1. 1. Carbohydrateconversion. Write a balanced equation for the formation of
glycogen from galactose.
2. If a little is good, a lot is better. -Amylose is an unbranched glucose polymer.
Why would this polymer not be as effective a storage form for glucose as
3. Telltale products. A sample of glycogen from a patient with liver disease is
incubated with orthophosphate, phosphorylase, the transferase, and the
-1,6-glucosidase). The ratio of glucose 1-phosphate to
glucose formed in this mixture is 100. What is the most likely enzymatic
deficiency in this patient?
4. Excessive storage. Suggest an explanation for the fact that the amount of
glycogen in type I glycogen-storage disease (von Gierke disease) is increased.
5. A shattering experience. Crystals of phosphorylase a grown in the presence of
glucose shatter when a substrate such as glucose 1-phosphate is added. Why?
6. Recouping an essential phosphoryl. The phosphorylgroup on
phosphoglucomutaseis slowly lost by hydrolysis. Proposea mechanism that
utilizes a known catalytic intermediate for restoring this essential phosphoryl
group. How might this phosphoryldonorbe formed?
7. Hydrophobia. Why is water excluded from the active site of phosphorylase?
Predict the effect of a mutation that allows water molecules to enter.
8. Removing all traces. In human liver extracts, the catalytic activity of glycogenin
-amylase (S -
amylase necessary to reveal the glycogenin activity?
9. Two in one. A single polypeptide chain houses the transferase and debranching
enzyme. Cite a potential advantage of this arrangement.
2. 10. How did they do that? A strain of mice has been developed that lack the
enzyme phosphorylasekinase. Yet, after strenuous exercise, the glycogen stores of
a mouse of this strain are depleted. Explain how this is possible.
11. Metabolic mutants. Predict the major consequenceof each of the following
(a) Loss of the AMP-binding site in muscle phosphorylase.
(b) Mutation of Ser 14 to Ala 14 in liver phosphorylase.
(c) Overexpression of phosphorylase kinase in the liver.
(d) Loss of the gene that encodes inhibitor 1 of protein phosphatase1.
(e) Loss of the gene that encodes the glycogen-targeting subunit of protein
(f) Loss of the gene that encodes glycogenin.
12. More metabolic mutants. Briefly, predict the major consequences of each of the
following mutations affecting glycogen utilization.
(b) Loss of the gene that encodes inhibitor 1 of protein phosphatase1.
(c) Loss of phosphodiesteraseactivity.
13. Multiple phosphorylation. Protein kinase A activates muscle phosphorylase
kinase by rapidly phosphorylating its
subunits susceptible to the action of protein phosphatase1. What is the
functional significance of the slow
14. The wrong switch. What would be the consequences to glycogen mobilization
of a mutation in phosphorylase kinase that leads to the phosphorylation -
3. 15. Family resemblance. Proposemechanisms for the two enzymes catalyzing
steps in glycogen debranching based on -
Chapter Integration and Data Interpretation Problems
16. Glycogen isolation 1. The liver is a major storage site for glycogen. Purified
from two samples of human liver, glycogen was either treated or not treated with
-amylase and subsequently analyzed by SDS-PAGE and Western blotting with
the use of antibodies to glycogenin. The results are presented in the adjoining
(a) Why are no proteins visible in the lanes without amylase treatment?
(b) What is the effect of treating the sampl -amylase? Explain the results.
(c) List other proteins that you might expect to be associated with glycogen. Why
are other proteins not visible?
17. Glycogen isolation 2. The gene for glycogenin was transfected into a cell line
that normally stores only small amounts of glycogen. The cells were then
manipulated according to the following protocol, and glycogen was isolated and
analyzed by SDS-PAGE and Western blotting using an antibody to glycogenin
- amylase treatment. The results are presented in the adjoining
The protocol:Cells cultured in growth medium and 25 mM glucose (lane 1) were
switched to medium containing no glucose for 24 hours (lane 2). Glucose-starved
cells were re-fed with medium containing 25 mM glucose for 1 hour (lane 3) or 3
-amylase, as indicated, before being loaded on the gel.
(a) Why did the Western analysis producea "smear" that is, the high-molecular-
weight staining in lane 1(-)?
(b) What is the significance of the decrease in high-molecular-weight staining in
(c) What is the significance of the difference between lanes 2(-) and 3(-)?
(d) Suggest a plausible reason why there is essentially no difference between lanes
3(-) and 4(-)?
(e) Why are the bands at 66 kd the same in the lanes treated with amylase, despite
the fact that the cells were treated differently?
4. 1. Galactose + ATP + UTP + H2O + glycogenn →glycogen n +1 + ADP + UDP +
2 Pi + H+
2. -amylose has only one nonreducing end.
amylose molecule. Because glycogen is highly branched, there are many
nonreducing ends per molecule. Consequently, many phosphorylase molecules can
release many glucose molecules per glycogen molecule.
3. The patient has a deficiency of the branching enzyme.
4. The high level of glucose 6-phosphate in von Gierke disease, resulting from the
absence of glucose 6-phosphataseor the transporter, shifts the allosteric
equilibrium of phosphorylated glycogen synthase toward the active form.
5. Glucose is an allosteric inhibitor of phosphorylase a. Hence, crystals grown in
its presence are in the T state. The addition of glucose 1-phosphate, a substrate,
shifts the R T equilibrium toward the R state. The conformational differences
between these states are sufficiently large that the crystal shatters unless it is
stabilized by chemical crosslinks.
6. The phosphoryldonoris glucose 1,6-bisphosphate, which is formed from
glucose 1-phosphate and ATP in a reaction catalyzed by phosphoglucokinase.
7. Water is excluded from the active site to prevent hydrolysis. The entry of water
could lead to the formation of glucose rather than glucose 1-phosphate. A site-
specific mutagenesis experiment is revealing in this regard. In phosphorylase, Tyr
573 is hydrogen bonded to the 2 -OH of a glucose residue. The ratio of glucose 1-
phosphateto glucose productis 9000:1 for the wild-type enzyme, and 500:1 for the
Phe 573 mutant. Model building suggests that a water molecule occupies the site
normally filled by the phenolic OH of tyrosine and occasionally attacks the
oxocarbonium ion intermediate to form glucose.
8. The amylase activity was necessary to remove all of the glycogen from the
glycogenin. Recall that glycogenin synthesizes oligosaccharides of about eight
5. glucose units, and then activity stops. Consequently, if the glucose residues are not
removed by extensive amylase treatment, glycogenin would not function.
9. The substrate can be handed directly from the transferase site to the debranching
10. During exercise, [ATP] falls and [AMP] rises. Recall that AMP is an allosteric
activator of glycogen phosphorylase b. Thus, even in the absence of covalent
modification by phosphorylase kinase, glycogen is degraded.
11. (a) Muscle phosphorylase b will be inactive even when the AMP level is high.
Hence, glycogen will not be degraded unless phosphorylase is converted into the a
form by hormone-induced or Ca2+-induced phosphorylation.
(b) Phosphorylase b cannot be converted into the much more active a form. Hence,
the mobilization of liver glycogen will be markedly impaired.
(c) The elevated level of the kinase will lead to the phosphorylation and activation
of glycogen phosphorylase.
Because glycogen will be persistently degraded, little glycogen will be present in
(d) Protein phosphatase1 will be continually active. Hence, the level of
phosphorylase b will be higher than normal, and glycogen will be less readily
(e) Protein phosphatase1 will be much less effective in dephosphorylating
glycogen synthase and glycogen phosphorylase. Consequently, the synthase will
stay in the less active b form, and the phosphorylase will stay in the more active a
form. Both changes will lead to increased degradation of glycogen.
(f) The absence of glycogenin will block the initiation of glycogen synthesis. Very
little glycogen will be
synthesized in its absence.
12. is always produced. Glycogen
degradation always occurs, and glycogen synthesis is always inhibited.
(b) Glycogen phosphorylasecannot be covalently activated. Glycogen degradation
is always inhibited; nothing can remain phosphorylated. Glycogen synthesis is
always active; nothing can remain phosphorylated.
(c) Phosphodiesterasedestroys cAMP. Therefore, glycogen degradation would
always be active and glycogen synthesis would always be inhibited.
6. See question
13. of phosphor-ylasekinase serves
to prolong the degradation of glycogen. The kinase cannot be deactivated until its
that the kinase and, in turn, phosphorylase stay active for an extended interval.
substrates for protein phosphatase. Thus, if the
and little glycogen degradation would take place.
7. 16. (a) The glycogen was too large to enter the gel and, because analysis was by
Western blot with the use of an antibody specific to glycogenin, one would not
expect to see background proteins.
-Amylase degrades glycogen, releasing the protein glycogenin so that it can
be visualized by the Western blot.
(c) Glycogen phosphorylase, glycogen synthase, and protein phosphatase1. These
proteins might be visible if the gel were stained for protein, but a Western analysis
reveals the presence of glycogenin only.
17. (a) The smear was due to molecules of glycogenin with increasingly large
amounts of glycogen attached to them.
(b) In the absence of glucose in the medium, glycogen is metabolized, resulting in
a loss of the high-molecularweight material.
(c) Glycogen could be resynthesized and added to the glycogenin when the cells
were fed glucose again.
(d) No difference between lanes 3 and 4 suggests that, by 1 hour, the glycogen
molecules had attained maximum size in this cell line. Prolonged incubation does
not apparently increase the amount of glycogen.
-Amylase removes essentially all of the glycogen, and so only the glycogenin