Question

. Explain how glycogen phosphorylase is regulated by AMP, ATP, G6Pand phosphorylation/dephosphorylation? What is the physiological significance of these control mechanisms? b. What are the effects of the following on the rates of glycogen synthesis and degradation? (a) increasing the Ca2+ ion concentration, (b) increasing the ATP concentration, (c) inhibiting adenylate cyclase, (d) increasing the epinephrine concentration, (e) increasing the AMP concentration. Explain.

Answer 1

Glycogen metabolism is precisely controlled by multiple interlocking mechanisms, and the focus of this control is glycogen phosphorylase. Phosphorylase is regulated by several allosteric effectors that signal the energy state of the cell as well as by reversible phosphorylation, which is responsive to hormones such as insulin, epinephrine, and glucagon. We will examine the differences in the control of glycogen metabolism in two tissues: skeletal muscle and liver. These differences are due to the fact that the muscle uses glucose to produce energy for itself, whereas the liver maintains glucose homeostasis of the organism as a whole.

Muscle Phosphorylase Is Regulated by the Intracellular Energy Charge

We begin by considering the glycogen phosphorylase from muscle. The dimeric skeletal muscle phosphorylase exists in two interconvertible forms: a usually active phosphorylase a and a usually inactive phosphorylase b. Each of these two forms exists in equilibrium between an active relaxed (R) state and a much less active tense (T) state, but the equilibrium for phosphorylase a favors the R state whereas the equilibrium for phosphorylase b favors the T state. Phosphorylase a and phosphorylase b differ by a single phosphoryl group in each subunit. Phosphorylase b is converted into phosphorylase a when it is phosphorylated at a single serine residue (serine 14) in each subunit. The regulatory enzyme phosphorylase kinase catalyzes this covalent modification. As will be described, increased levels of epinephrine (resulting from fear or from the excitement of exercise) and the electrical stimulation of muscle result in phosphorylation of the enzyme to the phosphorylase a form.

Comparison of the structures of phosphorylase a and phosphorylase b reveals that subtle structural changes at the subunit interfaces are transmitted to the active sites. The transition from the T state (represented by phosphorylase b) to the R state (represented by phosphorylase a) entails a 10-degree rotation around the twofold axis of the dimer. Most importantly, this transition is associated with structural changes in α helices that move a loop out of the active site of each subunit. Thus, the T state is less active because the catalytic site is partly blocked. In the R state, the catalytic site is more accessible and a binding site for orthophosphate is well organized.

The position of the equilibrium of phosphorylase b between the T and the R form is responsive to conditions in the cell. Muscle phosphorylase b is active only in the presence of high concentrations of AMP, which binds to a nucleotide-binding site and stabilizes the conformation of phosphorylase b in the R state. ATP acts as a negative allosteric effector by competing with AMP and so favors the T state. Thus, the transition of phosphorylase b between the T and the R state is controlled by the energy charge of the muscle cell. Glucose 6-phosphate also favors the T state of phosphorylase b, an example of feedback inhibition.Under most physiological conditions, phosphorylase b is inactive because of the inhibitory effects of ATP and glucose 6-phosphate. In contrast, phosphorylase a is fully active, regardless of the levels of AMP, ATP, and glucose 6-phosphate. In resting muscle, nearly all the enzyme is in the inactive b form. When exercise commences, the elevated level of AMP leads to the activation of phosphorylase b. Exercise will also result in hormone release that generates the phosphorylated a form of the enzyme. The absence of glucose 6-phosphatase in muscle ensures that glucose 6-phosphate derived from glycogen remains within the cell for energy transformation.

Liver Phosphorylase Produces Glucose for Use by Other Tissues

The regulation of liver glycogen phosphorylase differs markedly from that of muscle, a consequence of the role of the liver in glucose homeostasis for the organism as a whole. In human beings, liver phosphorylase and muscle phosphorylase are approximately 90% identical in amino acid sequence. The differences result in subtle but important shifts in the stability of various forms of the enzyme. In contrast with the muscle enzyme, liver phosphorylase a but not b exhibits the most responsive T-to-R transition. The binding of glucose shifts the allosteric equilibrium of the a form from the R to the T state, deactivating the enzyme . Why would glucose function as a negative regulator of liver phosphorylase a? The role of glycogen degradation in the liver is to form glucose for export to other tissues when the blood-glucose level is low. Hence, if free glucose is present from some other source such as diet, there is no need to mobilize glycogen. Unlike the enzyme in muscle, the liver phosphorylase is insensitive to regulation by AMP because the liver does not undergo the dramatic changes in energy charge seen in a contracting muscle. We see here a clear example of the use of isozymic forms of the same enzyme to establish the tissue-specific biochemical properties of muscle and the liver.

Phosphorylase Kinase Is Activated by Phosphorylation and Calcium Ions

The next upstream component of this signal-transduction pathway is the enzyme that covalently modifies phosphorylase. This enzyme is phosphorylase kinase, a very large protein with a subunit composition in skeletal muscle of (αβγδ)4 and a mass of 1200 kd. The catalytic activity resides in the γ subunit, whereas the other subunits serve a regulatory function. This kinase is under dual control. Like its own substrate, phosphorylase kinase is regulated by phosphorylation: the kinase is converted from a low-activity form into a high-activity one by phosphorylation of its β subunit. The enzyme catalyzing the activation of phosphorylase kinase is protein kinase A (PKA), which is switched on by a second messenger, cyclic AMP (Sections 10.4.2 and 15.1.5). As will be discussed, hormones such as epinephrine induce the breakdown of glycogen by activating a cyclic AMP cascade (Figure 21.13Activation of Phosphorylase Kinase. Phosphorylase kinase is activated by hormones that lead to the phosphorylation of the β subunit and by Ca2+ binding of the δ subunit. Both types of stimulation are required for maximal enzyme activity.Phosphorylase kinase can also be partly activated by Ca2+ levels of the order of 1 μM. Its δ subunit is calmodulin, a calcium sensor that stimulates many enzymes in eukaryotes. This mode of activation of the kinase is important in muscle, where contraction is triggered by the release of Ca2+ from the sarcoplasmic reticulum. Phosphorylase kinase attains maximal activity only after both phosphorylation of the β subunit and activation of the δ subunit by Ca2+ binding