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Q.1- Why do animals store energy as glycogen? Why not convert all excess fuel into fatty acids?

Answer- Glycogen is a readily mobilized storage form of glucose. It is a very large, branched polymer of glucose residues that can be broken down to yield glucose molecules when energy is needed. Most of the glucose residues in glycogen are linked by α-1,4-glycosidic bonds. Branches at about every tenth residue are created by α-1,6-glycosidic bonds (Figure 1).

 

Figure 1- Showing glycogen structure, the non reducing ends have been shown. R – Represents the remaining structure of glycogen molecule

Glycogen is not as reduced as fatty acids are and consequently not as energy rich. Glycogen is an important fuel reserve for several reasons-

 1) The controlled breakdown of glycogen and release of glucose increase the amount of glucose that is available between meals. Hence, glycogen serves as a buffer to maintain blood-glucose levels.

2) Glycogen’s role in maintaining blood glucose levels is especially important because glucose is virtually the only fuel used by the brain, except during prolonged starvation.

3) The glucose from glycogen is readily mobilized and is therefore a good source of energy for sudden, strenuous activity.

4) Unlike fatty acids, the released glucose can provide energy in the absence of oxygen and can thus supply energy for anaerobic activity.

The two major sites of glycogen storage are the liver and skeletal muscle. The concentration of glycogen is higher in the liver than in muscle (10% versus 2% by weight), but more glycogen is stored in skeletal muscle overall because of its much greater mass. Glycogen is present in the cytosol in the form of granules ranging in diameter from 10 to 40 nm. In the liver, glycogen synthesis and degradation are regulated to maintain blood-glucose levels as required to meet the needs of the organism as a whole. In contrast, in muscle, these processes are regulated to meet the energy needs of the muscle itself.

Q.2- What is Glycogenesis? Describe the steps and state under what conditions glycogenesis would be promoted in the body?

Or

Describe the separate roles of Glycogenin and Glycogen Synthase in glycogen synthesis. Summarize the reactions catalyzed by each enzyme.

Answer- Glycogenesis is the synthesis of glycogen from glucose. Glycogenesis mainly occurs in muscle and liver. Muscle glycogen provides a readily available source of glucose for glycolysis within the muscle itself. Liver glycogen functions to store and export glucose to maintain blood glucose between meals. After 12–18 hours of fasting, liver glycogen is almost totally depleted. Although muscle glycogen does not directly yield free glucose, (because muscle lacks glucose 6-phosphatase), pyruvate formed by glycolysis in muscle can undergo transamination to alanine, which is exported from muscle and used for gluconeogenesis in the liver.

Steps of Glycogen Synthesis

1) Activation of Glucose

Synthesis of glycogen from glucose is carried out by the enzyme glycogen synthase. This enzyme utilizes UDP-glucose as one substrate and the non-reducing end of glycogen as another. UDP-glucose, the glucose donor in the biosynthesis of glycogen, is an activated form of glucose, just as ATP and acetyl CoA are activated forms of orthophosphate and acetate respectively.

As in glycolysis, glucose is phosphorylated to glucose 6-phosphate, catalyzed by hexokinase in muscle and glucokinase in liver (Figure 2). Glucose 6-phosphate is isomerized to glucose 1-phosphate by Phosphoglucomutase. The enzyme itself is phosphorylated, and the Phospho-group takes part in a reversible reaction in which glucose 1,6-bisphosphate is an intermediate. Next, glucose 1-phosphate reacts with uridine triphosphate (UTP) to form the active nucleotide uridine diphosphate glucose (UDPGlc) and pyrophosphate (Figure 3). The reaction is catalyzed by UDPGlc pyro phosphorylase. The reaction proceeds in the direction of UDPGlc formation because pyrophosphatase catalyzes hydrolysis of pyrophosphate to 2 x phosphate, so removing one of the reaction products.

 

Figure 2- showing activation of glucose to UDP glucose and its incorporation in to the pre- existing glycogen fragment

 

Figure-3- Showing formation of UDP glucose, The C-1 carbon atom of the glucosyl unit of UDP glucose is activated because its hydroxyl group is esterified to the diphosphate moiety of UDP.

The essentially irreversible hydrolysis of pyrophosphate drives the synthesis of UDP-glucose.

 

2) Initiation- Glycogen synthase can add glucosyl residues only if the polysaccharide chain already contains more than four residues. Thus, glycogen synthesis requires a primer. This priming function is carried out by glycogenin, a protein composed of two identical 37-kd subunits, each bearing an oligosaccharide of alpha-1,4-glucose units (Figure-4).

Figure 4- showing diagrammatic representation of glycogenin dimer

Carbon 1 of the first unit of this chain, the reducing end, is covalently attached to the phenolic hydroxyl group of a specific tyrosine in each glycogenin subunit (Figure 5). Each subunit of glycogenin catalyzes the addition of eight glucose units to its partner in the glycogenin dimer. UDP-glucose is the donor in this autoglycosylation. At this point, glycogen synthase takes over to extend the glycogen molecule. In skeletal muscle, glycogenin remains attached in the center of the glycogen molecule (Figure-7) ; in liver the number of glycogen molecules is greater than the number of glycogenin molecules.

 

 

Figure 5- A glycosidic bond is formed between the anomeric C1 of the glucose moiety derived  from UDP-glucose and the hydroxyl oxygen of a tyrosine side-chain of Glycogenin. UDP is  released as a product.

3)  Elongation - New glucosyl units are added to the nonreducing terminal residues of glycogen. The activated glucosyl unit of UDPglucose is transferred to the hydroxyl group at a C-4 terminus of glycogen to form an α-1,4-glycosidic linkage. In elongation, UDP is displaced by the terminal hydroxyl group of the growing glycogen molecule. This reaction is catalyzed by glycogen synthase, the key regulatory enzyme in glycogen synthesis.

4) Glycogen branching- Glycogen synthase catalyzes only the synthesis of α-1,4 linkages. Another enzyme is required to form the α-1,6 linkages that make glycogen a branched polymer. Branching occurs after a number of glucosyl residues are joined in α-1,4 linkage by glycogen synthase. A branch is created by the breaking of an α-1,4 link and the formation of an α–1,6 link: this reaction is different from debranching. A block of residues, typically 7 in number, is transferred to a more interior site. The branching enzyme that catalyzes this reaction is quite exacting. The block of 7 or so residues must include the nonreducing terminus and come from a chain at least 11 residues long. In addition, the new branch point must be at least 4 residues away from a preexisting one. Branching is important because it increases the solubility of glycogen. Furthermore, branching creates a large number of terminal residues, the sites of action of glycogen phosphorylase and synthase (Figure 6). Thus, branching increases the rate of glycogen synthesis and degradation.

                                 

 Figure-6- showing the transfer of 7 glucosyl residue by the branching enzyme to create a branch point.                                               

 

Figure 7- Showing structure of glycogen, The glycogen molecule is a sphere approximately 10-40 nm in diameter that can be seen in electron micrographs. It has a molecular mass of 107 Da and consists of polysaccharide chains, each containing about 13 glucose residues. The chains are either branched or unbranched and are arranged in 12 concentric layers .The branched chains (each has two branches) are found in the inner layers and the unbranched chains in the outer layer. (G, glycogenin, the primer molecule for glycogen synthesis.)

Glycogen Synthase, The key regulatory enzyme of Glycogen synthesis is activated by glucose-6-phosphate. Thus Glycogen Synthase is active when high blood glucose leads to elevated intra cellular glucose-6-phosphate.

It is useful to a cell to store glucose as glycogen when the input to Glycolysis (glucose-6-phosphate), and the main product of Glycolysis (ATP), are adequate.

Q.3- Explain the pathway by which Glycogen is degraded in the body?

Answer- Glycogenolysis (degradation of glycogen) is not just a reverse of glycogenesis, it is a separate pathway. Glycogen degradation consists of three steps: (1) the release of glucose 1-phosphate from glycogen, (2) the remodeling of the glycogen substrate to permit further degradation, and (3) the conversion of glucose 1-phosphate into glucose 6-phosphate for further metabolism.

Enzymes required for glycogen degradation

The efficient breakdown of glycogen to provide glucose 6-phosphate for further metabolism requires four enzyme activities: one to degrade glycogen, two to remodel glycogen so that it remains a substrate for degradation, and one to convert the product of glycogen breakdown into a form suitable for further metabolism.

a) Phosphorylase- Glycogen phosphorylase, the key enzyme in glycogen breakdown, cleaves its substrate by the addition of orthophosphate (Pi) to yield glucose 1-phosphate. The cleavage of a bond by the addition of orthophosphate is referred to as phosphorolysis. 

b) Transferase and Debranching enzyme- The transferase shifts a block of three glucosyl residues from one outer branch to the other. This transfer exposes a single glucose residue joined by an alpha-1,6-glycosidic linkage. alpha-1,6-Glucosidase, also known as the debranching enzyme, hydrolyzes the alpha-1, 6-glycosidic bond, resulting in the release of a free glucose molecule.

c) Phosphoglucomutase- Glucose 1-phosphate formed in the phosphoroylytic cleavage of glycogen must be converted into glucose 6-phosphate to enter the metabolic mainstream. This shift of a phosphoryl group is catalyzed by Phosphoglucomutase.

 Steps of Glycogen degradation

 1) Release of Glucose-1-P- Phosphorylase catalyzes the sequential removal of glucosyl residues from the nonreducing ends of the glycogen molecule (the ends with a free 4-OH group. Orthophosphate splits the glycosidic linkage between C-1 of the terminal residue and C-4 of the adjacent on (Figure-8). Specifically, it cleaves the bond between the C-1 carbon atom and the glycosidic oxygen atom, and the a configuration at C-1 is retained.

 

 

 

Figure 8- Showing the release of Glucose-1-P by the action of Phosphorylase enzyme

Glucose 1-phosphate released from glycogen can be readily converted into glucose 6-phosphate an important metabolic intermediate, by the enzyme Phosphoglucomutase.

Advantages of phosphoroylytic cleavage- The phosphoroylytic cleavage of glycogen is energetically advantageous because the released sugar is already phosphorylated. In contrast, a hydrolytic cleavage would yield glucose, which would then have to be phosphorylated at the expense of the hydrolysis of a molecule of ATP to enter the glycolytic pathway. An additional advantage of phosphoroylytic cleavage for muscle cells is that glucose 1-phosphate, negatively charged under physiological conditions, cannot diffuse out of the cell.

 2) Remodeling of the glycogen substrate to permit further degradation- The alpha-1,6-glycosidic bonds at the branch points are not susceptible to cleavage by phosphorylase. Glycogen phosphorylase stops cleaving alpha-1,4 linkages when it reaches a terminal residue four residues away from a branch point (figure-9). Because about 1 in 10 residues is branched, glycogen degradation by the phosphorylase alone would come to a halt after the release of six glucose molecules per branch.

 

 

 

Figure 9- Remodeling of glycogen structure by Transferase and debranching enzyme, the transferase shifts a block of three glycosyl residues from one outer branch to the other. This transfer exposes a single glucose residue joined by an a-1,6-glycosidic linkage. a-1,6-Glucosidase, also known as the debranching enzyme, hydrolyzes the a-1, 6-glycosidic bond, resulting in the release of a free glucose molecule.

 Transferase and a-1,6-glucosidase, remodel the glycogen for continued degradation by the phosphorylase. The free glucose molecule (Figure-10)released by the action of debranching enzyme is phosphorylated by the glycolytic enzyme hexokinase. Thus, the transferase and alpha-1,6-glucosidase convert the branched structure into a linear one, which paves the way for further cleavage by phosphorylase.

In eukaryotes, the transferase and the a-1,6-glucosidase activities are present in a single 160-kd polypeptide chain, providing yet another example of a bifunctional enzyme

 

 

Figure 10- showing the action of debranching enzyme, free glucose is released by the action of debranching enzyme

3) Conversion of glucose 1-phosphate into glucose 6-phosphate for further metabolism

Phosphoglucomutase converts glucose 1-phosphate into glucose 6-phosphate in a reversible reaction. The catalytic site of an active mutase molecule contains a phosphorylated serine residue. The phosphoryl group is transferred from the serine residue to the C-6 hydroxyl group of glucose 1-phosphate to form glucose 1,6-bisphosphate. The C-1 phosphoryl group of this intermediate is then shuttled to the same serine residue, resulting in the formation of glucose 6-phosphate and the regeneration of the phosphoenzyme.

Glucose 6-phosphate derived from glycogen can (1) be used as a fuel for anaerobic or aerobic metabolism as in, for instance, muscle; (2) be converted into free glucose in the liver and subsequently released into the blood; (3) be processed by the pentose phosphate pathway to generate NADPH or ribose in a variety of tissues (Figure-11)

 

Figure 11- showing the fate of glucose-6-P

Conversion of Glucose-6-P to free Glucose

The liver contains a hydrolytic enzyme, glucose 6-phosphatase, which cleaves the phosphoryl group to form free glucose and orthophosphate.

Glucose 6-phosphatase is absent from most other tissues. Consequently, glucose 6-phosphate is retained for the generation of ATP. In contrast, glucose is not a major fuel for the liver. The liver releases glucose into the blood during muscular activity and between meals to be taken up primarily by the brain and skeletal muscle.

 Q.4- What is the role played by pyridoxal phosphate in glycogen metabolism?

Answer- Pyridoxal phosphate (PLP), a derivative of vitamin B6, serves as prosthetic group for Glycogen Phosphorylase. 

Pyridoxal phosphate is held at the active site of Phosphorylase enzyme by a Schiff base linkage, formed by reaction of the aldehyde of PLP with the e-amino group of a lysine residue (Figure-12).

In contrast to the role of this cofactor in other, the phosphate moiety of PLP is involved in acid/base catalysis by Phosphorylase. 

The Pi substrate binds between the phosphate of PLP and the glycosidic oxygen linking the terminal glucose residue of the glycogen substrate.

After the phosphate substrate donates a proton during cleavage of the glycosidic bond, it receives a proton from the phosphate moiety of PLP. PLP then takes back the proton as the phosphate oxygen attacks C1 of the cleaved glucose to yield glucose-1-phosphate.

 

Figure-12- showing Schiff base linkage A pyridoxal phosphate group (red) forms a Schiff base with a lysine residue (blue) at the active site of phosphorylase.

Q.5- What is the cost of converting glucose 6-phosphate into glycogen and back into glucose 6-phosphate?

Or

Q.6-Is the energy required to synthesize glycogen from glucose 6-phosphate the same as the energy required to degrade glycogen to glucose 6-phosphate?

Answer- No. More energy is required to synthesize glycogen from glucose 6-P.

The details of energy expenditure per reaction are as follows-

 

Thus, one ATP is hydrolyzed incorporating glucose 6-phosphate into glycogen. The energy yield from the breakdown of glycogen is highly efficient. About 90% of the residues are phosphorolytically cleaved to glucose 1-phosphate, which is converted at no cost into glucose 6-phosphate. The other 10% are branch residues, which are hydrolytically cleaved. One molecule of ATP is then used to phosphorylate each of these glucose molecules to glucose 6-phosphate.

Q.7-The phosphoroylytic cleavage of glycogen is key for glycogen metabolism. Why?

Answer- Hydrolysis of glycogen will generate glucose which is free to leave the cell and will require the additional input of energy to phosphorylate to glucose 6-P. Phosphoroylytic cleavage of glycogen produces glucose 1-P which can be converted to glucose 6 P and be utilized by several different pathways. This reaction does not require ATP and is therefore more efficient because it decreases the ATP investment.

Q.8-Why is it important to have different pathways for glycogenesis and glycogenolysis in liver and muscle cells?

Answer- The separate pathways for the synthesis and degradation of glycogen allow the synthesis of glycogen to proceed despite a high ratio of Pi to glucose 1 phosphate. The separate pathways allow the coordinated reciprocal control of glycogen synthesis and degradation by hormonal regulation.

 

 

 

 

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