Step 3: Branching of linear chains
B. De novo synthesis of glycogen
Glycogenin is a protein “primer which initiates synthesis of a new glycogen molecule. It is a 37 kDa, self-glycosylating protein that first attaches a glucosyl residue from UDPG to the hydroxyl group of a specific Tyrosine residue in its own peptide chain. Glycogenin then continues its glucosyltransferase activity till a linear chain of 8 glucosyl residues, joined by 1:4 α-glycosidic bonds, is obtained. The “primed” glycogenin can now be acted on by glycogen synthase and the
“branching” enzyme to obtain the final glycogen particle.
Fig 5.5.21. De novo synthesis of glycogen: a Tyrosine residue in the protein glycogenin (represented by the blue oval) attaches glucosyl residues borne by UDP. Subsequently glycogen synthase and the branching
enzyme help to produce the final product, glycogen (green oval).
Fig 5.5.22. Initial interaction between a molecule of UDP-Glucose and Tyrosine residue of glycogenin (Source: Nelson and Cox, 2005, p 570 fig 15-11)
Fig 5.5.23. Ribbon model of glycogenin: The red ball-and-stick structure represents the substrate, UDP-Glucose.
(Source: Nelson and Cox, 2005, p 569 fig 15-10)
Glycogen synthase is catalytically active only as long as it is in contact with glycogenin; this restricts the size of the final glycogen molecule
REGULATION OF GLYCOGENESIS
Glycogen synthase (GS) is the target enzyme for regulation of glycogenesis. The enzyme can exist in two forms (cf phosphorylase):
i. Glycogen synthase ‘a’ – non-phosphorylated and active
ii. Glycogen synthase ‘b’ – phosphorylated and relatively inactive
Notice that glycogen synthase is activated by dephosphorylation and inhibited by phosphorylation. This is in contrast to. phosphorylase.
The mechanisms of regulation are both hormonal and allosteric, and operate in response to blood and cellular levels of glucose:
Hormonal – reversible phosphorylation of glycogen synthase is the key to its control (cf phosphorylase). The effective hormones are insulin and epinephrine/glucagon.
Allosteric – by Glc 6-P, Ca2+ and possibly glycogen itself
Fig 5.5.24. Cascade mechanism for activation/inactivation of glycogen synthase. Refer to the text for details.
(Source: Murray et al, 2003, p 150 fig 18-7)
GS is unique in that it can be phosphorylated i.e. inactivated (GSa à GSb) on several serine residues by at least eleven different kinases! The most important among them is glycogen synthase kinase-3 (GSK3), which strongly inhibits glycogen synthase by phosphorylating three Ser residues near the carboxy-terminal end of the enzyme. GSK3 acts after prior “priming” of GS by another protein kinase, casein kinase II.
The kinases that phosphorylate GSa, are in turn regulated by small local molecules like cAMP, Ca2+ and DAG (diacylglycerol). High glycogen in tissues decreases GSa but the mechanism is not understood.
Fig 5.5.25. Phosphorylation of glycogen synthase by GSK3 and its dephosphorylation by PP-1. The effect of hormones on these two enzymes is also shown (Source: Nelson and Cox, 2005, p 586 fig 15-27)
In the liver, GS is dephosphorylated i.e. activated (GSb à GSa), by protein phosphatase-1 (PP- 1), which removes the same three phosphoryl groups attached by GSK3. The catalytic activity of PP-1 is facilitated by allosteric binding of Glc 6-P. Association with a glycogen-targeting protein, GM, brings PP-1 into close proximity with glycogen synthase and helps in catalysis. GM can be phosphorylated in two different sites (1 and 2), and its action depends on which of these sites has been phosphorylated.
In the muscle, a different phosphatase performs this function of dephosphorylating and activating glycogen synthase.
Insulin stimulates glycogenesis by promoting dephosphorylation of GS in two ways:
• binds to a membrane receptor, tyrosine kinase, and triggers a cascade involving protein kinase B (PKB) to phosphorylate and inactivate GSK3
Fig 5.5.26. Mechanism of insulin-induced activation of GSK3 and consequent inactivation of glycogen synthase.
(Source: Nelson and Cox, 2005, p 587 fig15-29)
• acts through an insulin-sensitive kinase to phosphorylate GM at site 1 so that it associates with the glycogen particle and activates PP-1; subsequently PP-1 dephosphorylates and activates glycogen synthase
Fig 5.5.27. Association of GM with enzymes in the glycogen particle: insulin (1) promotes association while epinephrine (2) causes dissociation of GM and PP-1 (Source: Nelson and Cox, 2005, p588 fig 15-30 )
Epinephrine/Glucagon inhibits glycogen synthesis by favoring phosphorylation of GS in two ways:
• binds to a β-adrenergic membrane receptor to initiate a cAMP-mediated cascade that stimulates protein kinase (PKA) (cf. phosphorylase regulation) (see Fig 5.5.22.).
Subsequently, PKA inactivates GS by:
phosphorylating GSa
phosphorylating inhibitor-1, which in turn inhibits PP-1
phosphorylating GM at sites 1 and 2, so that PP-1 dissociates from the glycogen particle and GS remains inactivated
• binds to an α-adrenergic membrane receptor, triggering release of IP3, DAG (diacylglycerol) and Ca2+, which inhibit GS. DAG, together with Ca2+, activates protein kinase C to phosphorylate and inactivate glycogen synthase
Fig 5.5.28. Inhibition of glycogen synthase by IP3, diacylglycerol (DG) and Ca2+ in a hepatocyte. Note the antagonistic action of epinephrine and glucagon in glycogen metabolism.
(Source: Voet and Voet, 1995, p 508 fig 17-22)
GLYCOGEN STORAGE DISEASES
The hereditary lack of any of the enzymes of glycogen metabolism may result in deposition of an abnormal type or quantity of glycogen in tissues. These disorders called “glycogenoses” may have serious consequences on health and even survival.
TABLE 5.5.1 GLYCOGEN STORAGE DISEASES
(Source: Murray et al, 2003, p 152 18-2)
Section 5.5.C
RECIPROCAL REGULATION OF GLYCOGENESIS AND GLYCOGENOLYSIS Synthesis and breakdown of glycogen are co-ordinated in such a way that when one is stimulated the other is inhibited. In this way steady levels of glucose are maintained both within the cell as well as in the body.
Regulatory mechanisms in glycogenolysis and glycogenesis respond to:
• Blood levels of glucose – which depends on the nutritional/metabolic state
• Muscular activity – which may suddenly demand energy fuel several hundred times that of the resting level
A balance in the activities of the two main enzymes viz. glycogen phosphorylase and glycogen synthase, is crucial in regulation of glycogen metabolism. From the preceding sub-sections you must have already realized that the actual controlling factors for glycogen synthase and phosphorylase are almost the same. It is therefore reasonable to assume that if a factor stimulates glycogenesis it should inhibit glycogenolysis synchronously and vice-versa.
In order to understand the reciprocal regulation of glycogenolysis and glycogenesis, it is useful to bear some fundamentals in mind:
• sites of glycogen metabolism are muscle and liver
• glycogen provides glucose as energy fuel when energy state of cell is low (i.e.
[ATP]:[AMP]↓)
• surplus glucose needs to be stored as glycogen
• regulation of the pathways is by reversible covalent modification (phosphorylation) and allosteric modulation of key enzymes
• key regulatory enzymes are glycogen phosphorylase and glycogen synthase
• enzymes common to both pathways are phosphorylase kinase A and protein phosphatase-1
• key hormones are epinephrine, glucagon and insulin; their secretion depends on the [blood glucose]
• second messengers in hormone action are cAMP, IP3, and DAG
• enzyme cascades which amplify the primary signal are mediated by cAMP and phosphoinositide
• other cellular molecules linked to both pathways are glucose, Ca2+, AMP and ATP Recall too the salient features in regulation of PL and GS:
• There is a basic difference in the effect of phosphorylation on PL and GS. Phosphorylation activates PL and inactivates GS
• PKA is the key enzyme in phosphorylation of PL and GS; it favors glycogenolysis and inhibits glycogenesis
• PP-1 is the main enzyme in dephosphorylation of PL and GS; it favors glycogenesis and inhibits glycogenolysis
• Cyclic AMP-mediated cascade simultaneously stimulates PKA and inhibits PP-1
• Epinephrine and insulin act on both muscle and liver; glucagon action is only on liver
Fig 5.5.29. Reciprocal regulation of glycogenolysis and glycogenesis. See text for details.
(Source: Murray et al, 2003, p 151 fig 18-8)
We can now summarise the reciprocal regulation of glycogenolysis and glycogenesis as follows:
1. Epinephrine/Glucagon stimulate glycogenolysis but simultaneously suppress glycogenesis:
Epinephrine/Glucagon à bound to β receptor on cell membrane à ↑ [cAMP] à activates PKA à phosphorylation promoted à PL activated; GS inhibited
Active PKA à inactivates PP-1 à dephosphorylation preventedà PL active; GS inactive
Epinephrine à bound to α receptor on cell membrane à phospholipase C active à ↑ [IP3]à cytosolic
↑ [Ca2+] à activates phosphorylase kinase à PL active
Phospholipase C activeà ↑ ([DAG] +↑ [Ca2+]) à activate protein kinase C à phosphorylation favored à GS inactive
2. Insulin stimulates glycogenesis but simultaneously suppresses glycogenolysis:
Insulin à ↑Glucose entry à ↑ [Glc 6-P]à inhibits phosphorylase kinase and activates PP-1à dephospohrylation favored à GS active; PL inactive
Insulin à inactivates GSK3 à dephosphorylation promotedà GS active
Insulin à activates PP-1 à dephosphorylation promoted à GS active; PL inactive
3. [Ca2+] stimulates glycogenolysis and simultaneously suppresses glycogenesis:
Muscle: the release of Ca2+ is the signal for muscle contraction and it simultaneously stimulates glycogenolysis to provide glucose as energy fuel
Neural/electrical stimulation of muscle à cytosolic ↑ [Ca2+] à stimulates phosphorylase kinase and à phosphorylation promoted à PL activated; GS inactive
Liver: Vasopressin, epinephrine etc.à ↑ [IP3] à cytosolic ↑ [Ca2+] à phosphorylation promoted à PL activated; GS inactive
4. [Glucose]: increased availability of glucose favors glycogenesis:
• ↑ [Glc 6-P] à allosteric binding to PL b à dephosphorylation favored à PL inactive
• ↑ [Glucose]à allosteric binding to PL a à dephosphorylation by PP-1 à GS active;
PL inactive
{Index: ↑ = increase; ↓ = decrease; GS = glycogen synthase; PL= phosphorylase; PKA
= protein kinase A; PP-1 = protein phosphatase -1}