Monday, December 24, 2012

Biochemistry Tutorial Essays: Glycogen Metabolism (Glycogenolysis and Glycogenogenesis)

Glucose, a major metabolic fuels used by living systems is degraded through glycolysis with a view to yield ATP. Nevertheless, until oxidised, glucose cannot stay dissolved in cytosol. This is due to the fact that, if glucose were to be dissolved in cytosol, the molar concentration of glucose in cytosol would be 0.4M, severely tampering with the cells’ osmotic properties. Instead, living organisms have developed two major ways of storing sugars.

Plants store sugars in the form of starch, a mixture of α-amylose and amylopectin, a polysaccharide with branches in the structure every 24 to 30 residues. Instead, animals have developed a way of keeping sugars in their cells, by storing them as glycogen, a chemical compound similar to amylopectin, with the only difference that the branches are to be found every 8 to 14 residues.

Glycogen is insoluble in cytosolic compounds and is therefore maintained in cytosol under the form of small glycogen β-particles, composed of around 55000 glucose residues each, with about 2000 non-reducing ends. Every 20 to 40 β-particles are clustered into α-rosettes. This means of keeping the glucose in a polymerised form so that is basically insoluble in cytosol, reduces the concentration from the 0.4M calculated in the case when all the glucose were to be dissolved in the cytoplasm, to a mere 0.01 μM.

An interesting fact about glycogen is that it basically exists only in skeletal muscles and liver, the latter containing glycogen up to 10% of its mass, as opposed to skeletal muscles, where the total mass of glycogen does not surpass 1-2% out of the muscular mass. Glycogen is an important resource for the human body, as the body would use its glycogen deposits after the glucose levels in the bloodstream have dropped. In this way, the glycogen deposits in muscle can be depleted in no more than one to two hours of intense physical effort and no more than in 24 hours of fasting if were are to talk about the glycogen reserves stocked in the liver.

Glycogenolysis - Glycogen Breakdown

Upon breakdown, the glycogen stored in muscles and liver undergoes three major steps. Firstly, the glycogen chain is shortened one glucose molecule at a time by the use of an enzyme called glycogen phosphorylase, which essentially cleaves the inter-glucose bonds. As a result glucose-1-phosphate is released. The reaction will only take place if the glucose unit is at least five units away from the branching point.

The mechanism for the action of glycogen phosphorylase involves the maintaining of configuration for the glucose units. Therefore, it has been suggested that this occurs via a double nucleophilic attack mechanism, each occurring with inversion of configuration. Here, pyridoxal-5-phosphate plays an essential role as a cofactor. It binds to the Lys 679 residue of the enzyme and it is part of the active site of the enzyme.

The enzyme catalysing this reaction is a dimer that catalyses the controlling step in glycogen breakdown. It comes in two forms, phosphorylase a and phosphorylase b, with the only difference that phosphorylase a has phosphoryl groups esterified to Ser 14 in both of the subunits. The enzyme might assume the inactive form T or the active form R. The reason for the difference in activity between the R and the T state is that the inactive T state has an active site buried into the molecule, inaccessible for the substrate, whereas the R state has an accessible catalytic site and a high affinity for substrate.

In the R form, the enzyme may only be subject to allosteric regulation; however, it may not undergo phosphorylation. For this, the enzyme has to be converted into the inactive T form that can be phosphorylated through the use of phosphorylase kinase, which will take 2 molecules of ATP and convert them to ADP, simultaneous to the phosphorylation of the inactive form of the enzyme. The reverse reaction is essentially a hydrolysis reaction catalysed by phosphoprotein phosphatase. This reaction will, however, not restore ATP as is only releases inorganic Pi. The b form of the active enzyme will suffer allosteric effects from ATP and G6P in what concerns the inhibition of it function, whereas the same form will be allostericaly activated by AMP.

In this specific case, when the action of the phosphorylase enzyme is controlled by phosphorylase kinase, its actions are also highly regulated by the so called glycogen phosphorylase bicyclic cascade. Phosphorylate kinase is controlled at its turn by the activity of two enzymes: an activator, cAMP-dependent protein kinase, which phosphorylates and thus activates phosphorylate kinase, and an inhibitor, phosphoprotein phosphatase-1, which dephosphorylates and thereby deactivates both phosphorylate kinase and glycogen phosphorylase.

Similarly, phosphorylase kinase is also controlled by the concentration of calcium ions in the cytosol. As calcium is an essential metal present in muscle contraction, its action on the glycogen breakdown enzymes would be expected to be one of activation, and in reality, this is also the case. Virtually, a calcium concentration as low as 10-7 M can activate the phosphorylase kinase enzyme, which is subject to covalent modification and significant conformational change. The phosphorylase kinase enzyme is essentially a tetramer with each unit made up of four different sub-units, one of which has full catalytic abilities (γ) and three of which play regulatory roles (α,β and δ). Nonetheless, the δ sub-unit, also known as calmodulin, is sensitive to calcium ions. When calcium ions bind to any of the four calmodulin sub-units of the protein, these undergo extensive conformational changes that will have as a major outcome, the activation of the phosphorylase kinase enzyme.

The next step of glycogen breakdown is the conversion of G1P to G6P by phosphoglucomutase, a reaction that involves glucose-1,6-bisphosphate as an intermediate. The mechanism involves the phosphorylation of the C6 on the G1P by the enzyme, followed by rephosphorylation of the enzyme by the substrate, thus removing the C1 phosphoryl group. It is known that the dissociation of G1,6P from the enzyme deactivates the enzyme. Therefore, we can draw the conclusion that catalytic quantities of G1,6P are necessary for keeping the enzyme in the fully active state.

Glycogen debranching enzyme, the next enzyme in this reaction chain acts as a glucosyl transferase, by transferring an α(1→4) trisaccharide unit from a branch of glycogen to the non-reducing end of another branch, in order for the phosphorylase to pursue the cleavage of sugar-sugar bonds. The remaining residue in the branch to the main chain is hydrolysed by the action of the same debranching enzyme and is set free into the cytosol as glucose.

Glycogenogenesis - Glycogen Synthesis

Glycogen breakdown and synthesis are both important metabolic processes. But when the question whether or not they are the same process just in reverse is raised, two approaches are available to answer the question. From a thermodynamic point of view it is better to have different pathways for glycogen synthesis and degradation for two reasons. First, reactions catalysed by different enzymes can be independently regulated which allows for a very accurate control of glycogen flux. Secondly, both the pathways may be required under similar concentration of cytosolic metabolites, an impossible situation in the case of product/reactant concentration dependent reversible reactions.

From a medical perspective, it has been shown that in the case of McArdle’s disease, the muscle tissue presents no glycogen phosphorylase activity and is therefore unable to perform glycogen breakdown, although glycogen is present in the tissue. This shows that in the case of glycogen, breakdown of glycogen is not associated with synthesis, since in the case of this specific disease, synthesis occurs without breakdown.

The steps of the glycogen synthesis pathway were elucidated after the discovery of an important compound, relevant in the process, uridine diphosphate glucose (UDPG), a high-energy compound capable of spontaneous donation go glucosyl units to the growing glycogen chain. The reaction is catalysed by UDP-glucose phosphorylase. What happens, is that the phosphoryl oxygen on G1P attacks the alpha phosphoryl group of UTP to form UDPG and release PPi, which is further converted to 2Pi by inorganic pyrophosphatase.

The subsequent reaction is the actual synthesis of glycogen, by glycogen synthase. This is achieved by the transferring of the glucosyl unit of UDPG to the C4 hydroxyl group on a non-reducing end to form a glycosidic bond. The mechanism is thought to involve a glucosyl oxonium ion since the reaction is inhibited by 1,5-gluconolactone, a similar compound that acts as a competitive inhibitor for this enzyme.

Interesting enough is the fact that glycogen synthase cannot initiate the reaction between the first two glucose units. For this, the first step towards the synthesis of glycogen is catalysed by glycogenin, an enzyme that binds to a glucose residue. Glycogenin extends the chain to up to seven units, thus allowing glycogen synthase to take the process from here.

Glycogen branching, the last step of glycogen synthesis, is an important step as it confers the 3D branched structure of glycogen as we know it in all living animals and some of the bacteria. This reaction occurs as a result of the action of the amylo-transglucosylase, better known as branching enzyme, which is distinct from the glycogen debranching enzyme. Branches are formed by the transfer of terminal chain glucose residues, consisting of about 7 glucosyl residues to the hydroxyl groups attached to the C6 of glucose residues. Each transferred segment must come from a chain at least 11 residues long and it must be at least 4 residues away from other branch points.

Hormone Regulation

Hormones also play an important role in the regulation of glycogen breakdown or synthesis. We have seen that several proteins, controlling each other’s actions as enzymes regulate the glycogen phosphorylase reaction, the starting point of the glycogen breakdown process. We have also underlined the importance of calcium as a regulatory agent, in strict connection to its relevance to muscle contraction.

Nonetheless, hormones play an equally important role in the assertion of control on processes such as glycogen breakdown or glycogen synthesis. This has to do with the fact that the two fore-mentioned processes directly affect the concentration of blood sugars, especially that of glucose. Moreover, as the body has to respond very quickly to the fight or flight stress situations, the exertion of an action of adrenergic hormones is beneficial for the organism as it mobilises the glycogen deposits to be rapidly converted into fuels that can undergo the glycolysis pathway, such as glucose or G6P.

First and foremost, when it comes to hormones, the most effect on the liver is exerted by glucagon, while in muscles control is imposed by the action of epinephrine (adrenalin) and noradrenalin (norepinephrine). It has been shown in this specific direction that hormones act at cell surfaces to stimulate adenylate cyclase, thus increasing the cytosolic levels of cyclic AMP. When the cytosolic levels of cAMP increase, the cAMP-dependent protein kinase activity increases, thus increasing the rate of phosphorylation and decreasing the rate of dephosphorylation at the same time.

Because of the amplifying properties of the cyclic cascades, a tiny change in the concentration of cAMP would cause a major change in the levels of phosphorylation of different proteins and enzymes, thus explaining the major impact that hormones have on the body over such a short period of time. The opposite is also true, meaning that when cAMP levels drop, glycogen synthase is activated through the same process, just in reverse.

An important task for the liver to achieve is the maintaining of blood sugar levels around a constant 5mM, so that the brain will have sufficient fuels so as to keep its activity in normal physiological ranges. The process is mediates by glucagon and insulin, the first of which acts according to the following process: low concentrations of blood glucose cause the secretion of glucagon by α cells of the pancreas (the islets of Langerhans). Glucagon receptors on the hepatocytes surfaces respond to the presence of the hormone by activating adenylate cyclase that will increase the levels of cAMP slightly, so that the breakdown of glycogen would be favoured with a view to restore blood sugar levels into normal ranges.

When the opposite situation occurs, meaning that the blood levels are too high and homeostasis is not achieved, insulin comes into action. It is produced by the β cells of the islets of Langerhans. In interesting effect of insulin on glucose is that it greatly increases the rate of glucose transport across cell membranes so that the concentration of sugar in blood will encounter a drop significant enough to see a physiologic effect. It is thought that this mechanism would also inhibit glycogen breakdown, as glucose inhibits phosphorylase and binds to the active site of the inactive T form of the enzyme, but in a manner different from that of the substrate. This would cause a conformational shift so that the Ser-14 residues would be exposed and subject to dephosphorylation. Above a concentration of 7mM this process takes place, almost totally reversing the flux of glycogen metabolism. In this way, excess glucose can be stored as glycogen.

Image from Biochemistry, Voet & Voet, Wiley

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