fermentation. The NADH produced in the glyceraldehyde 3- phosphate dehydrogenase reaction is recycled back to NAD+ in the alcohol dehydrogenase reaction. Thus, there is no net synthesis of NADH. The net yield of ATP is two—two used and four synthesized. The six carbons of glucose are converted into two molecules of ethanol and two molecules of carbon dioxide. 13 BioC 3021 Notes Robert Roon Slide 32. Net Yield for Ethanol Fermentation Anaerobic glycolysis in yeast has a similar energy balance to that in humans, giving a net yield of 2 ATP's with no net production of NADH. Slide 33. Invertase Reaction Now we will consider how other related monosaccharides can enter the glycolysis pathway. The first reaction of this sort involves the conversion of the disaccharide sucrose into glucose and fructose by the enzyme invertase. This reaction is important because a high percentage of the sugar we consume comes in the form of sucrose. The glucose formed in the invertase reaction enters directly into glycolysis. The fructose enters a short adaptive pathway before it can enter glycolysis. Slide 34. Entry of Fructose into Glycolysis. There is a short pathway that integrates fructose into glycolysis. The first step is catalyzed by fructokinase, which transfers a phosphoryl group from ATP to the number C-1 of fructose. Fructose 1-phosphate has a phosphoester bond with relatively low energy of hydrolysis, so the transfer of a phosphoryl group from ATP is thermodynamically favorable. The second step in this pathway is catalyzed by an aldolase, which is specific for fructose 1-phosphate. The products of this aldol cleavage reaction are dihydroxyacetone phosphate and glyceraldehyde. The glyceraldehyde is phosphorylated by a triose kinase to give glyceraldehyde 3-phosphate. The production of dihydroxyacetone phosphate and glyceraldehyde 3-phosphate completes the integration of fructose into glycolysis. Note that the energetics of glycolysis are the same for fructose as for glucose. It takes two ATP’s to integrate fructose into glycolysis—one to phosphorylate fructose and one to phosphorylate glyceraldehyde. 14 BioC 3021 Notes Robert Roon Slide 35. Lactase. The disaccharide lactose is the primary sugar in mammalian milk. The enzyme lactase cleaves lactose into galactose and glucose. Galactose is then converted to glucose 6-phosphate by a short but convoluted pathway. Entry of galactose into glycolysis involves an initial phosphorylation reaction followed by the transient conversion of galactose into a nucleotide sugar derivative. Slide 36. Entry of Galactose into Glycolysis. Galactose is first phosphorylated by galactose kinase. The product, galactose 1-phosphate, is converted into glucose 1-phosphate by an exchange reaction involving the sugar nucleotide, UDP-glucose. Phosphoglucomutase then catalyzes the isomerization of glucose 1-phosphate to glucose 6-phosphate. At that point, the compound is integrated into glycolysis. That sounds relatively easy, but there are some rather baroque reactions involved. Slide 37. Galactose 1-Phosphate Uridylyltransferase. The conversion of galactose 1-phosphate to glucose 1-phosphate is a bit complicated. It involves a transfer reaction in which glucose 1-phosphate is exchanged for galactose 1-phosphate. UDP-glucose (Uridine diphosphate glucose) picks up galactose 1-phosphate and releases glucose 1-phosphate. UDP-galactose is the other product. This is an even exchange—one sugar phosphate is exchanged for another. You can follow this reaction by the color coding—UDP- glucose releases the blue structure and gains the red. If this were politics, that would be a woefully uneven exchange, but this is chemistry so I guess everything balances. This is your first introduction to nucleotide sugars, but we will see more such derivatives as we progress through the course. Nucleotide compounds play an important role in both sugar and lipid metabolism. The formation of a nucleotide derivative is another (indirect) way of coupling ATP energy into 15 BioC 3021 Notes Robert Roon thermodynamically unfavorable biochemical reactions. For example, the formation of glycosidic bonds in the biosynthesis of disaccharides and polysaccharides is an uphill reaction that uses nucleotide sugars to achieve a favorable equilibrium. Slide 38. UDP-Galactose 4-Epimerase. The conversion of UDP-galactose back to UDP-glucose is catalyzed by UDP-galactose 4-epimerase. This reaction provides a continuing supply of UDP-glucose, which in turn allows the pathway to continue producing glucose 1-phosphate. This reaction is catalyzed by an epimerase that uses NAD+ as a cofactor. The mechanism involves the conversion of the 4-epimer (galactose) to a 4-ketone intermediate and back to another 4-epimer (glucose). The intermediate ketone has no chirality, and when it is reduced, it can produce either epimer. This is an easy mechanism (with low activation energy) to alter the chirality of an alcohol molecule. Slide 39. The Energetics of Glycolysis. This figure gives the standard free energies and the net free energies for all of the reactions of glycolysis. There are a couple of important points to be made here. 1. For any biological reaction to proceed in the forward direction, it must have a net negative free energy (ΔG) of hydrolysis under the metabolic conditions found in the cell. That is, only energy releasing reactions will give a net yield of product. 2. The figure shows that three reactions have net positive ΔG values. According to the laws of physics, this is impossible. If these ΔG values were indeed positive, the reactions would be going backwards. What it probably means is that our best estimates of the concentrations of certain glycolysis intermediates are wrong, and that if we had accurate values for these compounds, 16 BioC 3021 Notes Robert Roon all of the ΔG values would in fact be negative. (This may seem trivial to you, but your life depends on it.) Slide 40. Glycolysis vs Gluconeogenesis. We are now going to consider the reversal of glycolysis, which is called gluconeogenesis. In times when energy is needed, such as during vigorous exercise, glycolysis produces pyruvate at a faster rate than it can be used in the TCA cycle. We have seen that under such conditions, the extra pyruvate is diverted to form lactate. When exercise ceases, that lactate is converted back into pyruvate and then into glucose. That is the function of gluconeogenesis. Before looking at gluconeogenesis, let’s review what we learned about anaerobic glycolysis. The key points concerning anaerobic glycolysis are: 1. There are two ATP’s used and four produced for a net yield of two ATP’s. 2. There are three reactions in glycolysis that are essentially irreversible (the reactions with the arrows pointing in the forward direction only). I say “essentially” irreversible, because no reaction is totally irreversible. However, in these three reactions, the equilibrium is so far in favor of product that for practical purposes, there is little or no reverse reaction. The essentially irreversible reactions in glycolysis involve the conversion of glucose to glucose 6-phosphate, the conversion of fructose 6-phosphate to fructose 1-6-bisphosphate, and the conversion of phosphoenolpyruvate to pyruvate. One other key point is that, when the glycolysis pathway is functioning, all of the component reactions must have at least a slightly negative ΔG. That necessitates that the overall reaction pathway will also have a negative ΔG. 17 BioC 3021 Notes Robert Roon If the conversion of glucose to lactate is to be reversed, then this same thermodynamic reality must prevail. That is, the overall reaction pathway of gluconeogenesis and all of its component reactions must have a negative ΔG. This is accomplished by substituting alternate reactions for the three essentially irreversible reactions in glycolysis. The three new reactions either use additional ATP equivalents or substitute phosphatase reactions for kinase reactions. The net effect is that, whereas glycolysis produces a net of two ATP’s, gluconeogenesis uses a net of six ATP’s, and the ΔG of the overall pathway and its component reactions are shifted toward the synthesis of glucose. Slide 41. Gluconeogenesis Summary. This slide summarizes the essential components of gluconeogenesis: 1. The pathway involves the synthesis of glucose from pyruvate or lactate, and it occurs during recovery from exercise. 2. It utilizes many of the same enzymes from glycolysis (the reversible ones). 3. The essentially irreversible steps of glycolysis must be bypassed by substituting different reactions catalyzed by different enzymes. 4. Gluconeogenesis utilizes a net of six ATP equivalents. 5. When gluconeogenesis is occurring, each of the reactions and the net overall pathway all have a negative ΔG. Slide 42. The Cori Cycle. The Cori Cycle describes the physiological process that occurs when glucose is converted to lactate during intense exercise, and when the lactate is converted back to glucose during the recovery period. The cycle is named after its discoverers, Karl and Gerty Cori who studied glucose metabolism at Washington University in St Louis. The Cori Cycle is initiated by anaerobic glycolysis during exercise. The rapid utilization of glucose results in the 18 BioC 3021 Notes Robert Roon production of high levels of lactate. Most of the lactate is released from the muscle and travels in the blood to the liver, where is it taken into the cells. In the liver, the lactate is first converted to pyruvate. Some of the pyruvate is used to produce energy by oxidation in the TCA cycle. The rest is converted back to glucose by gluconeogenesis. The liver is unusual in that it will release a significant portion of its glucose into the blood. A high percentage of the released glucose finds its way back into muscle cells where it can serve in another round of glycolysis. Slide 43. Gluconeogenesis The slide shows the gluconeogenesis pathway with bypass enzymes coded in red. It takes two enzymes to bypass pyruvate kinase. Pyruvate carboxylase uses one ATP to convert pyruvate to oxaloacetate. Phosphoenolpyruvate carboxykinase uses one GTP to convert oxaloacetate to phosphoenolpyruvate. Collectively, these two bypass enzymes use two ATP equivalents to bypass pyruvate kinase, converting pyruvate to phosphoenolpyruvate in a thermodynamically favorable manner. In the second part of gluconeogenesis, two essentially irreversible enzymes, phosphofructokinase and hexokinase, are each replaced with a phosphatase enzyme (coded in red). If the kinase enzymes were used in gluconeogenesis, they would synthesize ATP, and the reactions would be thermodynamically unfavorable (They would have a positive ΔG). In contrast, the phosphatase enzymes, which bypass the kinases, hydrolyze the phosphate group from their substrates in reactions that are thermodynamically favorable (They have a negative ΔG). Slide 44. Conversion of pyruvate to phosphoenolpyruvate. The reversal of pyruvate kinase requires two enzyme catalyzed reactions, each of which requires an ATP equivalent. 19 BioC 3021 Notes Robert Roon The first step in this bypass is catalyzed by pyruvate carboxylase, an ATP-dependent reaction that uses biotin as a cofactor. The biotin cofactor is used in many carboxylation reactions, and we will look at it in more detail later in the course when we consider the carboxylation of acetylCoA in fatty acid biosynthesis. The product of the reaction is oxaloacetate, which we have seen before as the “sparking” compound in the TCA cycle. If oxaloacetate needs to be replenished in the TCA cycle, this reaction can provide it. In the sequence of gluconeogenesis, which we are looking at now, the oxaloacetate is converted to phosphoenolpyruvate. The second step in this bypass is catalyzed by phosphoenolpyruvate carboxykinase. That enzyme uses GTP instead of ATP (Which is a thermodynamically equivalent source of energy). The formation of phosphoenolpyruvate in this reaction is energized both by GTP cleavage to GDP and by the decarboxylation of oxaloacetate. The addition of a carboxyl group is a transient event, the function of which is to energize the formation of phosphoenolpyruvate. We will see that most biological decarboxylation reactions are energetically favorable in the direction of product formation. This same strategy of carboxylation followed by decarboxylation is used in the biosynthesis of fatty acids to provide extra energy. The bottom line is that it takes two ATP equivalents to convert pyruvate to phosphoenolpyruvate, whereas the reverse reaction in glycolysis only generates one ATP. That imbalance between the catabolic and anabolic reactions is one of the key features in giving both glycolysis and gluconeogenesis a net negative ΔG. Slide 45. Interconversion of fructose 1-6-bisphosphate and fructose 6-phosphate. The second reaction that must be bypassed in gluconeogenesis is phosphofructokinase. The reaction catalyzed by phosphofructokinase has a very favorable negative ΔG, so its 20 BioC 3021 Notes Robert Roon reversal requires a bypass. In this case, we see a second strategy for changing the energetics of a reaction. That is the substitution of a phosphatase for a kinase. In general, reactions that are coupled to ATP hydrolysis are energetically favorable. It is also true that the hydrolysis of an organic phosphate molecule to release inorganic phosphate is generally favorable. So, the release of the phosphate from fructose 1-6-bisphosphate as inorganic phosphate gives the formation of fructose 6-phosphate a negative ΔG. This allows that bypass reaction to proceed in the direction of product formation. Slide 46. Glucose-6-Phosphate Phosphatase. The last reaction that must be bypassed in gluconeogenesis is the hexokinase reaction. Again, the involvement of ATP hydrolysis in hexokinase gives the reaction a favorable negative ΔG, and that makes its reversal energetically unfavorable. To compensate for that, the ATP-dependent reaction is again bypassed by a phosphatase reaction. The same change in energetics applies to these two reactions—that is, the substitution of a phosphatase for a kinase gives the reverse reaction a favorable, negative ΔG. Slide 47. Reciprocal Regulation of Glycolysis and Gluconeogenesis. We have now looked at two pathways running between the same pair of compounds—glucose and lactate. In glycolysis, the catabolic direction, the process generates two ATP’s. In gluconeogenesis, the anabolic direction, there are six ATP’s used. It does not take higher math (lower math will suffice) to see that the uncontrolled simultaneous function of these two pathways would burn up ATP while producing no useful work. (Heat would be generated, but no work would be accomplished.) So it does not take a leap of faith to conclude that these two processes are regulated to prevent them from functioning rapidly at the same time—that glycolysis is allowed to function when extra ATP is 21 BioC 3021 Notes Robert Roon needed, and gluconeogenesis occurs when the supply of ATP is sufficient. Both glycolysis and gluconeogenesis are, in fact, subject to exquisite regulation at many levels. This process is still being intensely investigated, and there are a number of questions still to be answered. The “facts” about this regulation that appear in textbooks are sometimes inaccurate and often incomplete. For that reason, I would like you to assimilate a set of generalizations about the regulation of glycolysis and gluconeogenesis rather than learning detailed facts about which compound does what to a particular enzyme. So here are some generalizations: 1. While almost every reaction in glycolysis and gluconeogenesis is subject to some type of control, it appears as if the bypass sites are subject to the most stringent regulation. These are the essentially irreversible reactions in glycolysis involving the interconversion of glucose and glucose 6-phosphate, the interconversion of fructose 6-phosphate and fructose 1-6- bisphosphate, and the interconversion of phosphoenolpyruvate and pyruvate. 2. There is a reason that regulation is focused at the bypass steps. Because there are distinct enzymes functioning in each direction, it allows for regulation in one direction without exerting the same type of regulation on the other direction. For example, you can stimulate or inhibit gluconeogenesis without effecting glycolysis. In contrast, if the reversible steps were targeted for regulation, inhibition of glycolysis would also result in inhibition of gluconeogenesis. 3. The regulation of glycolysis and gluconeogenesis involves allosteric control mechanisms in which metabolites bind to target enzymes at regulatory sites and either activate or inhibit enzyme activity. 22 BioC 3021 Notes Robert Roon 4. Glycolysis generates ATP so it is activated when the ratio of ATP/ADP and AMP is low, and inactivated when that ratio is high. 5. Gluconeogenesis uses ATP so it is activated when the ratio of ATP/ADP and AMP is high, and inactivated when that ratio is low. 6. High levels of acetylCoA and citrate tend to inhibit glycolysis and stimulate gluconeogenesis. The buildup of these intermediates is a signal that the TCA cycle is overloaded and does not need more acetylCoA to be fed in from glycolysis. 7. There is a very interesting regulatory circuit involving fructose 2-6-bisphosphate. Note that this is “2-6” not “1-6”. This is a side product of glycolysis that is synthesized and broken down by a bifunctional enzyme. The regulation of this process is very complex. The fructose 2-6-bisphosphate stimulates phosphofructokinase and inhibits fructose 1-6-bisphosphate phosphatase. Slide 48. Energy Charge Formula. The “energy charge” concept was developed by Daniel Atkinson. What the formula does is to measure the percentage of adenylate material that carries a “high energy” phosphate. ATP gets two points in the numerator of the formula because it has two phosphoanhydride bonds that can be hydrolyzed to drive reactions. ADP gets one point because it has one phosphoanhydride bond. AMP gets no points because it has no phosphoanhydride bonds. The denominator is the sum of all three compounds—ATP, ADP, and AMP—the total of all of the phosphorylated adenylate material in the cell. The ½ term normalizes the energy charge to ATP, which is arbitrarily given a value of one. In the graph, the value of one would occur at the right edge when 100% of the adenylate material was in the form of ATP, and a value of zero would occur when it was 100% in the form of AMP. So, when a high energy charge prevails (ATP levels are high), glycolysis is inhibited and gluconeogenesis is stimulated. In 23 BioC 3021 Notes Robert Roon contrast, when the energy charge is low (ATP levels are low and AMP levels are high), glycolysis is stimulated and gluconeogenesis is inhibited. Slide 49. Sites in Glycolysis Subject to Negative Feedback. When an organism is resting, the three essentially irreversible enzymes are subject to negative feedback. Hexokinase is inhibited by its product, glucose 6-phosphate. Phosphofructokinase and pyruvate kinase are inhibited by a high energy charge (high ATP). Slide 50. Sites in Glycolysis Subject to Positive Feedback. During exercise, phosphofructokinase is subject to positive feedback by a low energy charge (high AMP). Pyruvate kinase is activated by feedforward stimulation by fructose 1-6-bisphosphate. 24