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Berg JM, Tymoczko JL, Stryer L. Biochemistry. 5th edition. New York: W H Freeman; 2002.

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Biochemistry. 5th edition.

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Section 21.1Glycogen Breakdown Requires the Interplay of Several Enzymes

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. We will examine each of these activities in turn.

21.1.1. Phosphorylase Catalyzes the Phosphorolytic Cleavage of Glycogen to Release Glucose 1-phosphate

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.

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Phosphorylase catalyzes the sequential removal of glycosyl residues from the nonreducing ends of the glycogen molecule (the ends with a free 4-OH groups; Section 11.1.3). Orthophosphate splits the glycosidic linkage between C-1 of the terminal residue and C-4 of the adjacent one. Specifically, it cleaves the bond between the C-1 carbon atom and the glycosidic oxygen atom, and the α configuration at C-1 is retained.

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Glucose 1-phosphate released from glycogen can be readily converted into glucose 6-phosphate (Section 21.1.3), an important metabolic intermediate, by the enzyme phosphoglucomutase.

The reaction catalyzed by phosphorylase is readily reversible in vitro. At pH 6.8, the equilibrium ratio of orthophosphate to glucose 1-phosphate is 3.6. The value of ΔG°´ for this reaction is small because a glycosidic bond is replaced by a phosphoryl ester bond that has a nearly equal transfer potential. However, phosphorolysis proceeds far in the direction of glycogen breakdown in vivo because the [Pi]/[glucose 1-phosphate] ratio is usually greater than 100, substantially favoring phosphorolysis. We see here an example of how the cell can alter the free-energy change of a reaction to favor the reaction's occurrence by altering the ratio of substrate and product.

The phosphorolytic 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 phosphorolytic cleavage for muscle cells is that glucose 1-phosphate, negatively charged under physiological conditions, cannot diffuse out of the cell.

21.1.2. A Debranching Enzyme Also Is Needed for the Breakdown of Glycogen

Glycogen phosphorylase, the key enzyme in glycogen breakdown, can carry out this process by itself only to a limited extent before encountering an obstacle. The α-1,6-glycosidic bonds at the branch points are not susceptible to cleavage by phosphorylase. Indeed, phosphorylase stops cleaving α-1,4 linkages when it reaches a terminal residue four residues away from a branch point. 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.

How can the remainder of the glycogen molecule be mobilized for use as a fuel? Two additional enzymes, a transferase and α-1,6-glucosidase, remodel the glycogen for continued degradation by the phosphorylase (Figure 21.4). 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 α-1,6-glycosidic linkage. α-1,6-Glucosidase, also known as the debranching enzyme, hydrolyzes the α-1, 6-glycosidic bond, resulting in the release of a free glucose molecule.

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Figure 21.4. Glycogen Remodeling.

Figure 21.4

Glycogen Remodeling. First, α-1,4-glycosidic bonds on each branch are cleaved by phosphorylase, leaving four residues along each branch. The transferase shifts a block of three glycosyl residues from one outer branch to the other. In this reaction, (more...)

This free glucose molecule is phosphorylated by the glycolytic enzyme hexokinase. Thus, the transferase and α-1,6-glucosidase convert the branched structure into a linear one, which paves the way for further cleavage by phosphorylase. It is noteworthy that, in eukaryotes, the transferase and the α-1,6-glucosidase activities are present in a single 160-kd polypeptide chain, providing yet another example of a bifunctional enzyme (Section 16.2.2). Furthermore, these enzymes may have additional features in common (Section 21.4.3).

21.1.3. Phosphoglucomutase Converts Glucose 1-phosphate into Glucose 6-phosphate

Glucose 1-phosphate formed in the phosphorolytic 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. Recall that this enzyme is also used in galactose metabolism (Section 16.1.11). To effect this shift, the enzyme engages in the exchange of a phosphoryl group with the substrate (Figure 21.5).

Figure 21.5. Reaction Catalyzed by Phosphoglucomutase.

Figure 21.5

Reaction Catalyzed by Phosphoglucomutase. A phosphoryl group is transferred from the enzyme to the substrate, and a different phosphoryl group is transferred back to restore the enzyme to its initial state.

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.

These reactions are like those of phosphoglycerate mutase, a glycolytic enzyme (Section 16.1.7). The role of glucose 1,6-bisphosphate in the interconversion of the phosphoglucoses is like that of 2,3-bisphosphoglycerate (2,3-BPG) in the interconversion of 2-phosphoglycerate and 3-phosphoglycerate in glycolysis. A phosphoenzyme intermediate participates in both reactions.

21.1.4. Liver Contains Glucose 6-phosphatase, a Hydrolytic Enzyme Absent from Muscle

A major function of the liver is to maintain a near constant level of glucose in the blood. The liver releases glucose into the blood during muscular activity and between meals to be taken up primarily by the brain and skeletal muscle. However, the phosphorylated glucose produced by glycogen breakdown, in contrast with glucose, is not readily transported out of cells. The liver contains a hydrolytic enzyme, glucose 6-phosphatase, which cleaves the phosphoryl group to form free glucose and orthophosphate. This glucose 6-phosphatase, located on the lumenal side of the smooth endoplasmic reticulum membrane, is the same enzyme that releases free glucose at the conclusion of gluconeogenesis. Recall that glucose 6-phosphate is transported into the endoplasmic reticulum; glucose and orthophosphate formed by hydrolysis are then shuttled back into the cytosol (Section 16.3.5).

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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.

21.1.5. Pyridoxal Phosphate Participates in the Phosphorolytic Cleavage of Glycogen

Let us now examine the catalytic mechanism of glycogen phosphorylase, which is a dimer of two identical 97-kd subunits. Each subunit is compactly folded into an amino-terminal domain (480 residues) containing a glycogen-binding site and a carboxyl-terminal domain (360 residues; Figure 21.6). The catalytic site is located in a deep crevice formed by residues from amino- and carboxyl-terminal domains. The special challenge faced by phosphorylase is to cleave glycogen phosphorolytically rather than hydrolytically to save the ATP required to phosphorylate free glucose. This cleavage requires that water be excluded from the active site. Several clues provide us with information about the mechanism by which phosphorylase achieves the exclusion of water. First, both the glycogen substrate and the glucose 1-phosphate product have an α configuration at C-1 (the designation α means that the oxygen atom attached to C-1 is below the plane of the ring; Section 11.1.3). A direct attack of phosphate on C-1 of a sugar would invert the configuration at this carbon because the reaction would proceed through a pentacovalent transition state. Because the resulting glucose 1-phosphate has an α rather than a β configuration, an even number of steps (most simply, two) is required. The most likely explanation for these results is that a carbonium ion intermediate is formed.

Figure 21.6. Structure of Glycogen Phosphorylase.

Figure 21.6

Structure of Glycogen Phosphorylase. Image mouse.jpg This enzyme forms a homodimer: one subunit is shown in white and the other in yellow. Each catalytic site includes a pyridoxal-phosphate (PLP) group, linked to lysine 680 of the enzyme. The binding site for the phosphate (more...)

A second clue to the catalytic mechanism of phosphorylase is its requirement for pyridoxal phosphate (PLP), a derivative of pyridoxine (vitamin B6, Section 8.6.1). The aldehyde group of this coenzyme forms a Schiff base with a specific lysine side chain of the enzyme (Figure 21.7). The results of structural studies indicate that the reacting orthophosphate group takes a position between the 5′-phosphate group of PLP and the glycogen substrate (Figure 21.8). The 5-phosphate group of PLP acts in tandem with orthophosphate by serving as a proton donor and then as a proton acceptor (that is, as a general acid-base catalyst). Orthophosphate (in the HPO42- form) donates a proton to the oxygen atom attached to carbon 4 of the departing glycogen chain and simultaneously acquires a proton from PLP. The carbonium ion intermediate formed in this step is then attacked by orthophosphate to form α-glucose 1-phosphate, with the concomitant return of a hydrogen atom to pyridoxal phosphate. The requirement that water be excluded from the active site calls for the special role of pyridoxal phosphate in facilitating the phosphorolytic cleavage.

Figure 21.7. PLP-Schiff-Base Linkage.

Figure 21.7

PLP-Schiff-Base Linkage. A pyridoxal phosphate group (red) forms a Schiff base with a lysine residue (blue) at the active site of phosphorylase.

Figure 21.8. Phosphorylase Mechanism.

Figure 21.8

Phosphorylase Mechanism. A bound HPO42- group (red) favors the cleavage of the glycosidic bond by donating a proton to the departing glucose (black). This reaction results in the formation of a carbocation and is favored by the transfer of a proton from (more...)

The glycogen-binding site is 30 Å away from the catalytic site (see Figure 21.6), but it is connected to the catalytic site by a narrow crevice able to accommodate four or five glucose units. The large separation between the binding site and the catalytic site enables the enzyme to phosphorolyze many residues without having to dissociate and reassociate after each catalytic cycle. An enzyme that can catalyze many reactions without having to dissociate and reassociate after each catalytic step is said to be processive—a property of enzymes that synthesize and degrade large polymers. We will see such enzymes again when we consider DNA and RNA synthesis.

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Copyright © 2002, W. H. Freeman and Company.
Bookshelf ID: NBK22467