Berg J.M., Tymoczko J.L., Stryer L. Biochemistry

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Glycogen is synthesized by a different pathway from that of glycogen breakdown. UDP-glucose, the activated

intermediate in glycogen synthesis, is formed from glucose 1-phosphate and UTP. Glycogen synthase catalyzes the

transfer of glucose from UDP-glucose to the C-4 hydroxyl group of a terminal residue in the growing glycogen

molecule. Synthesis is primed by glycogenin, an autoglycosylating protein that contains a covalently attached

oligosaccharide unit on a specific tyrosine residue. A branching enzyme converts some of the α-1,4 linkages into α-1,6

linkages to increase the number of ends so that glycogen can be made and degraded more rapidly.

Glycogen Breakdown and Synthesis Are Reciprocally Regulated

Glycogen synthesis and degradation are coordinated by several amplifying reaction cascades. Epinephrine and glucagon

stimulate glycogen breakdown and inhibit its synthesis by increasing the cytosolic level of cyclic AMP, which activates

protein kinase A. Elevated cytosolic Ca

levels stimulate glycogen degradation by activating phosphorylase kinase.

Hence, muscle contraction and calcium-mobilizing hormones promote glycogen breakdown.

The glycogen-mobilizing actions of PKA are reversed by protein phosphatase 1, which is regulated by several hormones.

Epinephrine inhibits this phosphatase by blocking its attachment to glycogen molecules and by turning on an inhibitor.

Insulin, in contrast, activates this phosphatase by triggering a cascade that phosphorylates the glycogen-targeting subunit

of this enzyme. Hence, glycogen synthesis is decreased by epinephrine and increased by insulin. Glycogen synthase and

phosphorylase are also regulated by noncovalent allosteric interactions. In fact, phosphorylase is a key part of the

glucose-sensing system of liver cells. Glycogen metabolism exemplifies the power and precision of reversible

phosphorylation in regulating biological processes.

Key Terms

glycogen phosphorylase

phosphorolysis

pyridoxal phosphate

phosphorylase kinase

protein kinase A

calmodulin

epinephrine (adrenaline)

glucagon

uridine diphosphate glucose (UDPglucose)

glycogen synthase

glycogenin

protein phosphatase 1

insulin

II. Transducing and Storing Energy 21. Glycogen Metabolism

Problems

Carbohydrate conversion. Write a balanced equation for the formation of glycogen from galactose.

See answer

If a little is good, a lot is better. α-Amylose is an unbranched glucose polymer. Why would this polymer not be as

effective a storage form for glucose as glycogen?

See answer

Telltale products. A sample of glycogen from a patient with liver disease is incubated with orthophosphate,

phosphorylase, the transferase, and the debranching enzyme (α-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?

See answer

Excessive storage. Suggest an explanation for the fact that the amount of glycogen in type I glycogen-storage disease

(von Gierke disease) is increased.

See answer

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?

See answer

Recouping an essential phosphoryl. The phosphoryl group on phosphoglucomutase is slowly lost by hydrolysis.

Propose a mechanism that utilizes a known catalytic intermediate for restoring this essential phosphoryl group. How

might this phosphoryl donor be formed?

See answer

Hydrophobia. Why is water excluded from the active site of phosphorylase? Predict the effect of a mutation that

allows water molecules to enter.

See answer

Removing all traces. In human liver extracts, the catalytic activity of glycogenin was detectable only after treatment

with α-amylase (Section 11.2.2). Why was α-amylase necessary to reveal the glycogenin activity?

See answer

Two in one. A single polypeptide chain houses the transferase and debranching enzyme. Cite a potential advantage

of this arrangement.

See answer

10.

How did they do that? A strain of mice has been developed that lack the enzyme phosphorylase kinase. Yet, after

strenuous exercise, the glycogen stores of a mouse of this strain are depleted. Explain how this is possible.

See answer

11.

Metabolic mutants. Predict the major consequence of each of the following mutations:

(a) Loss of the AMP-binding site in muscle phosphorylase.

(b) Mutation of Ser 14 to Ala 14 in liver phosphorylase.

(d) Loss of the gene that encodes inhibitor 1 of protein phosphatase 1.

(e) Loss of the gene that encodes the glycogen-targeting subunit of protein phosphatase 1.

(f) Loss of the gene that encodes glycogenin.

See answer

12.

More metabolic mutants. Briefly, predict the major consequences of each of the following mutations affecting

glycogen utilization.

(a) Loss of GTPase activity of the G protein α subunit.

(b) Loss of the gene that encodes inhibitor 1 of protein phosphatase 1.

See answer

13.

Multiple phosphorylation. Protein kinase A activates muscle phosphorylase kinase by rapidly phosphorylating its β

subunits. The α subunits of phosphorylase kinase are then slowly phosphorylated, which makes the α and β

subunits susceptible to the action of protein phosphatase 1. What is the functional significance of the slow

phosphorylation of α?

See answer

14.

The wrong switch. What would be the consequences to glycogen mobilization of a mutation in phosphorylase

kinase that leads to the phosphorylation of the α-subunit prior to that of the β subunit?

See answer

Mechanism Problem

15.

Family resemblance. Propose mechanisms for the two enzymes catalyzing steps in glycogen debranching based on

their potential membership in the α-amylase family.

See answer

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 α-amylase and subsequently analyzed by SDS-PAGE and Western

blotting with the use of antibodies to glycogenin. The results are presented in the adjoining illustration.

(a) Why are no proteins visible in the lanes without amylase treatment?

(b) What is the effect of treating the samples with α-amylase? Explain the results.

See answer

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 with and without α-

amylase treatment. The results are presented in the adjoining illustration.

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 hours (lane 4). Samples (12 µg of protein) were either treated or not treated with α-amylase, as

indicated, before being loaded on the gel.

(a) Why did the Western analysis produce a "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 lane 2(-)?

(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?

See answer

Media Problem

18.

Either/or. Mammalian glycogen phosphorylase is activated by phosphorylation of serine 14, and deactivated

by dephosphorylation. The binding of glucose to glycogen phosphorylase a inhibits the enzyme by

promoting the protein phosphatase-catalyzed dephosphorylation of serine 14, which converts the a form of

the enzyme to the b form. From the information presented in the Structural Insights module on glycogen

phosphorylase, provide an explanation how glucose binding might promote dephosphorylation of serine 14.

II. Transducing and Storing Energy 21. Glycogen Metabolism Problems

Glycogen Isolation 1. [Courtesy of Dr. Peter J. Roach, Indiana University School of Medicine.]

II. Transducing and Storing Energy 21. Glycogen Metabolism Problems

Glycogen Isolation 2. [Courtesy of Dr. Peter J. Roach, Indiana University School of Medicine.]

II. Transducing and Storing Energy 21. Glycogen Metabolism

Selected Readings

Where to start

E.G. Krebs. 1993. Protein phosphorylation and cellular regulation I Biosci Rep. 13: 127-142. (PubMed)

E.H. Fischer. 1993. Protein phosphorylation and cellular regulation II Angew. Chem. Int. Ed. 32: 1130-1137.

L.N. Johnson. 1992. Glycogen phosphorylase: Control by phosphorylation and allosteric effectors FASEB J. 6: 2274-

2282. (PubMed)

M.F. Browner and R.J. Fletterick. 1992. Phosphorylase: A biological transducer Trends Biochem. Sci. 17: 66-71.

(PubMed)

Books and general reviews

R.G. Shulman and D.L. Rothman. 1996. Enzymatic phosphorylation of muscle glycogen synthase: A mechanism for

maintenance of metabolic homeostasis Proc. Natl. Acad. Sci. USA 93: 7491-7495. (PubMed) (Full Text in PMC)

P.J. Roach, Y. Cao, C.A. Corbett, R.A. DePaoli, I. Farkas, C.J. Fiol, H. Flotow, P.R. Graves, T.A. Hardy, and T.W.

Hrubey. 1991. Glycogen metabolism and signal transduction in mammals and yeast Adv. Enzyme Regul. 31: 101-120.

(PubMed)

G.I. Shulman and B.R. Landau. 1992. Pathways of glycogen repletion Physiol. Rev. 72: 1019-1035. (PubMed)

X-ray crystallographic studies

E.D. Lowe, M.E. Noble, V.T. Skamnaki, N.G. Oikonomakos, D.J. Owen, and L.N. Johnson. 1997. The crystal structure

of a phosphorylase kinase peptide substrate complex: Kinase substrate recognition EMBO J. 16: 6646-6658. (PubMed)

D. Barford, S.H. Hu, and L.N. Johnson. 1991. Structural mechanism for glycogen phosphorylase control by

phosphorylation and AMP J. Mol. Biol. 218: 233-260. (PubMed)

S.R. Sprang, S.G. Withers, E.J. Goldsmith, R.J. Fletterick, and N.B. Madsen. 1991. Structural basis for the activation of

glycogen phosphorylase b by adenosine monophosphate Science 254: 1367-1371. (PubMed)

L.N. Johnson and D. Barford. 1990. Glycogen phosphorylase: The structural basis of the allosteric response and

comparison with other allosteric proteins J. Biol. Chem. 265: 2409-2412. (PubMed)

M.F. Browner, E.B. Fauman, and R.J. Fletterick. 1992. Tracking conformational states in allosteric transitions of

phosphorylase Biochemistry 31: 11297-11304. (PubMed)

J.L. Martin, L.N. Johnson, and S.G. Withers. 1990. Comparison of the binding of glucose and glucose 1-phosphate

derivatives to T-state glycogen phosphorylase b Biochemistry 29: 10745-10757. (PubMed)

L.N. Johnson, K.R. Acharya, M.D. Jordan, and P.J. McLaughlin. 1990. Refined crystal structure of the phosphorylase-

heptulose-2-phosphate-oligosaccharide-AMP complex J. Mol. Biol. 211: 645-661. (PubMed)

Priming of glycogen synthesis

M.D. Alonso, J. Lomako, W.M. Lomako, and W.J. Whelan. 1995. A new look at the biogenesis of glycogen FASEB J. 9:

1126-1137. (PubMed)

A. Lin, J. Mu, J. Yang, and P.J. Roach. 1999. Self-glucosylation of glycogenin, the initiator of glycogen biosynthesis,

involves an inter-subunit reaction Arch. Biochem. Biophys. 363: 163-170. (PubMed)

P.J. Roach and A.V. Skurat. 1997. Self-glucosylating initiator proteins and their role in glycogen biosynthesis Prog.

Nucleic Acid Res. Mol. Biol. 57: 289-316. (PubMed)

C. Smythe and P. Cohen. 1991. The discovery of glycogenin and the priming mechanism for glycogen biogenesis Eur. J.

Biochem. 200: 625-631. (PubMed)

Catalytic mechanisms

V.T. Skamnaki, D.J. Owen, M.E. Noble, E.D. Lowe, G. Lowe, N.G. Oikonomakos, and L.N. Johnson. 1999. Catalytic

mechanism of phosphorylase kinase probed by mutational studies Biochemistry 38: 14718-14730. (PubMed)

J.L. Buchbinder and R.J. Fletterick. 1996. Role of the active site gate of glycogen phosphorylase in allosteric inhibition

and substrate binding J. Biol. Chem. 271: 22305-22309. (PubMed)

D. Palm, H.W. Klein, R. Schinzel, M. Buehner, and E.J.M. Helmreich. 1990. The role of pyridoxal 5 -phosphate in

glycogen phosphorylase catalysis Biochemistry 29: 1099-1107. (PubMed)

Regulation of glycogen metabolism

B.A. Pederson, C. Cheng, W.A. Wilson, and P.J. Roach. 2000. Regulation of glycogen synthase: Identification of

residues involved in regulation by the allosteric ligand glucose-6-P and by phosphorylation J. Biol. Chem. 275: 27753-

27761. (PubMed)

R. Melendez, E. Melendez-Hevia, and E.I. Canela. 1999. The fractal structure of glycogen: A clever solution to optimize

cell metabolism Biophys. J. 77: 1327-1332. (PubMed)

J. Franch, R. Aslesen, and J. Jensen. 1999. Regulation of glycogen synthesis in rat skeletal muscle after glycogen-

depleting contractile activity: Effects of adrenaline on glycogen synthesis and activation of glycogen synthase and

glycogen phosphorylase Biochem. J. 344 (pt.1): 231-235. (PubMed)

J.B. Aggen, A.C. Nairn, and R. Chamberlin. 2000. Regulation of protein phosphatase-1 Chem. Biol. 7: R13-R23.

(PubMed)

M.P. Egloff, D.F. Johnson, G. Moorhead, P.T. Cohen, P. Cohen, and D. Barford. 1997. Structural basis for the

recognition of regulatory subunits by the catalytic subunit of protein phosphatase 1 EMBO J. 16: 1876-1887. (PubMed)

L. Ragolia and N. Begum. 1998. Protein phosphatase-1 and insulin action Mol. Cell. Biochem. 182: 49-58. (PubMed)

J. Wu, J. Liu, I. Thompson, C.J. Oliver, S. Shenolikar, and D.L. Brautigan. 1998. A conserved domain for glycogen

binding in protein phosphatase-1 targeting subunits FEBS Lett. 439: 185-191. (PubMed)

Genetic diseases

Chen, Y.-T., and Burchell, A., 1995. Glycogen storage diseases. In The Metabolic Basis of Inherited Diseases (7th ed.,

pp. 935

965) edited by C. R. Striver., A. L. Beaudet, W. S. Sly, D. Valle, J. B. Stanbury, J. B. Wyngaarden, and D. S.

Fredrickson, McGraw-Hill.

A. Burchell and I.D. Waddell. 1991. The molecular basis of the hepatic microsomal glucose-6-phosphatase system

Biochim. Biophys. Acta 1092: 129-137. (PubMed)

K.J. Lei, L.L. Shelley, C.J. Pan, J.B. Sidbury, and J.Y. Chou. 1993. Mutations in the glucose-6-phosphatase gene that

cause glycogen storage disease type Ia Science 262: 580-583. (PubMed)

B.D. Ross, G.K. Radda, D.G. Gadian, G. Rocker, M. Esiri, and J. Falconer-Smith. 1981. Examination of a case of

suspected McArdle's syndrome by

P NMR N. Engl. J. Med. 304: 1338-1342. (PubMed)

Evolution

L. Holm and C. Sander. 1995. Evolutionary link between glycogen phosphorylase and a DNA modifying enzyme EMBO

J. 14: 1287-1293. (PubMed)

J.W. Hudson, G.B. Golding, and M.M. Crerar. 1993. Evolution of allosteric control in glycogen phosphorylase J. Mol.

Biol. 234: 700-721. (PubMed)

V.L. Rath and R.J. Fletterick. 1994. Parallel evolution in two homologues of phosphorylase Nat. Struct. Biol. 1: 681-690.

(PubMed)

R. Melendez, E. Melendez-Hevia, and M. Cascante. 1997. How did glycogen structure evolve to satisfy the requirement

for rapid mobilization of glucose? A problem of physical constraints in structure building J. Mol. Evol. 45: 446-455.

(PubMed)

V.L. Rath, K. Lin, P.K. Hwang, and R.J. Fletterick. 1996. The evolution of an allosteric site in phosphorylase Structure

4: 463-473. (PubMed)

II. Transducing and Storing Energy

22. Fatty Acid Metabolism

We turn now from the metabolism of carbohydrates to that of fatty acids. A fatty acid contains a long hydrocarbon chain

and a terminal carboxylate group. Fatty acids have four major physiological roles. First, fatty acids are building blocks of

phospholipids and glycolipids. These amphipathic molecules are important components of biological membranes, as we

discussed in Chapter 12. Second, many proteins are modified by the covalent attachment of fatty acids, which targets

them to membrane locations (Section 12.5.3). Third, fatty acids are fuel molecules. They are stored as triacylglycerols

(also called neutral fats or triglycerides), which are uncharged esters of fatty acids with glycerol (Figure 22.1). Fatty

acids mobilized from triacylglycerols are oxidized to meet the energy needs of a cell or organism. Fourth, fatty acid

derivatives serve as hormones and intracellular messengers. In this chapter, we will focus on the oxidation and synthesis

of fatty acids, processes that are reciprocally regulated in response to hormones.

22.0.1. An Overview of Fatty Acid Metabolism

Fatty acid degradation and synthesis are relatively simple processes that are essentially the reverse of each other. The

process of degradation converts an aliphatic compound into a set of activated acetyl units (acetyl CoA) that can be

processed by the citric acid cycle (Figure 22.2). An activated fatty acid is oxidized to introduce a double bond; the

double bond is hydrated to introduce an oxygen; the alcohol is oxidized to a ketone; and, finally, the four carbon

fragment is cleaved by coenzyme A to yield acetyl CoA and a fatty acid chain two carbons shorter. If the fatty acid has

an even number of carbon atoms and is saturated, the process is simply repeated until the fatty acid is completely

converted into acetyl CoA units.

Fatty acid synthesis is essentially the reverse of this process. Because the result is a polymer, the process starts with

monomers

in this case with activated acyl group (most simply, an acetyl unit) and malonyl units (see Figure 22.2). The

malonyl unit is condensed with the acetyl unit to form a four-carbon fragment. To produce the required hydrocarbon

chain, the carbonyl must be reduced. The fragment is reduced, dehydrated, and reduced again, exactly the opposite of

degradation, to bring the carbonyl group to the level of a methylene group with the formation of butyryl CoA. Another

activated malonyl group condenses with the butyryl unit and the process is repeated until a C

fatty acid is synthesized.

II. Transducing and Storing Energy 22. Fatty Acid Metabolism

Figure 22.1. Electron Micrograph of an Adipocyte. A small band of cytoplasm surrounds the large deposit of

triacylglycerols. [Biophoto Associates/ Photo Researchers.]

II. Transducing and Storing Energy 22. Fatty Acid Metabolism

Figure 22.2. Steps in Fatty Acid Degradation and Synthesis. The two processes are in many ways mirror images of

each other.

II. Transducing and Storing Energy 22. Fatty Acid Metabolism

Fats provide an efficient means for storing energy for later use. (Right) The processes of fatty acid synthesis

(preparation for energy storage) and fatty acid degradation (preparation for energy use) are, in many ways, the reverse of

each other. (Above) Studies of mice are revealing the interplay between these pathways and the biochemical bases of