Phosphorylase Is Regulated by Allosteric Interactions and Reversible Phosphorylation

II. Transducing and Storing Energy
21. Glycogen Metabolism
21.1. Glycogen Breakdown Requires the Interplay of Several Enzymes
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 the protonated phosphate group of the bound pyridoxal phosphate PLP group (blue). The
combination of the carbocation and the orthophosphate results in the formation of glucose 1-phosphate.
II. Transducing and Storing Energy
21. Glycogen Metabolism
21.2. Phosphorylase Is Regulated by Allosteric Interactions and Reversible
Phosphorylation
Structural Insights, Glycogen Phosphorylase, looks closely at the structural
mechanisms of phosphorylase regulation, examining the efects of allosteric
effectors and serine phosphorylation.
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.
21.2.1. 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 (Figure 21.9). 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
(Figure 21.10). 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 (see Figure 21.9). 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 nucleotidebinding site and stabilizes the conformation of phosphorylase b in the R state (Figure 21.11). 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 6phosphate. 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.
21.2.2. 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 (Figure 21.12). 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.
21.2.3. 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 (α β γ δ)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 21.13).
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 (Section 15.3.2). 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.
II. Transducing and Storing Energy
21. Glycogen Metabolism
21.2. Phosphorylase Is Regulated by Allosteric Interactions and Reversible Phosphorylation
Figure 21.9. Structures of Phosphorylase A and Phosphorylase B . Phosphorylase a is phosphorylated on serine 14 of
each subunit. This modification favors the structure of the more active R state. One subunit is shown in white,
with helices and loops important for regulation shown in blue and red. The other subunit is shown in yellow, with
the regulatory structures shown in orange and green. Phosphorylase b is not phosphorylated and exists predominantly in
the T state.
II. Transducing and Storing Energy
21. Glycogen Metabolism
21.2. Phosphorylase Is Regulated by Allosteric Interactions and Reversible Phosphorylation
Figure 21.10. Phosphorylase Regulation. Both phosphorylase b and phosphorylase a exist as equilibria between an
active R state and a less-active T state. Phosphorylase b is usually inactive because the equilibrium favors the T state.
Phosphorylase a is usually active because the equilibrium favors the R state. Regulatory structures are shown in blue and
green.
II. Transducing and Storing Energy
21. Glycogen Metabolism
21.2. Phosphorylase Is Regulated by Allosteric Interactions and Reversible Phosphorylation
Figure 21.11. Allosteric Regulation of Muscle Phosphorylase. A low energy charge, represented by high
concentrations of AMP, favors the transition to the R state.
II. Transducing and Storing Energy
21. Glycogen Metabolism
21.2. Phosphorylase Is Regulated by Allosteric Interactions and Reversible Phosphorylation
Figure 21.12. Allosteric Regulation of Liver Phosphorylase. The binding of glucose to phosphorylase a shifts the
equilibrium to the T state and inactivates the enzyme. Thus, glycogen is not mobilized when glucose is already abundant.
II. Transducing and Storing Energy
21. Glycogen Metabolism
21.2. Phosphorylase Is Regulated by Allosteric Interactions and Reversible Phosphorylation