Fig 5.5.13. Schematic representation of the phosphorylation of glycogen phosphorylase by phosphorylase kinase and dephosphorylation by protein phosphatase-1. Organ-specific stimulatory effects (see text) of
epinephrine, glucagon, calcium ions and AMP on phosphorylase kinase have also been shown.
(Source: Nelson and Cox, 2003, p 584 fig 15-24)
The step-wise process of activation of phosphorylase depicted in Fig 5.5.12 (below) will now enable you to understand the enzyme cascade mechanism. Notice that there have been 4 amplification steps in phosphorylase activation. Adenylate cyclase, protein kinase, phosphorylase kinase and phosphorylase actions have followed in sequence, with each enzyme activating the next enzyme in the sequence. Follow the estimated number of molecules shown at each step in the figure. Remember that since an enzyme is a catalyst, every molecule of the enzyme is available again for action at the end of every reaction. Notice also that only the enzymes per se have undergone activation and remember that this is a reversible phenomenon.
Fig 5.5.14. The amplification of a primary hormonal signal by an enzyme cascade mechanism: An estimate of the number of molecules produced at each step in the cascade has been shown. One molecule of
epinephrine finally activates 1,0000 molecules of the target enzyme, glycogen phosphorylase.
(Source: Nelson and Cox, 2003, p 585 fig 15-25)
Superimposed on the hormonal regulation of muscle phosphorylase is faster allosteric regulation by:
• AMP – During muscle contraction, the breakdown of ATP increases [AMP]. An allosteric site in phosphorylase b attaches AMP and the enzyme is activated by a conformational change without conversion to phosphorylase a.
• Ca2+ – Calcium release is the signal for muscle contraction. Binding of Ca2+ activates dephosphorylated phosphorylase kinase. The muscle enzyme is represented as (αβγδ)4. Serine residues on the α and β subunits are phosphorylated by PKA. The δ subunit is calmodulin and it binds four Ca2. The enzyme is fully active only when both phosphorylation and Ca2+-binding have taken place
A. B. C.
Fig 5.5.15. Schematic diagram of the activation of phosphorylase kinase: the enzyme is only partly activated by covalent modification (A: phosphorylation) or allosteric modulation (B: attachment of Ca2+ ).
Full activation requires both the mechanisms of activation (C) to occur simultaneously.
(Adapted from Berg et al, 2002, fig 21.13)
• Glucose 6-P and ATP – High cellular concentrations of glucose 6-P and ATP inhibit PL b: they prevent conformational change of PL b by attaching to its AMP-binding site.
Thus, regulation of phosphorylase ensures that at times of increased muscle activity, glycogenolysis is stimulated and provides glucose. When muscle is at rest, ATP levels are adequate and glycogenolysis is not required.
2. Regulation of Phosphorylase in the Liver
The mechanism of activation of phosphorylase in the liver is similar to that in the muscle with the following differences that are in keeping with the role of the liver in carbohydrate metabolism:
• Glucagon, secreted by the pancreas when blood glucose falls, can also initiate the cAMP- dependent enzyme cascade pathway to activate phosphorylase. This ensures that hepatic cells release essential glucose into the blood when the nutritional state is low. Glucagon is ineffective in the muscle.
• Vasopressin, oxytocin, angiotensin II and epinephrine stimulate glycogenolysis through a phosphoinositide cascade pathway. The hormones bind to an α1-adrenergic receptor in the plasma membrane and activate a G protein that stimulates phospholipase C.
The resulting increase in inositol 1,4,5-triphosphate (IP3) induces Ca2+ mobilization from ER stores to the cytosol and activates phosphorylase kinase.
• Allosteric regulation of phosphorylase is by glucose and not by AMP. When blood glucose is high, glucose uptake in the hepatic cell is also increased. Glucose now binds to an allosteric site on PL a, causing a conformational change that results in dephosphorylation and inactivation of the enzyme by PP-1. When blood glucose falls, the allosteric inhibition is removed and glycogenolysis starts again. Thus PL a is a “glucose sensor” in liver cells.
Fig 5.5.16. Schematic diagram of the conformational change in phosphorylase a caused by allosteric binding of
glucose. (Source: Nelson and Cox, 2003, p 585 fig 15-26)
• Insulin, secreted by the pancreas when blood glucose is high, stimulates PP-1, which inactivates both phosphorylase kinase and PL a. Insulin also increases the action of phosphodiesterase so that cAMP is hydrolyzed. Thus insulin action is antagonistic to that of glucagon.
Section 5.5.B
GLYCOGENESIS
Glycogenesis is the synthesis of glycogen from glucose. After a meal when the levels of blood glucose are high and insulin is present, cells of the liver and muscle take up glucose to replenish their glycogen stores. The reactions of glycogenesis are cytosolic, well-regulated and co-ordinated with glycogenolysis.
Glycogen synthesis essentially involves step-wise attachment of glucose residues to a pre-existing
“glycogen primer” so that it increase in size. De novo synthesis of the glycogen molecule requires a protein “primer” called glycogenin. The reactions are essentially similar in liver and muscle though their regulation differs in detail in the two organs.
A. Steps in the synthesis of Glycogen by enlarging a “glycogen primer”
A “glycogen primer” is essentially a pre-existing α-1,4 glucan (i.e. amylose chains). There are three steps in expanding this structure to obtain the highly branched glycogen molecule:
GLYCOGEN( ) expanded
( primer )
+1 n P
UD n) GLYCOGEN(
glycogen synthase ( primer )
GLUCOSE
2 P i D A T P A P
- phospho glucomutase hexokinase/
glucokinase
Glucose DP-
U pyrophosphatase
T P U
PP i
pyrophophorylase
H2O
Glucose6-P Glucose1-P
Fig 5.5.17. Summary of the synthesis of glycogen from glucose by using ‘glycogen primer’.