Glycogen Metabolism Notes
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Glycogen is a polymer of glucose (up to 120,000 glucose residues) and is a primary carbohydrate storage form in animals. The polymer is composed of units of glucose linked alpha(1-4) with branches occurring alpha(1-6) approximately every 8-12 residues. The end of the molecule containing a free carbon number one on glucose is called a reducing end. The other ends are all called non-reducing ends. Related polymers in plants include starch (alpha(1-4) polymers only) and amylopectin (alpha (1-6) branches every 24-30 residues).
Glycogen provides an additional source of glucose besides that produced via gluconeogenesis. Because glycogen contains so many glucoses, it acts like a battery backup for the body, providing a quick source of glucose when needed and providing a place to store excess glucose when glucose concentrations in the blood rise. The branching of glycogen is an important feature of the molecule metabolically as well. Since glycogen is broken down from the "ends" of the molecule, more branches translate to more ends, and more glucose that can be released at once. Liver and skeletal muscle are primary sites in the body where glycogen is found.
The primary advantages of storage carbohydrates in animals are that
1) energy is not released from fat (other major
energy storage form in animals) as fast as from glycogen;
2) glycolysis provides a mechanism of anaerobic metabolism (important in muscle cells that cannot get oxygen as fast as needed); and
3) glycogen provides a means of maintaining glucose levels that cannot be provided by fat.
Breakdown of glycogen involves 1) release of glucose-1-phosphate (G1P), 2) rearranging the remaining glycogen (as necessary) to permit continued breakdown, and 3) conversion of G1P to G6P for further metabolism. Remember that G6P can be 1) broken down in glycolysis, 2) converted to glucose by gluconeogenesis, and 3) oxidized in the pentose phosphate pathway.
The concentration of reactants and products of glycogen breakdown are such that hydrolysis of glycogen to G1P in the cell is favored (though the reaction would not be favored if the ratios of products were not skewed).
Just as in gluconeogenesis, the cell has a separate mechanism for glycogen synthesis that is distinct from glycogen breakdown. As noted previously, this allows the cell to separately control the reactions, avoiding futile cycles, and enabling a process to occur efficiently (synthesis of glycogen) that would not occur if it were simply the reversal of glycogen breakdown. Synthesis of glycogen starts with G1P, which is converted to an 'activated' intermediate, UDP-glucose. This activated intermediate is what 'adds' the glucose to the growing glycogen chain. Once the glucose is added to glycogen, the glycogen molecule may need to be rearranged to make it available for metabolism.
Regulation is complex, occurring both allosterically and via hormone-receptor controlled events that result in protein phosphorylation or dephosphorylation.Glycogen Breakdown
Glycogen Phosphorylase catalyzes breakdown of glycogen into Glucose-1-Phosphate (G1P). The reaction (see HERE) that produces G1P from glycogen is a phosphorolysis, not a hydrolysis reaction. The distinction is that hydrolysis reactions use water to cleave bigger molecules into smaller ones, but phosphorolysis reactions instead use phosphate for the same purpose. Note that the phosphate is just that - it does NOT come from ATP. Since ATP is NOT used to put phosphate on G1P, the reaction saves the cell energy. In addition, the phosphate on the G1P helps prevent the molecule from leaving the cell as it is. Glycogen phosphorylase will only act on non-reducing ends of a glycogen chain that are at least 5 glucoses away from a branch point. A second enzyme, Glycogen Debranching Enzyme (GDE), is therefore needed to convert alpha(1-6) branches to alpha(1-4) branches (see HERE). GDE acts on glycogen branches that have reached their limit of hydrolysis with GP. It acts to transfer a trisaccharide from a 1,6 branch onto an adjacent 1,4 branch, leaving a single glucose at the 1,6 branch. Note that the enzyme also catalyzes the hydrolysis of the remaining glucose at the 1,6 branch point (see HERE). Thus, the breakdown products from glycogen are G1P and glucose (mostly G1P, however). Glucose can, of course, be converted to Glucose-6-Phosphate (G6P) as the first step in glycolysis by either hexokinase or glucokinase.
G1P can be converted to G6P by action of an enzyme called Phosphoglucomutase. Note that the mechanism of action (HERE) of phosphoglucomutase involves formation of a transient intermediate of glucose-1,6-bisphosphate before the G6P is produced. This reaction is readily reversible, allowing G6P and G1P to be interconverted as the concentration of one or the other increases. This is important, because phosphoglucomutase is needed to form G1P for glycogen biosynthesis.
As noted in the class previously, the liver is essential for monitoring and maintaining a relatively constant level of glucose in the bloodstream. Conditions leading to glucose concentrations being too high or too low are very detrimental. It was noted that the liver is involved in gluconeogenesis in the Cori cycle and it should come as no surprise that the liver is involved in glycogen breakdown and synthesis because these pathways allow the liver to remove glucose from the bloodstream for glycogen synthesis when blood glucose is high and to release glucose into the bloodstream from glycogen breakdown when blood glucose levels are too low. You may recall that the enzyme glucose-6-phosphatase (G6Pase) catalyzes the last step of gluconeogenesis - conversion of G6P to glucose + phosphate. This enzyme is necessary also for release of glucose into the bloodstream from glycogen metabolism (glycogen -> G1P -> G6P -> Glucose). It is interesting to note that G6Pase is ABSENT FROM MUSCLE. This is because muscle does NOT export glucose. the liver, on the other hand, DOES export glucose and thus has abundant supplies of the enzyme.
Mechanism of Glycogen Phosphorylase Action
Glycogen phosphorylase uses phosphate instead of water to break down glycogen. Glycogen phosphorylase manages to use phosphate to catalyze glycogen breakdown by employing the coenzyme pyridoxal phosphate (PLP). This coenzyme forms a Schiff base intermediate with a lysine residue of the enzyme (see HERE). The 5' phosphate of PLP act as a proton donor and then as a proton acceptor (acid-base catalyst). Orthophosphate acts to donate a proton to carbon 4 of the glycogen chain and simultaneously acquire a proton from PLP. The carbonium ion thus created is attacked by orthophosphate to form alpha-glucose-1-phosphate. The mechanism can be seen HERE.
Regulation of Glycogen Phosphorylase
In order to avoid a futile cycle of glycogen synthesis and breakdown simultaneously, cells have evolved an elaborate set of controls that ensure only one pathway is primarily active at a time. Regulation occurs on the enzymes glycogen phosphorylase and glycogen synthase, and involves allosterism, covalent modification of enzymes and, ultimately, hormonal control.
Allosteric factors - ATP, G6P, AMP.
Glycogen phosphorylase is regulated by both allosteric factors and by covalent modification (phosphorylation). Its regulation is consistent with the energy needs of the cell. High energy substrates (ATP, G6P, glucose) inhibit GP, while low energy substrates (AMP, others) activate it. The enzyme uses a cofactor, pyridoxal phosphate (PLP). Regulation of glycogen phosphorylase varies a bit, depending on the tissue in which it is found. For example, the liver makes glucose for the body, but muscles do not and depend on the liver for much of their glucose. Regulation of glycogen breakdown in these tissues is adjusted accordingly, as will be seen.
Muscle Glycogen Phosphorylase Regulation
In muscle, glycogen phosphorylase exists as a usually active form (I'll call GPa) and a usually inactive form (GPb). (See structures HERE). It is not readily apparent from the figure, but GPa and GPb differ chemically only in that GPa is phosphorylated (two phosphates), but GPb is not. GPb is converted to GPa by phosphorylation by an enzyme known as phosphorylase kinase. Note also that both GPa and GPb can exist in an 'R' state and a 'T' state (see HERE). For both GPa and GPb, the R state is the active form of the enzyme. this means, therefore, that the equilibrium between the T and R states differs for GPa and GPb. GPa's equilibrium of forms favors the R state (thus usually active) and GPb's equilibrium of forms favors the T state (thus usually inactive).
Conversions between the T and R states of GPb involve allosteric interactions. GPb can convert from the T state to the GPb R state by binding AMP (see HERE). Thus, a low energy state of the cell (signalled by AMP) can ACTIVATE GPb. Normally this does not happen. Thus, the R state of GPb is strongly favored by AMP. On the other hand, ATP and/or G6P are usually present at high enough concentration in the cell that binds to GPb (instead of AMP) and favors the T state (inactive). Binding of ATP or G6P to GPb thus favors the inactive state of GPb. GPa is NOT affected by AMP, ATP, or G6P and is usually found in the R state (active).
Remember that muscles do not have G6Pase so that when G6P is produced from glycogen breakdown, it can enter glycolysis. When you begin exercise, most of the glycogen phosphorylase is in the GPb form and inactive. As AMP builds up from the use of ATP, GPb is converted from the T to the R state. Further exercise results in hormonal stimulation that results ultimately in phosphorylation of GPb to form GPa.
Differences in Liver Glycogen Phosphorylase
Glycogen phosphorylase is very similar to, but not identical to the one found in muscle. Related enzymes like these are called isozymes, for the fact that they are different forms of the same enzyme. The subtle changes in liver glycogen phosphorylase cause GPa to have a property that muscle glycogen phosphorylase does not - namely that GPa is allosterically inhibited by the accumulation of glucose. Glucose binding to liver GPa causes it to convert into the T form (inactive) (see HERE). This does not happen in muscle and is an important control in the liver, allowing it to shut down when glucose accumulates faster than it is needed. In addition, the GPb form of the enzyme is insensitive to AMP, unlike the muscle GPb.
Activation of Glycogen Phosphorylase
Because the relative amounts of GPa and GPb largely govern the overall process of glycogen breakdown, it is important to understand the controls on the enzymes that interconvert GPa and GPb. Interconversion of GPa and GPb is accomplished by the enzyme Phosphorylase Kinase, which transfers phosphates from 2 ATPs to GPb to form GPa. Phosphorylase kinase is present in a low activity form and a high activity form. The enzyme can be activated by two mechanisms (see HERE). First, it can be phosphorylated by Protein Kinase A (PKA). Phosphorylation of phosphorylase kinase ACTIVATES the enzyme. (Recall that PKA is activated by cAMP). Another way to activate the enzyme is with calcium. Remember that calcium is also a second messenger in the cell (in addition to cAMP) and can bind to the protein calmodulin. Calmodulin, it turns out, is a subunit of phosphorylase kinase. Activation of phosphorylase kinase by calcium is VERY important in muscle, which uses the ion to trigger musclular contraction. Thus, the same ion that stimulates muscular contraction also activates phosphorylase kinase, which activates glycogen phosphorylase, which releases G1P from glycogen, which can be used to make ATP to support muscular contraction.
Overall Glycogen Breakdown Regulation
As noted above, phosphorylase kinase is activated by PKA. PKA is, of course, activated by cAMP, which is, in turn produced by adenlyate cyclase after activation by a G protein. G proteins are activated ultimately by binding of ligands to specific 7TM receptors. Common ligands for receptors include epinephrine (binds beta-adrenergic receptor) and glucagon (bind glucagon receptor). Epinephrine exerts it greatest effects on muscle and glucagon works preferentially on the liver. The overall regulatory pathway (called a regulatory cascade) is shown HERE. Regulatory cascades are extraordinarily effective for 1) amplifying signals - producing a LARGE response to a SMALL signal) and 2) working quickily.
Epinephrine (or glucagon) -> receptor -> stimulate adenylate cyclase -> cAMP ->
activates PKA -> activates PbK -> converts GPb to GPa -> cleaves glycogen to release G1P.
Note in this cascade that putting on phosphates has the effect of activating enzymes. In the similar regulation of glycolysis/gluconeogenesis, putting phosphate onto PFK2/F2,6BPase favored gluconeogenesis. Thus, a VERY important point to recognize is that putting phosphates onto proteins favors glucose production/release.
Turning Off Glycogen Breakdown
As you should recall, turning OFF signals is as important, if not more so, as turning them ON. The steps in the glycogen breakdown regulatory pathway can be reversed at several levels. First, the ligand can leave the receptor. Second, the G proteins have an inherent GTPase activity that serves to turn them off over time. Third, cells have phosphodiesterase (inhibited by caffeine) for breaking down cAMP. Fourth, an enzyme known as protein phosphatase (also called phosphoprotein phosphatase) can remove phosphates from phosphorylase kinase (inactivating it) AND from GPa, converting it to the much less active GPb.Glycogen Synthesis
The anabolic pathway contrasting with glycogen breakdown is that of glycogen synthesis. Just as cells reciprocally regulate glycolysis and gluconeogenesis to prevent a futile cycle, so too do cells use reciprocal schemes to regulate glycogen breakdown and synthesis. Let us first consider the steps in glycogen synthesis. 1) Glycogen synthesis from glucose involves phosphorylation to form G6P, and isomerization to form G1P (using phosphoglucomutase common to glycogen breakdown). There is an energetic barrier to the direct incorporation of G1P into glycogen due to concentrations of factors that favor breakdown of glycogen under normal conditions over synthesis of glycogen. Cells provide a pathway around the glycogen synthesis barrier with the use of UTP. This is akin to the barrier of reversing Pyruvate to PEP in gluconeogenesis, which is overcome by going through oxaloacetate. The first reaction, catalyzed by UDP-Glucose Pyrophosphorylase (UGPP), converts G1P and UTP into uridine diphosphoglucose (UDPG) (see HERE).
G1P + UTP <=> UDPG + PPi
The high energy phosphate bonds of UTP make the formation of UDPG energetically favorable. In addition, note that the product on the right side include pyrophosphate. Pyrophosphate can readily be broken down to Pi + Pi by the enzyme pyrophosphatase (in a hydrolysis reaction). Breaking the PPi product down in this manner helps to pull the reaction even further to the right.
2) The second step of glycogen synthesis is catalyzed by the enzyme Glycogen Synthase. The reaction combines carbon #1 of the UDPG-derived glucose onto the carbon #4 of the non-reducing end of a glycogen chain. to form the familiar alpha(1,4) glycogen links. Another product of the reaction is UDP. This reaction too, is energetically favorable. The UDPG "side step" in glycogen synthesis thus uses the energy of UTP and UDP to make energetically favorable a reaction (addition of glucose to glycogen) that otherwise would not be. Thus by inputting energy via a side-stepping reaction, the cell manages to overcome a significant energy barrier. It is also worth noting in passing that Glycogen Synthase will only add glucose units from UDPG onto a preexisting glycogen chain that has at least four glucose residues. Linkage of the first few glucose units to form the minimal "primer" needed for glycogen synthase recognition is catalyzed by a protein called Glycogenin, which attaches to the first glucose and catalyzes linkage of the first eight glucoses by alpha(1,4) bonds.
3) The characteristic alpha(1,6) branches of glycogen are the products of an enzyme with the unwieldy name Amylo-(1,4 to 1,6)-transglycosylase. I will refer to it as Branching Enzyme. Branching Enzyme breaks alpha(1,4) chains and carries the broken chain to the carbon #6 and forms an alpha(1,6) linkage. A few structural parameters of the chain must be considered.
Branching enzyme prefers chains about 7 glucose residues in length from a branch at least 11 glucose residues long, and the new branch must be at least 4 residues from the previous one. Interestingly, hydrolysis of alpha(1,4) bonds releases more energy than hydrolysis of alpha(1,6) bonds. Thus branching, which breaks alpha(1,4) bonds and forms alpha(1,6) bonds is energetically favored. Remember that the reverse reaction, debranching (catalyzed by GDE) involves transfer of a trisaccharide (cleaved at alpha(1,4)) to another alpha(1,4) bond. This reaction is energetically neutral. The last glucose, linked alpha(1,6) from the original branch is cleaved to release glucose, which is also energetically favored. If instead, the reaction involved transfer of the entire branch, which would require an alpha(1,6) cleavage and an alpha(1,4) linkage, the reaction would not be energetically favored. Thus, puzzling reactions inside cells, such as formation of UDPG in glycogen synthesis and cleavage one glucose residue from a glycogen alpha(1,6) branch point followed by hydrolysis of the remaining glucose at the alpha(1,6) joint, have simple explanations as mechanisms for working around energy barriers.Regulation of Glycogen Synthesis
The pattern of regulation of glycogen biosynthesis is similar to that of glycogen breakdown. It also has a cascading covalent modification system similar to the glycogen breakdown system described above. In fact, part of the system is identical to glycogen breakdown. Epinephrine or glucagon stimulates adenylate cyclase to make cAMP, which activates PKA, which activates phosphorylase kinase. As you should recall, this is the same as for glycogen breakdown. In glycogen breakdown, phosphorylase kinase phosphorylates GPb to the more active form, GPa. In glycogen synthesis, phosphorylase kinase phosphorylates the active form of Glycogen Synthase (GSa), and converts it into the usually inactive b form (called GSb). Note the conventions for glycogen synthase and glycogen phosphorylase. For both enzymes, the more active forms are called the 'a' forms (GPa and GSa) and the less active forms are called the 'b' forms (GPb and GSb). One MAJOR difference, however, is that GPa has a phosphate, but GSa does not and GPb has no phosphate, but GSb does. Thus phosphorylation and dephosphorylation have OPPOSITE EFFECTS on the enzymes of glycogen metabolism. This is the hallmark of reciprocal regulation, just as you saw in glucose metabolism (see HERE). It is of note that the less active glycogen synthase form, GSb, can be activated by G6P. Recall that G6P had the exact opposite effect on GPb. It is also worth noting that glycogen synthase can be phosphorylated by kinases other than phosphorylase kinase. One of these is Calmodulin-dependent protein kinase, yet another protein that can be turned on by calcium. Thus, calcium activates glycogen breakdown and inhibits glycogen synthesis.
Reversing Effects of Kinases
Glycogen synthase, glycogen phosphorylase (and phosphorylase kinase) can be dephosphorylated by several enzymes called phosphatases. One of these is called Protein Phosphatase 1 (PP - note to avoid confusion with PP-In below, I refer to the enzyme as PP instead of PP1 ). When PP is active, glycogen breakdown is inhibited (because GPa is converted to GPb) and glycogen synthesis is favored (because GPb is converted to GPa). PP has three subunits. One of these, called RG1, which provides a link between the phosphatase portion of the enzyme and glycogen (see HERE). PP is in turn regulated by another inhibitor called Phosphoprotein Phosphatase Inhibitor (PP-In). As seen in the figure, both RG1 and PP-In can be phosphorylated (by PKA). Phosphorylation of RG1 causes PP to dissociate in a less active form. Phosphorylation of PP-In causes it to bind PP, completely inactivating it. Again, note that phosphorylation by PKA is completely consistent with favoring glycogen breakdown. PKA phosphorylation turns on glycogen phosphorylase and turns off glycogen synthase. It also activates PP-In, which turns off the phosphatase (PP) that would normally activate glycogen synthase by dephosphorylating it.
Action by Insulin
How, you might wonder, is PP activated? Another hormone, insulin, is released when blood glucose concentration is high, initiating a cascade (see HERE). Part of this cascade activates an insulin-sensitive protein kinase. Note that it too phosphorylates RG1, but notably this occurs at a different site on the subunit than phosphorylation by PKA. Phosphorylation of RG1 by insulin-sensitive protein kinase causes PP to be activated, stimulating dephosphorylation, and thus activating glycogen synthesis and inhibiting glycogen breakdown. Again, there is reciprocal regulation of glycogen synthesis and degradation.
Maintaining Blood Glucose Levels
After a meal, blood glucose levels rise and glycogen synthesis in the liver is stimulated. GPa in the liver rises and falls with changing blood glucose levels. It turns out that GPa acts as a glucose sensor in the liver. Remember that binding of glucose to GPa in the liver converts it from the R to the T state (inactive). This conformational change also enhances the ability of PP to dephosphorylate GPa, converting it to the GPb form. Interestingly, PP binds GPa tightly normally, but is only active when GPa is in the T state. Conversion of GPa to GPb by the PP causes the PP to be released, which then goes to activate glycogen synthase (converting GSb to GSa) (see HERE). Since GPa is usually in excess compared to PP, PP is only free to dephosphorylate GSb AFTER GPa is converted to GPb. Thus a futile cycle is avoided.
Glycogen Storage Diseases
Numerous diseases relating to glycogen metabolism are known. von Gierke's disease results from absence of G6Pase in the liver. In this disease, the liver glycogen is normal in structure, but is present in VERY large amounts. Remember that G6P is essential in the liver for release of glucose into the bloodstream. If G6P accumulates, it will be ultimately made into glycogen. As shown in the Table (HERE), glycogen storage diseases are traced to numerous enzymes relating to glycogen (and related) metabolism. Note that McArdle's disease involves loss of the glycogen phosphorylase in muscle, yet these patients survive. They do experience painful muscle cramps on strenuous exercise, but are otherwise normal. The figure (HERE) illustrates the reason for the discomfort.