Lecture 15: Glycogen Metabolism and the Pentose Pathway PDF

BioC 3021 Notes Robert Roon Lecture 15: Glycogen Metabolism and the Pentose Pathway Slide 1. Glycogen Metabolism and the Pentose Pathway In this lecture, we will consider the synthesis and breakdown of glycogen, the major carbohydrate storage compound in hu...

BioC 3021 Notes Robert Roon Lecture 15: Glycogen Metabolism and the Pentose Pathway Slide 1. Glycogen Metabolism and the Pentose Pathway In this lecture, we will consider the synthesis and breakdown of glycogen, the major carbohydrate storage compound in humans. We will also look at the pentose pathway, which provides the mechanism for generating NADPH. Slide 2. Glycogen. The glucose polymer, glycogen, serves as the major form of carbohydrate storage in the liver and muscle of humans. Glycogen has a backbone of α-1,4 linked glucose units with α-1,6 branches occurring about every 10-12 glucose units. The largest stores of glycogen are found in liver and muscle. Slide 3. Glycogen Structure. The glycogen polymer contains about 90% 1-4-linked glucose units and 10% 1-6-linked glucose units. The extensive branching of glycogen provides many non-reducing ends that are subject to enzymatic hydrolysis reactions. There is only one reducing end where a glucose unit could undergo mutarotation to the open chain form. Slide 4. Phosphorylase a Cleaves Glycogen. The enzyme phosphorylase a attacks the non-reducing ends of glycogen, releasing 1,4-linked glucose units as glucose-1-P. This reaction proceeds in the direction of product formation. Slide 5. Glycogen degradation. Phosphorylase a cannot attack glucose units that are near to 1,6- branch points, so a transferase enzyme and an α-1,6-glucosidase enzyme convert the 1,6-branches into additional linear 1,4-linked 1 BioC 3021 Notes Robert Roon glucose units. That transformation allows phosphorylase a action to continue further into the glycogen molecule. Slide 6. Phosphoglucomutase. The glucose-1-P units produced by phosphorylase a are converted to glucose-6-P by the enzyme phosphoglucomutase. The glucose- 6-P can then enter into the glycolysis pathway. Slide 7. Glycogen Synthesis. Glycogen can be synthesized from glucose using different anabolic enzymes than those used for glycogen breakdown. -The synthesis of glycogen is the reversal of glycogen breakdown. The cleavage of glycogen is an energy releasing reaction, so energy input is necessary to make the reverse reaction proceed. -Glycogen synthesis employs a set of enzymes to reverse the action of phosphorylase a. -Glycogen synthesis proceeds through an activated (UDP-glucose) sugar nucleotide intermediate. Slide 8. UDP-Glucose Pyrophosphorylase The first step in glycogen synthesis is catalyzed by UDP-glucose pyrophosphorylase. In that reaction, glucose-1-P reacts with UTP to form UDP-glucose and release an inorganic pyrophosphate (PPi). Slide 9. Inorganic Pyrophosphatase. The pyrophosphate (PPi) released in the previous reaction can be hydrolyzed to form two Pi's. The hydrolysis of PPi is an energy releasing process that removes the product of the previous reaction and helps to drive the overall reaction sequence toward product formation. Slide 10. Glycogen synthetase. 2 BioC 3021 Notes Robert Roon In the second step in glycogen synthesis, a synthase enzyme transfers a glucose unit from UDP-glucose to the non-reducing end of glycogen. UDP is released as the other product. Slide 11. Summary of Reactions in Glycogen Breakdown and Synthesis. From this overview slide, it is clear that glycogen breakdown and synthesis involve different enzyme systems. There are a number of important reasons why this is necessary. -The use of different enzymes allows both breakdown and synthesis of glycogen to be energy releasing processes. This is necessary so that both reaction sequences can proceed in the direction of product formation. -The use of alternate enzyme systems allows both systems to be subject to hormonal regulation. The net result of hormonal regulation is that both systems do not function simultaneously. If they did, the net result would be a futile cycle of enzyme activity that would waste energy and yield no net product. One cycle around the system results in the net hydrolysis of one ATP equivalent. Slide 12. Hormone Definition. Hormones are organic compounds that are synthesized in one tissue and are transported through the blood to another tissue, where they exert an effect of metabolic processes. Slide 13. Adrenaline is Derived from Tyrosine. Adrenaline (also called epinephrine) is an example of a low molecular weight hormone that is derived from an amino acid (tyrosine). Slide 14. Epinephrine Cascade. Epinephrine stimulates a series of reactions that result in the activation of the catabolic enzyme phosphorylase a, which 3 BioC 3021 Notes Robert Roon degrades glycogen. The regulatory cascade includes a number of features that you will encounter often in biochemistry: -The binding of a hormone to activate an extracellular receptor protein -The activation of a G protein by interaction with an activated extracellular receptor -The activation of adenylate cyclase by interaction with a G protein -The conversion of ATP to Cyclic AMP by the adenylate cyclase enzyme -The activation of a protein kinase enzyme by cyclic AMP -The activation of phosphorylase kinase by a phosphorylation reaction catalyzed by the cyclic AMP sensitive kinase -The conversion of phosphorylase b to phosphorylase a through phosphorylation by the phosphorylase kinase -The cleavage of glycogen by phosphorylase a Slide 15. Inactivation of Glycogen Synthase. Epinephrine also stimulates an enzyme cascade that results in the inactivation of the anabolic enzyme, glycogen synthase, which adds glucose units to glycogen. This regulatory cascade includes most of the same features seen in the activation of glycogen catabolism, except that the end of this cascade results in the inactivation of an enzyme and the suppression of glycogen synthesis. The dual function of the epinephrine cascade, in activating glycogen breakdown and simultaneously inactivating glycogen synthesis, prevents these reactions from catalyzing a futile cycle in which energy is wasted. Slide 16. Adenylate Cyclase. Adenylate cyclase converts ATP to the second messenger, cyclic- AMP. Second messengers are intracellular signaling agents that 4 BioC 3021 Notes Robert Roon are often synthesized or degraded in response to the action of an extracellular hormone. In the cell, the adenylate cyclase reaction is essentially irreversible because one of the products, inorganic pyrophosphate, is rapidly degraded by a pyrophosphatase enzyme. This strategy (the breakdown of inorganic pyrophosphate) is used often in biochemical pathways to force a reaction to go in the direction of product formation. Slide 17. Pentose Phosphate Pathway. The pentose phosphate pathway is an alternate pathway to glycolysis that provides a source of NADPH for use in reductive biosynthesis. This pathway also generates pentose sugars. Some of the reactions of the pentose pathway are integrated with glycolysis and gluconeogenesis. All the reactions of the pentose phosphate pathway take place in the cytosol. Slide 18. Biosynthetic Pathways Requiring NADPH. Most anabolic (biosynthetic) pathways are also reductive. That is, the products of biosynthesis are generally more reduced (have a higher % of hydrogen atoms) than the starting materials. The reducing power for anabolic pathways comes from NADPH. Fatty acid biosynthesis and nucleotide biosynthesis are examples of anabolic pathways that utilize NADPH. Slide 19. Mode 4 of the Pentose Phosphate Pathway. This graphic shows one way of representing the pentose phosphate pathway. This is a simplified version of mode 4 of the pathway with a number of the reactions omitted. Mode 4 of the pathway is operative when the cell needs NADPH and ATP, but does not need ribose-5-phosphate for nucleic acid biosynthesis. We can break this form of the pathway down into two phases: 5 BioC 3021 Notes Robert Roon -The first phase of the pathway, which runs from glucose 6-P to ribose 5-P, involves the oxidative generation of NADPH. -The second phase of the pathway involves the non-oxidative conversion of five-carbon sugars to intermediates of the TCA cycle, such as fructose 6-P, glyceraldehydes 3-P, and pyruvate. Slide 20. The Oxidative Stage of the Pentose Pathway. In the oxidative phase, the starting material, glucose 6-P, is oxidized to ribulose 5-P. In two of the reactions, the cofactor NADP+ is reduced to NADPH. In fact, all of the NADPH produced by the pentose phosphate pathway is synthesized in the oxidative phase. The net reaction of the oxidative phase is: glucose 6-phosphate + 2 NADP+ + H20  ribulose 5-phosphate + 2 NADPH + 2 H+ + C02 The pathway could stop right here if it were not for the fact that the cell only needs a limited supply of ribulose 5-P—probably much less than is produced to meet the need for NADPH. So, further reactions are needed to convert excess ribulose 5-P into usable intermediates such as pyruvate, which ultimately are oxidized to carbon dioxide. In other modes of the pentose pathway (not shown), excess ribulose 5-P is converted back to glucose 6-P by gluconeogenesis. Slide 21. The Non-Oxidative Stage of the Pentose Pathway. In the non-oxidative phase, the five carbon sugars xylulose 5-P and ribose-5 P are converted to fructose 6-P and glyceraldehyde 3-P. This phase of the pathway involves a convoluted anastomosing Sandy Johng 7/19/11 7:55 PM series of reactions, which nature designed to confound the naïve. Comment: I’m unfamiliar with this term, but google tells me this is how it’s spelled The products of this phase are mainstream metabolites that can enter glycolysis or gluconeogenesis. Slide 22. Glucose 6-P Dehydrogenase. The first reaction of the oxidative phase is catalyzed by glucose 6- P dehydrogenase. In this reaction, glucose 6-P (in the hemiacetal 6 BioC 3021 Notes Robert Roon form) is oxidized to 6-phosphoglucano-δ-lactone (a cyclic ester), coupled to the reduction of NADP+ to NADPH. It may be a little hard to visualize, because both the substrate and the product are in the cyclic form, but the reaction is the equivalent to oxidizing an aldehyde to an acid. Slide 23. Lactonase. The second reaction in the pathway, catalyzed by lactonase, is a ring opening that converts the lactone to the open chain carboxylic acid, 6-P-gluconate. Slide 24. 6-P-Gluconate Dehydrogenase. In the next reaction, the 6-P-gluconate is decarboxylated and oxidized to ribulose 5-P. The reaction is coupled to the reduction of a second molecule of NADP+ to NADPH. Slide 25. The Oxidative Stage of the Pentose Pathway At this point, we can consider that the oxidative phase of the pathway is finished because the synthesis of NADPH is completed. If the synthesis of NADPH were the only consideration, the pathway could stop here. The problem is that stopping at this point would leave a big pool of ribulose 5-P in the cell. This would not only waste energy, but the accumulation of so much ribulose 5-P within the cell would probably be toxic. For that reason, the pathway continues into the much more complicated non-oxidative stage. Slide 26. The Non-Oxidative Stage of the Pentose Pathway The purpose of the non-oxidative phase is to convert the five carbon intermediate, ribulose 5-P, into other useful intermediates that can be used in glycolysis. It takes a series of five convoluted reactions to accomplish that task. The ribulose 5-P is first converted into two different five carbon compounds, ribose 5-P and xylulose 5-P, by two separate enzymes. These two five carbon 7 BioC 3021 Notes Robert Roon intermediates are then converted to glyceraldehyde 3-P and fructose 6-P by a series of three reactions. Slide 27. Phosphopentose Isomerase. As a first step in the non-oxidative phase, the ketone, ribulose 5-P, can be isomerized to the aldehyde, ribose 5-P. The reaction involves the formation of an unstable endiol intermediate, in which two adjacent carbon-atom-carrying hydroxyl groups are connected by a double bond. The endiol decays into an aldehyde, which is stable. This reaction completes the oxidative phase of the pathway. Slide 28. Phosphopentose Isomerase Mechanism The isomerase reaction goes through an enediol intermediate. Similar enediol intermediates occur in two isomerization reactions that we have seen previously in glycolysis. The mechanism as shown here is exactly analogous to that of phosphoglucose isomerase and triose phosphate isomerase in glycolysis. Learning to recognize such similarities in mechanism will help you memorize new reactions as variations on a theme rather than completely new phenomena. Slide 29. Mode 2 of Pentose Phosphate Pathway. The top part of the pentose pathway to this point is sometimes referred to as the oxidative phase, or Mode 2. (We are not including the formation of xylulose 5-phosphate in this mode.) The products are two molecules of NADPH, one molecule of carbon dioxide, and one molecule of ribose 5-phosphate. If the cell always needed both NADPH and ribose 5-phosphate in the exact ratio of 2 to 1, then this could be the end of the pathway. However, the need for these two substances in that precise ratio is not generally the case. Often, the two products are needed in different ratios, or one is needed, and the other is not. Thus, there is a necessity for modes of the pathway other than mode 2. That is where the non-oxidative phase of the pathway comes into play. 8 BioC 3021 Notes Robert Roon Slide 30. Phosphopentose epimerase. At the beginning of the non-oxidative phase of the pentose pathway, ribose 5-P is converted into xylulose 5-P by an epimerase reaction. This reaction simply changes the chirality of the hydroxyl group at the number three carbon, converting ribulose 5-P to xylulose 5-P. Slide 31. Transketolase. Now we launch into the non-oxidative phase of the pathway. Up to this point, the sequence of reactions has been relatively linear and more-or-less easy to follow. With the exception of the conversion of ribulose 5-phosphate to xylulose 5-phosphate, the reactions are all in an exact linear order. We will now show you a series of reactions that are individually straight-forward, but when we put them all together, it creates a convoluted pattern. There are bifurcations and conjunctions designed to confuse the unwary. In the transketolase reaction, a two carbon fragment (coded in red) is transferred from xylulose 5-phosphate to ribose 5-phosphate. The products of this reaction are glyceraldehyde-3-phosphate and sedoheptulose 7-phosphate. (In this reaction, two C-5 compounds are converted to one C-3 and one C-7 compound.) The sedoheptulose 7-phosphate is a new bird—and a big bird at that with seven carbons. Its stereochemistry is derived from the ribose 5-phosphate, with the C-1 carbonyl carbon of the ribose 5- phosphate becoming a hydroxyl group on C-3 of sedoheptulose 7- phosphate with an orientation to the left. The reaction is reversible, and under some modes of the pentose phosphate pathway, it will actually function in the reverse direction. Slide 32. Thiamine Pyrophosphate. This structure of thiamine pyrophosphate is here to remind you that transketolase uses this coenzyme to transfer the two carbon fragment from xylulose 5-phosphate to ribose 5-phosphate. The site of attachment to the coenzyme is coded in red. This is the 9 BioC 3021 Notes Robert Roon same cofactor that was used to transfer a two carbon fragment in the pyruvate dehydrogenase reaction. Slide 33. Transaldolase. The next reaction involves the two products of the previous reaction-- sedoheptulose 7-phosphate and glyceraldehyde 3- phosphate. The transaldolase enzyme transfers a three carbon fragment (coded in red) from sedoheptulose 7-phosphate to glyceraldehyde 3-phosphate, producing erythrose 4-phosphate and fructose 6-phosphate. In this step, one C-7 and one C-3 compound are converted into one C-4 and one C-6 compound. Slide 34. Transketolase II. Finally (and it can't come too soon), the erythrose 4-phosphate product of the previous step reacts with a second xylulose 5- phosphate (from the phosphopentose epimerase reaction) to give glyceraldehyde-3-phosphate and fructose 6-phosphate. This reaction is catalyzed by the same transketolase enzyme we talked about before, but one of the substrates (erythrose 4-phosphate) and one of the products (fructose 6-phosphate) are different. The reason for all of these bizarre manipulations is to produce stuff that can be metabolized in core reaction pathways (eg. glycolysis.), instead of building up a product such as ribose 5-P that is a useful metabolite, but only in limited quantities. The glyceraldehyde-3-P and fructose 6-P can be used in virtually unlimited amounts because they are intermediates of glycolysis. Slide 35. Stoichiometry of the Non-Oxidative Phase. Chemists mirror nature in that they have a compulsion to balance reaction pathways. This diagram gives a carbon balance for the non-oxidative phase of the pathway. If you do your bookkeeping, you will see that there are three five carbon compounds going into the process for a total of fifteen carbons. There is one three carbon product and two six carbon products for a total of fifteen carbons. 10 BioC 3021 Notes Robert Roon OK. I can relax now and move on. Slice 36. The Non-Oxidative Stage of the Pentose Pathway Take one last look at the non-oxidative stage of the pentose pathway. Try to identify where and how the three five carbon compounds enter into the pathway. Then identify the three carbon product and the two six carbon products. 11