Formationof liver glycogenfrom lactic acid is thus seento establish

an importantconnectionbetweenthe metabolismof the muscleand

that of the liver.Muscle glycogenbecomesavailableas blood sugar
throughthe interventionof the liver,and blood sugarin turn is con-
vertedinto muscleglycogen.Thereexiststhereforea completecycle
o f t h e g l u c o s em o l e c u l ei n t h e b o d y . . . E p i n e p h r i nw e asfoundto
a c c e l e r a t et h i s c y c l e i n t h e d i r e c t i o no f m u s c l eg l y c o g e nt o l i v e r
glycogen. . . Insulin,on the other hand, was found to acceleratethe
cycle in the directionof blood glucoseto muscleglycogen.
-C. F.Cori and G. T.Cori,
articlein Journalof BiologicalChemistry,1929

ples0fMetabolic
Princi Regulation
15.1 Regulation
ofMetabolic
Pathways570 be partially degradedto provide acetyl-CoAfor fatty
acid and sterol synthesis. And the bacterium .os-
15.2 Analysis ofMetabolic (ontrol 577 cheri,chi,acoli, can use glucose to produce the carbon
skeleton of euery one of its severalthousand types of
15.3 (oordinated RegulationofGlycolysisand
molecules.When any cell usesglucose6-phosphatefor
Gluconeogenesis 582 one purpose,that "decision"affectsall the other path-
15.4 TheMetabolism ofGlycogen inAnimals 594 waysfor which glucose6-phosphateis a precursoror in-
termediate: any change in the allocation of glucose
15.5 (oordinated RegulationofGlyrogenSynthesis 6-phosphateto one pathway affects, directly or indi-
andBreakdown602 rectly, the flow of metabolitesthrough all the others'
Suchchangesin allocationare conunonin the life of
a cell. Louis Pasteurwas the first to describethe more
etabolicregulation,a centraitheme in biochem- than l0-fold increasein glucoseconsumptionby a yeast
istry, is one of the most remarkablefeaturesof culture when it was shifted from aerobic to anaerobic
Iiving organisms.Of the thousandsof enzyme- conditions.This "Pasteureffect"occurswithout a signif-
catalyzedreactionsthat can take place in a cell, there is icant changein the concentrations of ATP or most of the
probablynot one that escapessomeform of regulation. hundredsof metabolichtermediatesand productsde-
This need to regulateevery aspectof cellularmetabo- rived from glucose.A similar effect occursin the cells of
Iism becomesclear as one examinesthe complexityof skeletat muscle when a sprinter Ieavesthe starting
metabolicreactionsequences. Althoughit is convenient blocks. The ability of a cell to carry out all these inter-
for the student of biochemistry to divide metabolic lockingmetabolicprocessessimultaneously-obtaining
processesinto "pathways"that play discreterolesin the every product in the amount needed and at the right
no
cell's economy, such separation exists i.n the living time, in the face of major perturbationsfrom outside,
cell. Rather,every pathway in
we discuss this book is and without generatingleftovers-is an astoundi'ng
inextricably intertwined with all the other cellular accomplishment.
pathwaysin a multidimensionalnetwork of reactions In this chapterwe use the metabolismof glucoseto
(FiS. 15-1). For example,in Chapter14 we discussed illustrate somegeneralprinciplesof metabolicregula-
four possiblefatesfor glucose 6-phosphate in a hepato- tion. First we look at the generalroles of regulationin
cy[e: breakdownby glycolysisfor the production of AIP, achievingmetabolichomeostasisand introduce meta-
breakdorn"'ri in the pentose phosphate pathway for the bolic control analysis,a system for analyzingcomplex
production of NADPH and pentosephosphates,use in metabolic interactions quantitatively.We then describe
the sl,nthesisof complexpolysaccharides of the extra- the speciflc regulatory properties of the individual en-
cellularmatrix, or hydrolysisto glucoseand phosphate zymes of glucose metabolism;for glycolysisand gluco-
to replenishbloodglucose.In fact, glucose6-phosphate neogenesis, we described the catalyticactivitiesof the
has other possiblefates in hepatocytes,too; it may,for enzyrnes in Chapter 14. Here we also discussboth the
example,be used to synthesizeother sugars,such as catalytic and regulatory properties of the enz;.'rnesof
glucosamine, galactose,galactosamine, fucose,and neu- glycogen synthesis and breakdowrl, one of the best-
raminic acid, for use in protein glycosylation,or it may studied cases of metabolic regulation. Note that in
T l
569 I
 ICttl Principtes
ofMetabotic
Regutation
FIGURE 15-1 Metabolismasa three-dimensional meshwork.A typical
eukaryoticcell has the capacityto make about 30,000 differentpro-
teins,which catalyzethousands of differentreactions involvingmany
hundredsof metabolites,most sharedby more than one ,,pathway.,,
Thisoverviewimageof metabolicpathways is from the onlineKECC
selectingcarbohydratemetabolismto illustratethe prin-
ciples of metabolic regulation,we have artificially sepa-
rated the metabolismof fats and carbohydrates. In fact,
these two activities are very tightly integrated, as we
shallseein Chapter23.
15.1Regulation
ofMetabolic
Pathways
The pathways of glucose metabolism provide, in the
catabolic direction, the energy essential to oppose the
forces of entropy and, in the anabolic direction, biosyn-
thetic precursors and a storage form of metabolic en-
ergy. These reactions are so important to survival that
very complex regulatory mechanisms have evolved to
(KyotoEncyclopedia of Cenesand Cenomes)PATHWAYdatabase
(www genomead jplkegg/pathway/map/map0.l 100 html). Eacharea
can be furtherexpandedfor increasingly
detailedinformation,to the
levelof specificenzymesand intermediates.
ensurethat metabolitesmove through each pathway in
the correct direction and at the correct rate to match
exactly the cell's or the organism'schangingcircum-
stances.By a variety of mechanismsoperatingon differ-
ent time scales,adjustmentsare made in the rate of
metaboliteflow through an entire pathway when exter-
nal circumstances change.
Circumstancesdo change,sometimesdramatically.
For example,the demandfor ATP in insect flrght muscle
increases100-foldin a few secondswhen the insect
takes flight. In humans,the availability of oxygen may
decreasedue to hypoxia (diminisheddelivery of oxygen
to tissues)or ischemia(diminishedflow of blood to tis-
sues).The relativeproportionsof carbohydrate, fat, and

protein in the diet vary from mealto meal,and the sup-
ply of fuels obtainedin the diet is intermittent, requiring
metabolicadjustmentsbetweenmealsand during peri-
ods of starvation.Woundhealingrequireshuge amounts
of energyand biosyntheticprecursors.
(ellsand0rganisms
Maintain
a Dynamic
Steady
State
Fuels such as glucoseenter a cell, and wasteproducts
suchas CO2leave,but the massand the grosscomposi-
tion of a typical cell, organ, or adult animal do not
changeappreciablyover time; cells and organismsexist
in a dynamicsteadystate.For eachmetabolicreaction
in a pathway,the substrateis providedby the preceding
reaction at the same rate at which it is convertedto
product. Thus, althoughthe rate (u) of metaboliteflow,
or flux, through this step of the pathway may be high
and variable,the concentrationof substrate,S, remains
constant.So,for the two-stepreaction
u',
A ") s P mec
when ai : az, [S] is constant.For example,changesin
a1 for the entry ofglucosefromvarioussourcesinto the
blood are balancedby changesin u2 for the uptake of
glucosefrom the blood into varioustissues,so the con-
centrationof glucosein the blood ([S]) is held nearly
constantat 5 mnt.This is homeostasis at the molecular
level. The failure of homeostaticmechanismsis often at
the root of human disease.In diabetesmellitus,for ex-
ample,the regulationof blood glucoseconcentrationis
defective as a result of the lack of or insensitivity to in-
sulin,with profoundmedicalconsequences.
When the external perturbation is not merely tran-
sient,or when onekind of cell developsinto another,the
adjustmentsin cell compositionand metabolismcan be
more dramatic and may require signiflcant and lasting
changesin the allocation of energy and synthetic pre-
cursorsto bring abouta new dynamicsteadystate.Con-
sider, for example, the differentiation of stem cells in
the bone marrow into erythrocytes.The precursorcell
containsa nucleus,mitochondria,and little or no hemo-
globin, whereasthe fully differentiatederythroclte con-
tains prodigiousamountsof hemoglobinbut has neither
nucleus nor mitochondria;the cell's compositionhas
permanentlychangedin responseto externaldevelop-
mental signals,with accompanyingchangesin metabo-
lism. This cellular differentiation requires precise
regulationof the levelsof cellularproteins.
In the courseof evolution,organismshaveacquireda
remarkablecollectionof regtrlatorymechanismsfor main-
taininghomeostasis at the molecular,cellular,and organis-
mal levels,as reflected in the proportion of genesthat
encoderegulatorymachinery.In humans,about 4,000
genes(-12% of all genes)encoderegulatoryproteins,in-
cludinga variety of receptors,regr.rlators of gene expres-
sion,and morethan 500differentprotein kinases!In many
cases,the regulatorymechanismsoverlap:one erz5.'rne is
subjectto regulationby severaldifferentmechanisms.
M e t a b oP
1 5 . 1R e g u l a t ioof n l i ca t h w a y[st t t - l
BoththeAmount Activity
andtheCatalytic
ofan (an
Enzyme BeRegulated
The flux through an enzyrne-catalyzedreaction can be
modulatedby changesin the number of enzymemole-
cules or by changesin the cataLgticacti'ui'tAof each
enzyrnemoleculealreadypresent.Such changesoccur
on time scalesfrom millisecondsto many hours, in re-
sponseto signalsfrom within or outside the cell' Very
rapid allostericchangesin enzl'rneactivity are generally
triggered locally, by changesin the local concentration
of a small molecule-a substrate of the pathway in
which that reaction is a step (say, glucosefor glycoly-
sis), a product of the pathway(ATPfrom glycolysis),or
a key metabolite or cofactor (such as NADH) that indi-
cates the cell's metabolic state. Second messengers
(such as cyclic AMP and Ca2*) generatedintracellularly
in response to extracellular signals (hormones, cy-
tokines, and so forth) alsomediate allostericregulation,
on a slightly slower time scale set by the rate of the
e-Chapter12).
signal-transduction
Extracelluiar srgnals @) maybe hormonal
(insutin or eptnephrine, for example) or neuronal (acetyl-
choline),or maybe gowth factorsor c1'tokines. The num-
ber of moleculesof a given erzyme in a cell is a function of
the relativerates of q,nthesis and degradation of that en-
25.'rne.The rate of syrrthesiscanbe adjustedby the activa-
tion (in responseto someoutsidesrgnal)of a transcription
factor (Fig. l5-2,@; descnbedin more detail in Chapter
28). Tbanscription factors are nuclear proteins that,
whenactivated,bind speci-flc DNA regions(response ele-
ments) near a gene'spromoter (its transcriptionalstartirg
point) andactivateor repressthe transcriptionofthat gene,
leadingto increasedor decreasedsyrrthesisofthe encoded
protein. Activationof a transcriptionfactor is sometimes
the result of its binding of a specificligand and sometimes
the result of its phosphorylationor dephosphorylation.
Eachgeneis controlledby one or moreresponseelements
that are recognizedby speciflctranscription factors. Some
geneshave severalresponseelementsand are therefore
controlled by severaldifferent transcription factors, re-
spondingto severaldifferent signals.Groupsof genesen-
codingproteinsthat act together,suchas the enz1rnesof
glycolysisor gluconeogenesis, often share cornmonre-
sponseelementsequences,so that a singlesignal,acting
through a particulartranscriptionfactor,turns all ofthese
geneson and off together.The regulationof carbohydrate
metabolismby speciflctranscriptionfactorsis describedin
Section15.3.
The stabilityof messengerRNAs-their resistanceto
degradationby cellular ribonucleases(Fig. 15-2, @)-
varies, and the amount of a given mRNA in the cell is a
function of its rates of synthesisand degradation(Chap-
ter 26). The rate at which an mRNA is translatedinto a
protein by ribosomes(Fig. l5_2, @) is also regulated,
and dependson severalfactors describedin detail in
Chapter27.Note that ann-fold increasein an mRNAdoes
not alwaysmeanan z-fold increasein its protein product'
 fl

j72- P r i n c i p loefsM e t a b oR
l i ce g u l a t i o n

Protein degradation
mRNe translation on ribosome (ubiquitin; proteasome)
@

FIGURE15-2 Factorsaffectingtheactivityof enzymes. Thetotalactiviry through@), or by modulatingthe activityof existingmolecules(@

of an enzyme canbe changed by alteringthenumberof itsmolecules through @), asdetailedin the text.An enzymemay be influencedby
in the cell,or iIs effective
activityin a subcellular
compartment (@ a combinationof suchfactors.

Once synthesized, protein molecules have a finite
lifetime, which may range from minutes to many
days (Table_15-1). The rate of protein degradation
(Fig. 15-2, @; Aiffers from one enz).rne to another and
depends on the conditions in the cell. Some proteins are
tagged by the covalent attachment of ubiquitin for
degradation in proteasomes, as discussed in Chapter 2g
(see, for example, the case of cyclin, in Fig. 12_46).
Rapid turnover (s;,nthesis followed by degradation) is
energetically expensive, but proteins with a short half_
life can reach new steady state levels much faster than
those with a long half-life, and the beneflt of this quick
responsiveness must balance or outweigh the cost to
the cell.
fissue
Liver
Kidney
Heart
Brain
Muscle
Yet another way to alter the elfectzueactivity of an
enzymeis to sequesterthe enz}rmeand its substratein
different compartments(Fig. I5-2, @).In muscle,for
example,hexokinasecannot act on glucoseuntil the
sugarentersthe myocytefrom the blood, and the rate at
which it entersdependson the activityof glucosetrans-
porters (see Table 11-3) in the plasma membrane.
Within cells,membrane-bounded compartmentssegre-
gate certain enzymesand enzyme systems,and the
transport of substrateacrossthese intracellularmem-
branesmay be the limiting factor in enzlirneaction.
By these severalmechanismsfor regulatingenz),Tne
level, cells can dramaticallychangetheir complementof
en4,.rnesin responseto changesin metabolic circum-
stances.In vertebrates,liver is the most adaptabletis-
sue; a changefrom a high-carbohydrateto high-lipid
diet, for example,affects the transcription of hundreds
of genesand thus the levels of hundreds of proteins.
Half-life (days) Theseglobal changesin gene expressioncan be quanti-
0.9 fled by the use of DNA microarrays (seeFig. g-22) that
I7
display the entire complement of mRNAs present in a
given cell type or organ (the transcriptome) or by two-
4.7 dimensionalgel electrophoresis(see Fig. B-21) that
4.6 displaysthe protein complementof a cell type or organ
t0.7 (its proteome). Both techniquesoffer great insights
into metabolic regulation. The effect of changesin the
 't5.1
M e t a b oP
R e g u l a t ioof n l i ca t h w a ylst t {

proteome is often a change in the total ensemble of low

molecular weight metabolites, the metabolome.
Once the regulatory mechanisms that involve pro-
tein synthesis and degradation have produced a certain
number of molecules of each enzyme in a cell, the
activity of those enz).rnes can be further regulated in
several other ways: by the concentration of substrate,
the presence of allosteric effectors, covalent modiflca-
tions, or binding of regulatory proteins-all of whrch can
change the activity of an individual enz;rme molecule
(Fig. l5-2, @ to @).
All enzymes are sensitive to the concentration of
their substrate(s) (Fig l5-2,@). Recall that in the sim-
plest case (an enzyme that follows Michaelis-Menten ki-
netics), the initial rate of the reaction is half-maximal
when the substrate is present at a concentration equal
to K- (that is, when the enz;'rne is half-saturated with
substrate). Activity drops off at lower [S], and when [S]
11 K^, the reaction rate is linearly dependent on [S].
This is important because intracellular concentrations
of substrate are often in the same range as, or lower
than, K-. The activity of hexokinase, for example,
changeswith [glucose],and intracellular [glucose]varies
with the concentration of glucose in the blood. As we
will see, the different forms (isozymes) of hexokinase
have different K- values and are therefore differently
affected by changes in intracellular [glucose], in ways
that make sense physiologically.
I W0RKED
EXAMPIE
15-1 Activityof
aGlucose
Transporter
If K, (the equivalentof K-) for the glucosetransporter
in liver (GLUT2) is 40 mlt, calculate the effect on the
rate of glucoseflux into a hepatocyteof increasingthe
bloodglucoseconcentrationfrom 3 mu to 10 mu.
Solution:We use Equation11-1 (p. 393) to find the ini-
tial velocity (flux) of glucoseuptake.
V-.*[SJo16
- changesthat translate into changesin 7^u* or K-. For
"o K, I [sL*
At 3 mu glucose
Vo : V-"* (3 mu)/(40 mlr + 3 mttl)
: V-., (3 mrt/43 mu) : 0.07V-.*
Requiredchangein [S]
Hill coeffieient to increase76from
(nn) l0o/otog0%V^n*
0.5 x6,600
1.0 x81
2.0 X9
3.0 x4.3
4.0 X3
to sigmoidkinetics,or vice versa (see Fig. 15-14b,for
example).In the steepestpart of the sigmoidcurve, a
small changein the concentrationof substrate,or of
allostericeffector,can have a large impact on reaction
rate.Recallfrom Chapter5 (p. 164)that the cooperativ-
ity of an allostericenzymecan be expressedas a Hill
coefficient,with higher coefficientsmeaning greater
cooperativity.For an allosteric enzyme with a Hill
coefflcientof 4, actiletyincreasesfrom 10%o V^u*to 90o/o
I/.,u, with only a 3-fold increasein [S], comparedwith
the 81-fold rise in [S] needed by an enzymewrth no
cooperativeeffects(Hill coefflcientof l; Table15-2).
Covalentmodificationsof enz;.'mesor other proteins
(Fig. 15-2,@) occurwithin secondsor minutesof a reg-
ulatory si.gnal,fupicallyan extracellttlarsignal.By far the
most commonmodiflcationsare phosphorylationand de-
phosphorylation(Fig. f 5-3); up to half the proteinsin a
eukaryotic cell are phosphorylatedunder somecircum-
stances.Phosphorylationby a specific protein kinase
may alter the electrostaticfeaturesof an enz}rme'sactive
site, causemovementof an inhibitory region of the en-
in-
z;.'rneprotein out of the active site, alter the enz}.'rne's
teraction wrth other proteins,or force conformational
Protein
substrate
' Ser/Ihr/Iyr;OH

At 10 mvt glucose

Vo: V^u' (10 mn'l)/(40mn + 10 mu)

: V-^* (10 mu/50 mu) : 0.20V-"*

So a rise in blood glucose from 3 mlt to 10 mM increases

the rate ofglucose influx into a hepatocyte by a factor of
0 . 2 0 / 0 . :037
Enzy'rneactivity canbe either ircreasedor decreased
by an allostericeffector (Fig. 15-2, @; se" Fig. 6-34).
Allosteric effectorstypically convert hyperbolickinetics
-P-O-
I
o-
FIGURE 15-3 Protein phosphorylationand dephosphorylation. Pro-
tein kinasestransfera phosphorylgroupfrom ATPto a Ser,Thr,orTyr
residuein an enzymeor otherproteinsubstrate.Proteinphosphatases
removethe phosphorylgrouPas P;.
 l:,1 P r i n r i p loefsM e t a b oR
l i ce g u l a t i o n
covalentmodi-flcationto be useful in regulation,the cell
must be able to restorethe altered enzyrneto its original
activity state.A family of phosphoproteinphosphatases,
at least someof which are themselvesunder regulation,
catalyzesthe dephosphorylation of proteins.
Finally, many enzyrnesare regulatedby association
with and dissociationfrom another,regulatoryprotein
(Fig. 15-2,@;. f'or example,the cyclicAMP-dependent
protein kinase(PKA; seeFig. 12-6) is inactiveuntil cAMP
binding separatescataly'ticfrom regulatory subunits.
These several mechanismsfor altering the flux
through a step in a metabolicpathway are not mutually
exclusive.It is very corrunonfor a single enz;rmeto be
regulatedat the level of transcriptionand by both al-
losteric and covalent mechanisms.The combination
providesfast, smooth,effectiveregulationin response
to a very wide array of perturbationsand signals.
In the discussionsthat follow,it is usefirlto think of
changesin enzl'rnatic activity as serving two distinct
though complementaryroles.Weusethe term metabolic
regulation to refer to processesthat serveto mamtarn
homeostasis at the molecularlevel-to hold somecellular
parameter(concentrationof a metabolite,for example)at
a steadyIevel over time, even as the flow of metabolites
through the pathwaychanges.The term metabolic con-
trol refers to a processthat ieadsto a changein the out-
put of a metabolicpathwayovertime, in responseto some
outside signal or changein circumstances.The distinc-
tion, althoughuseful,is not alwayseasyto make.
Reartions
FarfromEquilibrium
inCells
Are
(ommonPointsofRegulation
For some steps in a metabolicpathway the reaction is
closeto equilibrium,with the cell in its dynamic steady
state (Fig. 154). The net flow of metabolitesthrough
these steps is the small differencebetweenthe rates of
the forward and reverse reactions, rates that are very
similarwhen a reactionis near equilibrium.Smallchanges
in substrateor product concentrationcan produce large
o@@
y: 10.01 v : 200 y: 500
-!n-
v=0.01 v=190 V=490
net rate: 10 10 10
FIGURE 15-4 Near-equilibriumand nonequilibriumstepsin a meta-
bolicpathway. Steps@ and@ of thispathwayarenearequilibriumin
the cell; for each step,the rate(V) of the forwardreactionis only
slightlygreaterthan the reverserate,so the netforwardrate(10) is rel-
ativelylow and the free-energy change,AC,, is closeto zero.An in-
creasein [C] or [D] can reversethe directionof thesestepsStep@ is
maintained in the cell far fromequilibrium;itsforwardrategreatlyex-
ceedsits reverserate.The net rateof stepO ftO) is much largerthan
the reverserate(0 01) and is identicalto the net ratesof steps@and@
when the pathwayis operatingin the steadystate.Step@ hasa large,
neeativeAC'
changesin the net rate, and can even changethe direc-
tion of the net flow We can identify these near-equilib-
rium reactionsin a cell by comparingthe mass action
ratio, @,with the equilibrium constantfor the reaction,
K!o. Recallthat for the reaction A + B -+ C * D, Q :
tcltDl/tAltBl.WhenI andKlo are within I to 2 ordersof
magnitudeof eachother,the reactionis near equil_rbrium.
This is the casefor 6 of the 10 stepsin the glycolS,'tic
path-
way (Table15-3).
Other reactions are far from equilibrium in the
cell. For example,K'.o for the phosphofructokinase-l
(PFK-1) reactionis about 1,000,but Q ([fructose 1,6-
bisphosphatel[ADP]/[fructose 6-phosphate][ATP]) in a
hepatocytein the steadystateis about0.1 (Table15-3).
It is becausethe reaction is so far from equilibrium that
the processis exergonicunder cellularconditionsand
tendsto go in the forwarddirection.The reactionis held
far from equilibrium because,under prevailing cellular
conditionsof substrate,product, and effector concen-
trations,the rate of conversionof fructose6-phosphate
to fructose1,6-bisphosphate is limited by the activity of
PFK-1,which is itself limited by the number of PFK-1
moleculespresentand by the actionsof allostericeffec-
tors. Thus the net forward rate of the enzyme-catalyzed
reaction is equalto the net flow of glycoly-ticintermedi-
atesthrough other stepsin the pathway,and the reverse
flow throughPFK-1remainsnearzero.
The cell cannot allow reactionswith large equilib-
rium constantsto reachequilibrium.If [fructose6-phos-
phatel, [ATP],and [ADP]in the cell were held at typical
levels (low millimolar concentrations)and the PFK-1re-
action were allowedto reach equilibrium by an increase
in [fructose 1,6-bisphosphate], the concentration of
fructose 1,6-bisphosphate would rise into the molar
range,wreakingosmotichavocon the cell. Consideran-
other case:if the reaction ATP -+ ADP * P, were al-
lowed to approachequiiibrium in the cell, the actual
free-energychange (AG') for that reaction (AGo; see
Worked Example I3-2, p. 503) would approachzero,
and ATP would lose the high phosphorylgroup transfer
potential that makesit valuableto the cell. It is therefore
essentialthat enzS,.rnes catalyzingATP breakdown and
other highly exergonicreactionsin a cell be sensitiveto
regulation, so that when metabolic changesare forced
by externalcircumstances, the flow through these en-
zymeswill be adjustedto ensurethat [ATP] remainsfar
above its equilibrium level. When such metabolic
changesoccur, the activities of enz;rmesin all intercon-
nectedpathwaysadjustto keepthesecriticalstepsaway
from equilibrium. Thus, not surprisingly,many enzlnnes
(such as PFK-I) that catalyzehighly exergonic reac-
tions are subject to a variety of subtle regulatory mech-
anisms.The multiplicity of theseadjustmentsis so great
that we cannot predict by examiningthe properties of
any one enzyrnein a pathway whether that enzymehas
a strong influence on net flow through the entire path-
way.This complexproblem can be approachedby meta-
bolic controlanalysis,as describedin Section15.2.
 M e t a b oP
15 . 1R e g u l a t ioofn
t--J
l i ca t h w a yLs ) / ) l

Reaction
ne&r AG'
Massactionratio,0 equilibrium LG'" (kJ/tnol)
Enzyme KLs Liver IIeart in vivo?* (kJ/mol) in heart

Hexokinase 1x103 2 x l0-2 8 x 10-2 No _1t7 -27

PFK-1 1 . 0x 1 0 3 g x 10-2 3 x 10-2 No 1l -
Zt)

Aldolase 1 . 0x 1 0 - 4 1 . 2x 1 0 - 6 g x 10-6 Yes LDA -6.0

Tliose phosphateisomerase 4 x 1.0*2 2.4 x l0*r Yes +7.5 +3.8
Glyceraldehyde3-phosphate
dehydrogenase*
phosphoglyceratekinase 2x103 6x102 9.0 Yes -13 f it.D

Phosphoglyceratemutase 1 x 10-1 1 x 10-1 1 . 2x 1 0 - 1 Yes LAA +0.6

Enolase 2.9 1.4 Yes -6.d -0.5
Pyruvatekinase 2xr04 7 x 10-1 40 No _DI -17
Phosphoglucose
isomerase 4 x 10-r 3 . 1x 1 0 - 1 2.4 x r}-L Yes Ta.a
-r.4
Pyruvatecarboxylase
+ PEP carboxykinase 7 1 x 10-3 No -D.U -23
Glucose6-phosphatase 8 . 5x 1 0 2 1.2 x r02 Yes -17 -D.U

Source:K:qandQ fromNewsholme, E.A.& Start,C.(1973)Regulationin Metabolism,WtleyPress,NewYork, fromthesedata.

pp.97,263.AG' andAG'' werecalculated
+Forsimplicity,
anyreaction
forwhichtheabsolutevalueof thecalculated nearequilibrium.
AG' is lessthan6 is considered

Adenine
Nucleotides
Play Roles
Special in
Metabolic
Regulation
After the protection of its DNA from damage,perhaps
nothing is more important to a cell than maintaininga
constant supply and concentrationof ATP. Many ATP-
using enzyrneshave K* values between 0.1 and I mrr,t,
and the ATP concentrationin a typical cell is about5 mtr,l.
If [ATP]were to drop significantly,these enzyrneswould
be lessthan fully saturatedby their substrate(ATP), and
the rates of hundreds of reactions that involve ATP
woulddecrease(FiS. f 5-5); the cellwouldprobablynot
survivethis ki,netzceffect on so many reactions.
There is also an important thermodynamzc effect
of lowered tATPl. BecauseATP is convertedto ADP
or AMP when "spent" to accomplish cellular work,
determinesthe magnitudeand sign of AG' and therefore
the drivingforce,AG', of the reaction:
IADPI[glucoseG-phosphateJ
AG':AG'"+ft?ln
lATPllglucosel
Becausean alteration of this driving force profoundly
influences every reaction that involves ATP, organ-
isms have evolved under strong pressure to develop
regulatorymechanismsresponsiveto the IATPI/tADPl
ratio.
AMP concentration is an even more sensitiveindi-
cator of a cell'senergeticstate than is [ATP].Normally
!1
d

the [ATP]/[ADP]ratio profoundly affects all reactions h

that employ these cofactors. (The same is true for h

other important cofactors,such as NADH/NAD* and

NADPHI\TADP*.)For example,considerthe reaction
catalyzedby hexokinase:
ATP + glucose-----+ADP f glucoseG-phosphate
6
't

IADPI 6-phosphatel
KLq "nlglucose "o : 2 x 1 0 3
[ATP]"o[glucosel
"o
510152025303540
Note that this expressionholds true only when reac-
AIP concentration [mu]
tants and products are at their equi,Li,bri,umconcentra-
tions,where LG' : 0. At any other set of concentrations, FIGURE 15-5 Effectof ATPconcentrationon the initial velocityof a
AG' is not zero.Recall(from Chapter13) that the ratio typicalATP-dependent enzyme.Theseexperimental datayield a K- for
of products to substrates(the mass action ratio, Q) ATPof 5 mv. Theconcentrationof ATPin animaltissuesis -5 mtt.
 EZt- P r i n c i p loefsM e t a b oR
l i ce g u l a t i o n

Concenfoation
before Concentrationafter
Adenine ATP depletion ATP depletion
nueleotide (ntu) (mu) Relativechange
ATP 5.0 4.5 I0o/o
ADP 1.0 1.0 0
AMP 0.1 0.6 600o/o

cells have a far higher concentration of ATP (5 to by a reducednutrient supply or by increasedexercise.

10 mu) than of AMP (<0.1 mtr). When sorrreprocess
(say,musclecontraction) consumesATP,AMP is pro-
duced in two steps.First, hydrolysisof ATP produces
ADP,then the reaction catalyzedbyadenylate kinase
producesAMP:
2ADP ------+
AMp + ATp
If ATP is consumedsuch that its concentrationdrops
10%, the reLatiue increasein [AMP] is much greater
than that of [ADP] (Table I5-4).It is not surprising,
therefore,that many regulatoryprocessesare keyed to
changesin [AMP].Probablythe most importantmedia-
tor of regulation by AMP is AMP-activated protein
kinase (AMPK), which responds to an increase in
[AMP] by phosphorylatingkey proteinsand thus regu-
lating their activities.The rise in [AMP]may be caused
The actionof AMPK (not to be confusedwith the cycli,c
AMP-dependentprotein kinase;see Section 15.5) in-
creasesglucosetransport and activatesglycolysisand
fatty acid oxidation,while suppressingenergy-requiring
processessuch as the synthesisof fatty acids,choles-
terol, and protein (Fig. f 5-6). We discussAMPK fur-
ther, and the detailed mechanismsby which it effects
thesechanges,in Chapter23.
In addition to ATP,hundreds of metabolicinterme-
diates also must be present at appropriateconcentra-
tions in the cell.To takejust one example:the glycolytic
intermediates dihydroxyacetone phosphate and 3-
phosphoglycerate are precursorsof triacylglycerolsand
serine,respectivelyWhen these products are needed,
the rate of glycolysismust be adjustedto providethem
without reducing the glycolytic production of ATP.The

Brain r IAMP]
(hypothalamus)
J IATP]
Leptin,
adiponectin Exercise

Skeletal muscle
\

r-\r
SNS
-------

lFood intake Fatty acid uptake, oxidation

Glucoseuptake
Heart Mitochondrial biogenesis

Pancreatic B cell
--f

Ir'/

Fatty acid oxidation

secretion
Glucoseuptake
Glycolysis

Fatty acid synthesis

Cholesterol svnthesis

FIGURE15-6 Roleof AMP-activated proteinkinase(AMpK)in carbo- varietyof tissuesawayfrom energy-consuming processes suchas the
hydrateand fat metabolism.AMPK is activatedby elevated[AMp] or synthesisof glycogen, fattyacids,andcholesterol;shiftsmetabolism in
decreasedIATPI,by exercise,by the sympatheticnervcrus sysrem extrahepatic tissuesto the use of fatty acidsas a fuel; and triggers
(SNS),
or by peptidehormones producedin adiposetissue(leptinand gluconeogenesis in the Iiverto provideglucosefor the brain In the
adiponectin,
describedin moredetailin Chapter23).Whenactivated, hypothalamus, AMPK stimulates feedingbehaviorto providemore
AMPK phosphorylates targetproteinsand shiftsmetabolismin a dietaryfuel.

sameis true for maintaining the levels of other impor-
tant cofactors,such as NADH and NADPH:changesin
their massaction ratios (that is, in the ratio of reduced
to oxidized cofactor) haveglobal effects on metabolism.
Of course, priorities at the organi,smaLlevel have
also driven the evolution of regulatory mechanisms,In
mammals,the brain has virtually no stored source of
energy,dependinginstead on a constant supply of glu-
cosefrom the blood.If bloodglucosedropsfrom its nor-
mal concentrationof 4 to 5 mv to half that level, mental
confusionresults,and a fivefoldreductionin blood glu-
cose can lead to coma and death. To buffer against
changesin blood glucoseconcentration,releaseof the
hormonesinsulin and glucagon,elicited by high or low
blood glucose,respectively,triggersmetabolicchanges
that tend to return the blood glucoseconcentrationto
normal.
Other selectivepressuresmust also have operated
throughout evolution, selecting for regulatory mecha-
nismsthat accomplishthe following:
1. Maxrmizethe efficiencyof fuel utilization by
preventingthe simultaneousoperation of pathways
in oppositedirections(suchasglycolysisand
gluconeogenesis).
2. Partitionmetabolitesappropriatelybetween
alternativepathways(such as glycolysisand the
pentosephosphatepathway).
3. Draw on the fuel best suitedfor the immediate
needsof the organism(glucose,fatty acids,
glycogen,or aminoacids).
4. Slow down biosyrrtheticpathwayswhen their
productsaccumulate.
The remaining chapters of this book present many ex-
amplesof eachkind of regulatorymechanism.
S U M M A R1Y5 . 1 R e g u l a t i o on f
M e t a b o l i cP a t h w a y s
r In a metabolically active cell in a steady state,
intermediates are formed and consumed at equal
rates. When a transient perturbation alters the rate
of formation or consumption of a metabolite,
compensating changes in enz;.rne activities return
the system to the steady state.
r Cells regulate their metabolism by a variety of
mechanisms over a time scale ranging from less
than a millisecond to days, either by changing the
activity of existing enzyrne molecules or by
changing the number of molecules 6f 3 snonifie
enzyme.
r Various signals activate or inactivate transcription
factors, which act in the nucleus to regulate gene
expression. Changesin the transcriptome Iead to
changes in the proteome, and ultimately in the
metabolome of a cell or tissue.
15.2Analysis Control
ofMetabolic frrr)
In multistepprocessessuchas glycolysis,certain
reactionsare essentiallyat equilibrium in the
steadystate;the ratesofthese reactionsrise and
fall with substrateconcentration.Other reactions
are far from equilibrium;these stepsare typically
the points of regulationof the overallpathway.
Regulatorymechanismsmaintain nearly constant
levels of key metabolitessuch as ATP and NADH in
cells and glucosein the blood, while matchingthe
use or productionof glucoseto the organism's
changingneeds.
The levelsof ATP and AMP are a sensitive
reflection of a cell'senergystatus,and when the
[ATP]/[AMP]ratio decreases, the AMP-activated
protein kinase (AMPK) triggers a variety of cellular
responsesto raise[ATP]and lower [AMP].
"15,2Analysis Control
ofMetabolic
Detailed studies of metabolic
regulation were not feasible
until the basic chemical steps
in a pathwayhad been clarified
and the responsibleenzymes
characterized.Beginning with
Eduard Buchner's discovery
(c. 1900) that an extract of
broken yeast cells could con-
vert glucose to ethanol and
CO2, a major thrust of bio-
chemicalresearchwas to de-
EduardBuchner, duce the steps by which this
1860-1917 transformation occurred and
to purify and characterizethe enzyrnesthat catalyzed
eachstep. By the middle of the twentieth century,all 10
enz).rnesof the glycolytic pathway had been purified
and characterized.In the next 50 years much was
learned about the regulation of these enzyrnesby intra-
cellular and extracellular signals,through the kinds of
allosteric and covalentmechanismsdescribedin this
chapter. The conventionalwisdom was that in a linear
pathway such as glycolysis,catalysisby one enzyme
must be the slowestand must therefore determine the
rate of metaboliteflow, or flux, through the whole path-
way. For glycolysis,PFK-I was consideredthe rate-lim-
iting enzyme, because it was known to be closely
regulatedby fructose 2,6-bisphosphateand other al-
Iostericeffectors.
With the advent of genetic engineeringtechnology,
it becamepossibleto test this "single rate-determining
step" hypothesisby increasingthe concentrationof the
enz).rnelhat catalyzesthe "rate-limiting step" in a path-
way and determiningwhether flux through the pathway
increasesproportionally.Most often it doesnot; the sim-
ple solution (a singlerate-determiningstep) is wrong. It
hasnow becomeclear that in most pathwaysthe control
of flux is distributed among several enzyrnes,and the
fI
578 Principles
ofMetabolic
Regulation
extent to which each contributes to the control varies
with metaboliccircumstances-the supplyof the start-
ing material (say,glucose),the supply of oxygen,the
need for other productsderivedfrom intermediatesof
the pathway (say,glucose6-phosphatefor the pentose
phosphatepathway in cells synthesizinglarge amounts
of nucleotides),the effectsof metaboliteswith regula-
tory roles, and the hormonal status of the organism
(such as the levels of insulin and glucagon),among
other factors.
Why are we interested in what limits the flux
through a pathway?To understandthe action of hor-
monesor drugs,or the pathologythat resultsfrom a fail-
ure of metabolic regulation, we must know where
control is exercised.If researcherswish to developa
drug that stimulates or inhibits a pathway, the Iogical
target is the enz;.'rnethat has the greatestimpact on the
flux through that pathway.And the bioengineeringof a
microorganismto overproducea product of commercial
vaiue (p. 312) requiresa knowledgeof what limits the
flux of metabolitestowardthat product.
TheContribution
ofEach
EnzymetoFlux
through
a circumstances, Iie at the center of metabolic eontrol
Fathway
ls[xperimentally
Measurable
There are several ways to determine experimentally
how a change in the activity of one enz).me in a pathway
affects metabolite flux through that pathway. Consider
the experimental results shown in Figure 15-7. When a
sample of rat liver was homogenized to release all solu-
ble enz5,'mes,the extract carried out the glycolytic con-
version of glucose to fructose 1,6-bisphosphateat a
measurable rate. (This experiment, for simplicity, fo-
cused on just the first part of the glycolytic pathway.)
0.10
0.08
X
0.06
6 004
0.02
0.00'
0 0.5 1.0 1.5 2.0 2.5
Enzymeadded(arbitrary units)
FI6URE15-7 Dependence of glycolyticflux in a rat liver homogenate
on addedenzymes.Purifiedenzymesin the amountsshownon the x
axiswereaddedto an extractof livercarryingout glycolysis
Theincrease
in flux throughthe pathwayis shownon the y axls.
When increasing amounts of purified hexokinase IV
(glucokinase) were added to the extract, the rate ofgly-
colysis progressively increased. The addition of purrfied
PFK-1 to the extract also increased the rate of glycoly-
sis, but not as dramatically as did hexokinase. Purifled
phosphohexose isomerasewas without effect. These re-
sults suggest that hexokinase and PFK-I both con-
tribute to setting the flux through the pathway
(hexokinase more than PFK-l), and that phosphohex-
ose isomerase does not.
Similar experiments can be done on intact cells or
organisms, using specific inhibitors or activators to
change the activity of one enzyrne while observing the
effect on flux through the pathway. The amount of an
enz).rne can also be altered genetically; bioengineering
can produce a cell that makes extra copies of the enz}.'rne
under investigation or has a version of the enzyrne that is
less active than the normal enzyrne. Increasing the
concentration of an enzyrne genetically sometimes has
significant effects on flux; sometimes it has no effect.
Three critical parameters, which together describe
the responsivenessof a pathway to changesin metabolic
analysis. We turn now to a qualitative description of
these parameters and their meaning in the context of a
living cell. Box 15-1 pror,rdes a more rigorous quantita-
tive discussion.
The(ontrol
(oefficient theEffect
Quantifies ofaChangein
EnzymeActivity
onMetabolite
Flux through
a Pathway
Quantitative data on metabolic flux, obtained as de-
scribed in Figure 15-7, can be used to calculatea flux
control coefficient, C, for each enzyrnein a pathway.
This coefficientexpressesthe relative contribution of
each enz;.rneto setting the rate at which metabolites
flow through the pathway-that is, the flux, J. C can
have any value from 0.0 (for an enz).rnewith no impact
on the flux) to 1.0 (for an enzyrnethat wholly determines
the flux). An enzyrnecan also have a negatiue flux con-
trol coefficient.In a branchedpathway,an enzyrnein one
branch, by drawing intermediatesaway from the other
branch, can have a negativeimpact on the flux through
that otherbranch(Fig. f5-8). C is not a constant,andit
E
1l
c4: -o.2ll
u
Cr : 0.3 Cz:0.0 C : r: 0 . 9
tIGURE15-8 Flux control coefficient,C, in a branchedmetabolic
3.0
pathway,In thissimplepathway,the intermediate B hastwo alternative
fates.To the extentthat reactionB --+EdrawsB awayfrom the pathway
A + D, it controlsthat pathway,which will resultin a negativellux
controlcoefficientfor the enzymethat catalyzesstepB --+E.Notethat
in vitro. the sum of all four coefficientsequals1 0, as it mustfor any defined
svstemof enzvmes.

The factors that influence the flow of intermediates
(flux) through a pathwaymay be determinedquantita-
tively by experimentand expressedin terms usefulfor
predicting the change in flux when some factor in-
volvedin the pathwaychanges.Considerthe simplere-
actron sequencein Figure 1, in which a substrate X
(say,glucose)is convertedin severalstepsto a prod-
uct Z (perhapspyruvate,formedglycolytically).An en-
zyme late in the pathway is a dehydrogenase(ydh)
that acts on substrateY. Becausethe action of a dehy-
drogenaseis easilymeasured(seeFig. lB-24), we can
use the flux (J) through this step (JvarJto measure
the flux through the wholepath. We manipulateexper-
imentally the level of an early enzyme in the pathway
(xase,which acts on the substrateX) and measurethe
flux through the path (JvorJ for several levels of the
enzymexase.
The relationship between the flux through the
pathwayfrom X to Z in the intact cell and the concen-
tration of each enzymein the path should be hyper-
bolic, with virtually no flux at infinitely low enzyme
activity and near-maximumflux at very high enzyme
activity. In a plot of Jya1againstthe concentrationof
xase,Errr", the changein flux with a small changein
enzymelevel is AJydhfAExase, which is simply the slope
of the tangent to the curve at any concentrationof en-
zyme,E*^"., and which tends toward zero at saturating
.o*ase.At low ,E*u.",the slope is steep; the flux in-
creaseswith eachincrementalincreasein enzymeac-
tivity. At very high .O*u.",the slope is much smaller;
the systemis lessresponsiveto addedxasebecauseit
is alreadypresentin excessover the other enzymesrn
the pathway.
To show quantitatively the dependence of flux
through the pathway,dJyor,,on dE"u.",we could use
the ratio 0Jy4n/dE*u"u. However, its usefulness is
limited becauseits value dependson the units used to
expressflux and enzyme activity. By expressingthe
JractzonaL changes in flux and enzyme activity,
dJy6n/Jyon, and \E*u""fE*u"e,w€ obtain a unitless ex-
pressionfor the flux control coeffieient, C, in this
caseC{5gi:
c*as - $q/gtt" i'r\
" J"an / E*u".
This canbe rearrangedto
- g*'+*
cdat:
" dE*.". J"ar,
which is mathematically identical to
15.2
A n a l y s i s o f M e t a b o l i cFC
t tol n t r o l
.)
y -:t::- S,- /-er!'.t"1-y jgf-* So.-*r!!!t!st--- Z
rvdh
4'"" \
tIGURE
1 Fluxthrougha hypothetical pathway
multienzyme
This equation suggestsa simple graphical means for
determining the flux control coefficient: Ct';93is tfre
slope of the tangent to the plot of ln Jya1versus ln
.O"u.",which can be obtainedby replotting the experi-
mental data in Figure 2a to obtain Figure 2b. Notice
that C{#Eis not a constant;it dependson the starting
.O*u."from which the change in enzyme level takes
place. For the casesshown in Figure 2, Ct"X#is about
1.0at the lowest.E"""",but only about 0.2 at high.O*r"".
A valuenear 1.0for CIXBE meansthat the enzyme'scon-
centration wholly determines the flux through the
pathway;a valuenear 0.0meansthat the enzyme'scon-
centrationdoesnot limit the flux through the path. Un-
less the flux control coefficient is greater than about
0.5, changesin the activity of the enzlirnewill not have
a strong effect on the flux.
The elasticity, e, of an enz).Tneis a measureof how
that enz;.'rne'scataly'ticactivity changeswhen the con-
centrationof a metabolite-substrate,product,or effec-
tor-changes. It is obtained from an experimentalplot
of the rate of the reaction catalyzedby the enzymever-
sus the concentrationof the metabolite,at metabolite
concentrationsthat prevail in the cell. By arguments
analogous to thoseusedto deriveC, we canshowe to be
the slopeof the tangentto a plot of ln Z versusIn [sub-
strate,or product,or effector]:
dv*u".. s
e.s___:
aS y*
_ d ln iV""".l
dlnS
For an enzyme with typical Michaelis-Mentenkine-
tics, the value of e rangesfrom about 1 at substrate
concentrationsfar below K^ to near 0 as 7*u* is ap-
proached. Allosteric enzJ,rynes can have elasticities
greater than 1.0, but not larger than their Hill coeffi-
cient (p. 164).
Finally,the effect of controllersoutsidethe pathway
itself (that is, not metabolites)canbe measuredand ex-
pressedas the response coefficient, R. The changein
flux through the pathwayis measuredfor changesin the
concentration of the controlling parameterP, and .R is
definedin a form analogousto that ofEquation 1, yield-
ing the expression
R{"un:#
*
(contznued on nert page)
t
580 P r i n c i pol ef M
s e t a b oRl i ec g u l a t i o n

(a)

\
X
\

Concentration of enzyme, E*u"" In E*u""

FIGURE 2 Theflux controlcoefficient. (a)Typicalvariation of the pathwayflux,/161,measured by the

at the stepcatalyzed
enzymeydh,asa functionofthe amountofthe enzymexase,f"",., whichcatalyzes an earlierstepin the pathway.Theflux
andthe ratio(scalingfacto0el
controlcoefficientat (el ) is the slopeof the productof the tangentto the curve,0lr6tnl6E*a5s,
(b) On a double-logarithmic plotof thesamecurve,the flux controlcoefficient isthe slopeof thetangentto thecurve.

Using the same logic and graphicai methods as de-
scribedabovefor determiningC, we can obtainfi as the
slopeof the tangentto the plot of ln J versusln P.
The three coefficientswe have describedare re-
lated in this simpleway:
R{'o":c1#8."f""
Thus the responsiveness of eachenzyrnein a pathwayto
a changein an outsidecontrollingfactor is a simplefunc-
tion of two things: the control coefflcient,a variablethat
expressesthe extent to which that enzlrne influences
the flux under a given set of conditions,and the elastic-
ity, an intrinsic property of the enz;rne that reflects its
sensitiutvto substrateand effectorconcentrations.

is not intrinsicto a singleenzi,me;it rs a functionof the Michaelis-Menten kinetics shows a hyperbolic response
whole systemof enzJ,'mes, and its vaiue dependson the to increasing substrate concentration (Fig. 15-9). At
concentrations of substratesand effectors.
Whenreal datafrom the experi.ment on glycolysisin
a rat Iiver extract (Fig. i5-7) were subjectedto this kind V-"" -
of analysis,investigatorsfound flux control coefflcients
(for en4.'rnes at the concentrations foundin the extract)
of 0.79for hexokinase, 0.21for PFK-I, and 0.0for phos-
phohexoseisomerase.It is not just fortuitousthat these
valuesadd up to 1.0;we can showthat for any complete
pathway, the sum of the flux control coefficientsmust
equalunity.

TheElasticity lsRel*ted
{*effreiesrt tnanHn;yrne's
fiespe
nsl'.'en*ss
t{.} iruMetabolite
{haffifies nr K^ tsl
Regulat*r
{*nrenfratin*s
FIGURI15-9 Elasticitycoefficient,e, of an enzymewith typical
A second parameter, the elasticity coefficient, €, ex- Michaelis-Menten kinetics.At substrateconcentrationsfar below the
presses quantitatively the responsiveness of a single K-, eachincrease in [S]producesa correspondingly largeincrease in
enzyme to changes in the concentration of a metabolite the reactionvelocity,v Forthisregionof the curve,theenzymehasan
or regulator; it is a function of the enzyme's intrinsic ki- s of about1.0.At tsl ) K-, increasing [SJhaslittleeffecton v; s here
netic properties. For exampie, an enzyme with typical i s c l o s et o 0 . 0 .

Iow concentrationsof substrate(say,0.1 K-) each in-
crementin substrateconcentrationresultsin a compa-
rable increasein enz;.'rnaticactivity, yielding an a near
1.0.At relativelyhigh substrateconcentrations(say,10
K-), increasingthe substrateconcentrationhas little
effect on the reaction rate, becausethe enzymeis al-
ready saturatedwith substrate.The elasticity in this
caseapproacheszero.For allostericenzymesthat show
positivecooperativity,e may exceed1.0,but it cannot
exceedthe Hill coefflcient,which is typically between
1.0and4.0.
TheResponse
Coefficient
Expresses
theEffect
ofan
Outside
Controller
onFlux
through
a Pathway
We can also derive a quantitative expression for the rel-
ative impact of an outside factor (such as a hormone or
growth factor), which is neither a metabolite nor an en-
z;'rne in the pathway, on the flux through the pathway.
The experiment would measure the flux through the
pathway (glycolysis, in this case) at various levels of the
parameter P (the insulin concentration, for example) to
obtain the response coefficient, R, which expresses
the change in pathway flux when P ([insulin]) changes.
The three coefflcients C, e, and .R are related in a
simple way: the responsiveness (l?) of a pathway to an
outside factor that affects a certain enz).rne is a function
of (1) how sensitive the pathway is to changesin the ac-
tivity ofthat enz).rne (the control coefflcient, C) and (2)
how sensitive that speciflc enzyme is to changes in the
outside controlling factor (the elasticity, e):
R:C.E
Each enzyme in the pathway can be examined in this
way, and the effects of any of several outside factors on
flux through the pathway can be separately determined.
Thus, in principle, we can predict how the flux of sub-
strate through a series of enz;'rnatic steps will change
when there is a change in one or more controlling fac-
tors external to the pathway. Box 15-1 shows howthese
qualitative concepts are treated quantitatively.
Metabolic
Control
Analysis
HasBeen
Applied
to
Carbohydrate
Metabolism,with
Surprising
Results
Metaboliccontrol analysisprovides a framework within
which we can think quantitatively about regulation, in-
terpret the significanceof the regulatory properties of
each en4rme in a pathway,identifii the steps that most
affect the flux through the pathway,and distinguishbe-
tween regulatory mechanismsthat act to maintain
metaboliteconcentrations and controLmechanisms that
actually alter the flux through the pathway.Analysis of
the glycolytic pathway in yeast, for example,has re-
vealed an unexpectedlylow flux control coefflcient for
PFK-l, which,aswe havenoted,hasbeenviewedasthe
main point of flux control-the "rate-determining
step"-in glycolysis.Experimentallyraisingthe level of
15.2
A n a l y s i s o f M e t a b o l i cI sCgornI t r o l
PFK-1 fivefold led to a changein flux through glycolysis
of lessthan 10%,suggestingthat the real role of PFK-I
regulationis not to control flux through glycolysisbut to
mediate metabolite homeostasis-to prevent large
changesin metabolite concentrationswhen the flux
through glycolysisincreasesin responseto elevated
blood glucoseor insulin. Recallthat the study of glyco-
lysis in a liver extract (Fig. 15-7) alsoyielded a flux con-
trol coefficient that contradicted the conventional
wrsdom;it showedthat hexokinase,not PFK-I, is most
influential in setting the flux through glycolysis. We
must note here that a liver extract is far from equivalent
to a hepatoclte; the ideal way to study flux control is by
manipulatingone en4/rneat a time in the living cell. This
is alreadyfeasiblein many cases.
Investigatorshave used nuclear magneticresonance
(NMR) as a noninvasivemeansto determinethe concen-
tration of glycogenand metabolitesin the flve-steppath-
way from glucosein the blood to $ycogen in myocytes
(Fig. 15-f0) in rat and human muscle.They found that
the flux control coefflcient for $ycogen syrthase was
smallerthan that for the steps catalyzedby the $ucose
transporter and hexokinase.(We discussglycogens1n-
thase and other en4.'rnesof glycogen metabolism in
Sections15.4and 15.5.)This finding contradictsthe con-
ventionalwisdom that $ycogen slmthaseis the locus of
flux controland suggeststhat the importanceof the phos-
phorylatiorVdephosphorylation of glycogen s;mthaseis
related rnsteadto the maintenanceof metabolitehome-
ostasis-that is,regulation, not control. TWometabolites
in this pathway,glucoseand $ucose 6-phosphate,are key
intermediatesin other pathways,including$ycolysis,the
Capillary
Plasma Myocyte
membrane
Glucose
Jr'"*or.i'.."
Glucose6-phosphate
I
UDP-glucose
. lgly"og"t
Jsvnthase
Glycogen
tIGUREl5-10 Control of glycogensynthesisfrom blood glucosein
muscle.Insulinaffectsthreeof the five stepsin this pathway,but it is
the effectson transportand hexokinaseactivity,not the changein
glycogensynthaseactivity,that increasethe flux towardglycogen.
rG'l P r i n c i p loefsM e t a b oRl i ce g u l a t i o n
pentose phosphatepathway, and the syrrthesisof glu-
cosamine.Metaboliccontrol analysissuggeststhat when
the blood $ucose levei rises,insulin actsin muscleto (1)
increase$ucose transportinto cellsby conveyingGLUT4
to the plasmamembrane,(2) inducethe synthesisof hex-
okinase,and (3) activateglycogensyrthase by covalent
alteration(seeFig. 15-39).The first two effectsof insuhn
increase$ucose flux through the pathway (control), and
the third servesto adaptthe activity of $ycogen syrrthase
so that metaboLiteIevels(glucose6-phosphate,for exam-
ple) will not changedramaticallywith the increasedflr.x
(regulation).
Metabolic
Control
Analysis
Suggests Method
aGeneral for
Increasing
Flux
through
a Pathway
How could an investigatorengineera cell to increasethe
flr.rxthrough onepathwaywithout alteringthe concentra-
tions of other metabolites or the fluxes through other
pathways?More than three decadesago Henrik Kacser
predicted,on the basisof metaboliccontrol analysis,that
this conld be accomplishedby increasingthe concentra-
tions of every en4,nnein a pathway.The prediction has
been con-firmedin severalexperimentaltests, and it also
flts with the way cells normally control fluxes through a
pathway.For example,rats fed a high-proteindiet dispose
of excessaminogroupsby convertingthem to ureain the
urea cycle (Chapter 1B). After such a dietary shifl, the
urea output increasesfourfold, and the amount of all
eight enzy'rnes
rn the urea cycle ircreasestwo- to three-
fold. Similarly, when increased fatty acid oxidation is
triggered by activation of peroxisome proliferator-
activatedreceptor 7 (PPAR7,a ligand-activatedtran-
scriptionfactor;seeFig. 2I-22), synthesisof the whole
set of fatty acid oxrdativeenz).rnesis increased.With the
growinguse of DNA microarraysto study the expression
of whole sets of genesin responseto various perturba-
tions, we should soon learn whether this is the general
mechanismby which cellsmakelong-termadjustmentsin
flux through speci-flcpathways.
SUMMAR
1Y5 . 2 A n a l y s iosf
M e t a b o l i(co n t r o l
r Metaboliccontrol analysisshowsthat control of
the rate of metaboliteflux through a pathwayis
distributed amongseveralof the enzymesin that
path.
r The flux control coefficient,C, is an experimentally
determinedmeasureof the effect of an en4.rne's
concentrationon flux through a multienzy'rne
pathway.It is characteristicof the whole system,
not intrinsic to the enz;.'rne.
r The elasticity coefflcient,e, of an en4.'rneis an
experimentallydeterminedmeasureof its
responsiveness to changesin the concentration
of a metaboliteor reAulatormolecule.
r The responsecoefflcient,R, is a measureof the
experimentallydeterminedchangein flux through
a pathwayin responseto a regulatoryhormone
or secondmessenger. It is a function of C and e:
R:C'e.
r Someregulatedenz).rnescontrol the flux through
a pathway,while othersrebalancethe level of
metabolitesin responseto the changein flux. The
first activity is control; the second,rebalancing
activity is reguLat'ion.
r Metabohccontrol analysispredicts,and experiments
have confirmed,that flux toward a speci-flcproduct
is most effectivelyincreasedby raising the
concentrationof all enz).rnesin the pathway.
Regulation
15.3Coordinated ofGlycolysis
andGluconeogenesis
In mammals,gluconeogenesis occurs primarily in the
liver, where its role is to pronde glucosefor export to
other tissueswhen glycogenstoresare exhaustedand
when no dietary glucoseis available.As we discussedin
Chapter14,gluconeogenesis employsseveralofthe en-
zymesthat act in glycolysis,but it is not simply the re-
versalofglycolysis.Sevenofthe glycoly'ticreactionsare
freely reversible,and the en4'rnesthat catalyzethesere-
actionsalsofunction in gluconeogenesis (Fig. 15-1f ).
Three reactionsof glycolysisare so exergonicas to be
essentiallyirreversible:those catalyzedby hexokinase,
PFK-I, and pyruvatekinase.All three reactionshave a
Iarge, negative AG' (Table 15-3 shows the values in
heart muscle). Gluconeogenesis uses detours around
each of these irreversiblesteps;for example,the con-
versionoffructose 1,6-bisphosphate to fructose6-phos-
phate is catalyzed by fructose 1,6-bisphosphatase
(FBPase-1).Each of these bypassreactionsalso has a
large,negativeLG'.
At eachof the three pointswheregiycol:,'ticreactions
are bypassedby alternative,gluconeogenicreactions,
simultaneousoperationof both pathwayswould consurne
ATP without accomplishingany chemical or biological
work. For example,PFK-I and FBPase-l catalyzeoppos-
ing reactions:
ATP + fructose 6-phosphate
ADP + fructose 1,6-bisphosphate
Fructose 1,6-bisphosphate f H2O
fructose 6-phosphate + P1
The sum of thesetwo reactionsis
ATP + H2O -----+ADP + P1* heat
that is, hydrolysis of ATP without any useful metabolic
work being done. Clearly, if these two reactions were al-
lowed to proceed simultaneously at a high rate in the
same cell, a large amount of chemical energy would be
dissipated as heat. This uneconomical process has been
 15 . 3C o o r d i n aR
t eedg u l a t ioofG
n l y c o l yasni sdG l u c o n e o g e n [etsuirs- - ]

isomerase
lfon".r-n*""e

(2) 1,3-Bisphosphoglycerate

I ohosohorlvcerat
e k i nase
ll
{l
(2) 3-Phosphoglycerate

I
l l p h o s p h o g l v c e r a tm
e ulase
'll
(2) 2-Phosphoglycerate

11.
enoluse
ll
{I
olnrmvate
\ PEPcarboxvkinase
\
(2) Oxaloacetate
T pyruvate carboxylase
tvate

FIGURE 15-11 Glycolysisand gluconeogenesis. Opposingpath- (the"bypassreactions") sevenstepsarecatalyzedby

and glycolysis;
ways of glycolysis(pink) 1n6 gluconeogenesis
(blue)in rat liver. the sameenzymesin the two pathways. Cofactorshavebeenomit-
Threestepsare catalyzedby differentenzymesin gluconeogenesis ted for simplicity.

called a futile cycle. However, as we shall see later, havefour isoz;rmes(designatedI to IVJ, encodedby four
such cycles may provide advantagesfor controlling different genes.Isoz;.'rnes
are different proteins that cat-
pathways,and the term substrate eycle is a better de- alyze the same reaction (Box 15-2). The predominant
scription. Similar substrate cycles also occur with the hexokinaseisozSrme of myoc),'tes(hexokinase II) has a
other two sets of blpass reactionsof gluconeogenesis high affinity for glucose-it is half-saturated at about
(Fig.15-11). 0.1 mnr.Becauseglucoseentering myocytesfrom the
We look now in somedetail at the mechanismsthat blood (where the glucose concentrationis 4 to 5 mu)
regulate glycolysisand gluconeogenesis at the three produces an intracellular glucose concentration high
points where thesepathwaysdiverge. enoughto saturate hexokinaseII, the enzlrne normally
acts at or near its maximal rate. Muscle hexokinase I
Hexokinase
lsozymesofMusde
andLiver
AreAffected and hexokinaseII are allosterically inhibited by their
product, glucose6-phosphate,so wheneverthe cellular
Differently
byTheir
Produrt,
Glucose
6-Phosphate
concentrationofglucose6-phosphaterisesaboveits nor-
Hexokinase,which catalyzesthe entry of glucose into mal level, these isozSrmesare temporarily and reversibly
the glycolytic pathway,is a regulatory enzyrne.Humans inhibited, bringing the rate of glucose 6-phosphate
f-
584 P r i n c i p loefsM e t a b oR
l i ce g u l a t i o n
The four fonns of hexokinasefound rn mammaliantissues
are but one exampleof a corunon biologicalsituation:the
samereactioncatalyzedby two or more different molec-
ular forms of an enzy'rne.These mr-rltipleforms, called
isoz5.'rnes
or isoenzy'rnes,may occurin the samespecies,
in the sametissue,or evenin the samecell. The differ-
ent forms (isoforms) of the enz;rne generally differ in
kinetic or regulatoryproperties,in the cofactorthey use
(NADH or NADPH for dehydrogenaseisoz;rmes,for ex-
ample), or in their subcellulardistribution (solubleor
membrane-bound). Isozymesmay have similar,but not
identical,aminoacid sequences, and in many casesthey
clearly sharea conunonevolutionaryorigin.
One of the first enzymesfound to have isozymes
waslactatedehydrogenase (LDH;p.547), which in ver-
tebrate tissues exists as at least five different isoz;.'mes
separableby electrophoresis. All LDH isozl'rnescontain
four polypeptidechains(eachof M,33,500), eachtype
containinga different ratio of two kinds of polypeptides.
The M (for muscle) chain and the H (for heart) chain
are encodedby two differentgenes.
In skeletalmuscle the predominantisozl,rnecon-
tains four M chains, and in heart the predominant
isoz5,'rne
containsfour H chains.Other tissueshavesome
combinationof the five possibletypesof LDH isozy'rnes:
Tfpe Composition Location
LDHl HHHH Heart and erythrocy'te
LDH2 HHHM Heart and erythrocyte
LDHs HHMM Brain and kidney
LDH4 HMMM Skeletalmuscleand liver
LDH5 MMMM Skeletalmuscleand liver
The differences in the isozyme content of tissues
can be used to assess the timing and extent of
heart damage due to myocardial infarction (heart at-
tack). Damageto heart tissue resultsin the releaseof
heart LDH into the blood. Shortly after a heart attack,
formation into balancewith the rate of its utilization and
reestablishingthe steadystate.
The different hexokinase isozymes of liver and
musclereflectthe differentroles of theseorgansin car-
bohydratemetabolism:muscle consumesglucose,us-
ing it for energy production, whereasliver maintains
blood glucosehomeostasisby consumingor producing
glucose,dependingon the prevailingbloodglucosecon-
centration. The predominant hexokinaseisozyme of
liver is hexokinase IV (glucokinase),which differs in
three importantrespectsfrom hexokinasesI-III of mus-
cle. First, the glucoseconcentrationat which hexoki-
naseIV is half-saturated(about 10 mu) is higher than
the bloodlevelof total LDH increases,and there is more
LDH2than LDH1.After 12 hours the amountsof LDHI
and LDH2 are very similar, and after 24 hours there is
moreLDHI than LDH2.This switchin the [LDH1]/[LDH2]
ratio, combinedwith increasedconcentrationsin the
blood of anotherheart enzyrne,creatinekinase,is very
strong evidenceof a recent myocardialinfarction. r
The different LDH isozymeshave signiflcantly dif-
ferent values of 7-u* and K-, particularly for pyruvate.
The propertiesof LDHafavor rapid reduction of very low
concentrationsof pyruvate to lactate in skeletalmuscle,
whereasthoseof isozymeLDHI favorrapid oxidationof
lactate to pyruvate in the heart.
In general,the distribution of different iso4.rnesof a
given en4rmereflects at least four factors:
1. Di,fJerentmetabol'icpatterns i,n di,fJerentorgans.
For glycogenphosphorylase, the isozl'rnesin skele-
tal muscleand liver have different regulatoryprop-
erties,reflectingthe differentrolesof glycogen
breakdownin thesetwo tissues.
2. Di,fJerentlocat'ionsand metaboli,crolesfor
'isozymeszn the same cell The isocitrate dehydro-
genaseisozymesof the cytosoland the mitochon-
drion are an example(Chapter16).
3. Di,fJerentstagesoJdeue\opmenti,n embryonic or
Jetal ti,ssuesand i,n adult t'issues For example,
the fetal liver has a characteristicisoz;'rnedistribu-
tion of LDH, which changesas the organdevelops
into its adult form. Someenzymesof glucosecatab-
olism in malignant (cancer) cells occur as their
fetal, not adult, isoz;.'rnes.
4. Di,lferent responsesof i,sozymesto allosteri,cmod-
ulators. This differenceis usefulin fine-tuningmeta-
bolic rates.HexokinaseIV (glucokinase)of Jiverand
the hexokrnaseisoz;rmesof other tissuesdiffer in
their sensitivrtyto inhibition by glucose6-phosphate.
the usual concentrationof glucosein the blood. Be-
causean efficient glucosetransporter in hepatoc;,tes
(GLUTZ) rapidly equilibratesthe glucoseconcentra-
tions in cytosoland blood (seeFig. 11-30for the kinet-
ics of the sametransporter,GLUT2,in erythrocytes),
the highK^ of hexokinaseIV allowsits direct regulation
by the level of bloodglucose(F'ig. f 5- 12) . Whenblood
glucoseis high, as it is after a meal rich in carbohy-
dtates,excessglucoseis transportedinto hepatoc;,'tes,
where hexokinaseIV converts it to glucose 6-phos-
phate.BecausehexokinaseIV is not saturatedat l0 mlt
glucose,its activity continuesto increaseasthe glucose
concentrationrisesto 10 mnror more.Under conditions
 1 5 . 3C o o r d i n aR
t eedg u l a t ioofG F"l
n l y c o l yasni sdG l u c o n e o g e n e s i s

afler a carbohydrate-rich meal,whenblood$ucoseis high,

glucoseenters the hepatoclte via GLUT2 and activates
hexokinaseIV by this mechanism.During a fast, when
blood$ucosedropsbelow5 nM, fTuctose6-phosphate trig-
gers the inhibition of hexokinaseIV by the regulatorypro-
tein, sothe liver doesnot competewith otherorgansfor the
scarce$ucose.The mechanismof intribitionby the regula-
o tory proteinis interesting:the protein anchorshexokinase
xN IV insidethe nucleus,whereit is segregated from the other
enzyrnesof glycolysisin the cytosol(Fig. 15-13).Whenthe
Hexokinase IV $ucose concentrationin the cytosolrises,it equilibrates
(glucokinase) with $ucose rL the nucleus by transport through the nu-
e
clear pores.Glucosecausesdissociationof the regulatory
protein,andhexokinaseN entersthe cytosolandbeginsto
phosphorylate glucose.

lV(6lucokinase)
Hexokinase andGlucose
6-Phosphatase
05101520 Transcriptionally
Are Regulated
Glucoseconcentration(mM) HexokinaseIV is also regulated at the level of protein
FIGURE15-12 Comparisonof the kineticproperties
of hexokinasetV
synthesis.Circumstancesthat call for greater energy
(glucokinase)
and hexokinasel. Notethe sigmoidicity
for hexokinase
production(low [ATP],high [AMP],vigorousmusclecon-
lV andthe muchlowerK. for hexokinase l. Whenbloodglucoserises traction) or for greaterglucoseconsumption(high blood
above5 mv, hexokinase lV activityincreases, but hexokinase I is al- glucose,for example)causeincreasedtranscriptionof
readyoperatingnearV-u* and cannotrespondto an increase in glu- the hexokinaseIV gene.Glucose6-phosphatase, the glu-
c o s ec o n c e n t r a t i o H
n e x o k i n a s eI ,s l l , a n d l l l h a v es i m i l a rk i n e t i c coneogenicenzlrne that bypassesthe hexokinasestep of
propenres. glycolysis,is transcriptionallyregulated by factors that
call for increasedproductionof glucose(low blood glu-
cose,glucagonsignaling).The transcriptionalregulation
of low bloodglucose,the glucoseconcentrationin a he- of thesetwo enzl.rnes(alongwith other enz),Tnes of gly-
patoc),teis low relativeto the K- of hexokinaseIV, and colysisand gluconeogenesis) is describedbelow.
the glucosegeneratedby gluconeogenesis leavesthe
cell beforebeingtrappedby phosphorylation.
Phosphofructokinase-1
andFru(tose
1,6-bisphosphatase
Second,hexokinaseIV is not inhibited by glucose
6-phosphate,and it can therefore continueto operate AreReciprocally
Regulated
when the accumulationof $ucose6-phosphatecompletely As we havenoted,glucoseG-phosphate can flow either
inlLibitshexokinasesI-III. Finally,hexokinaseIV is subject into glycolysisor through any of severalother pathways,
to inhibition by the reversiblebindmg of a regulatory pro- including glycogensynthesisand the pentosephosphate
tein specificto liver (Fig. 15-13). The bindrngis much pathway.The metabolicallyirreversible reaction cat-
tighter in the presenceofthe allostericeffectorfructose6- alyzedby PFK-I is the step that commitsglucoseto gly-
phosphate.Glucosecompeteswrth fructose 6-phosphate colysis.In addition to its substrate-bindingsites, this
for bindingand causesdissociationof the regulatoryprotein complex en4rrne has several regulatory sites at which
from the hexokinase,relieving the inhibition. Immedtately allostericactivatorsor inhibitors bind.

Capillary

FIGURE 15-13 Regulationof hexokinase

lV (glucokinase)by sequestrationin the
nucleus.The proteininhibitorof hexoki-
naselV is a nuclearbindingproteinthat
drawshexokinase lV intothe nucleuswhen
the fructose6-phosphateconcentrationin
liver is high and releasesit to the cytosol
when the glucoseconcentration is high.
tl
586 P r i n c i p loefsM e t a b oR
l i ce g u l a t i o n

FIGURE 15-14 Phosphofructokinase-l (PFK-I)and its regulation.

(a) Ribbondiagramof E coli PFK-I,showingtwo of itsfour identical
subunits (PDBID .lPFK).Eachsubunithasitsown catalyticsite,where
theproducts ADPandfructose1,6-bisphosphate (blueandyellowball-
and-stick models,respectively) arealmost in contact,
and itsown bind-
ing sitesfor the allosteric
regulatorADP (blue),locatedat the interface
betweensubunits(b) Allostericregulationof musclePFK-Iby ATP,
shownby a substrate-activity curve.At low IATP],the Ke,5for fructose
6-phosphate is relativelylow, enablingthe enzymeto functionat a
highrateat relatively low [fructose6-phosphatel. (RecallfromChapter
6 that K05 is the K. term for regulatoryenzymes.) When [ATP]is high,
Ke5 for fructose6-phosphateis greatlyincreased, as indicatedby the
sigmoidrelationship betweensubstrate concentration andenzymeac-
tivity.(c) Summaryof the regulators affectingPFK-1activity.

production, act allostericallyto relieve this inhibition

by ATP.These effects combineto produce higher en-
zyme activity when ADP or AMP accumulatesand lower
activity when ATP accumulates.
Citrate (the ionized form of citric acid), a key in-
(a) termediatein the aerobic oxidation of pyruvate, fatty
acids,and aminoacids,is alsoan allostericregulatorof
PFK-1; high citrate concentrationincreasesthe in-
hibitory effect of ATR further reducingthe flow of glu-
cose through glycolysis.In this case, as in several
others encounteredlater, citrate servesas an intracel-
lular signalthat the cell is meetingits cunent needsfor
h
energy-yieldingmetabolism by the oxidation of fats
and proteins.
The correspondingstep in gluconeogenesis is the
J conversionof fructose 1,6-bisphosphate to fructose 6-
phosphate(Fig. 15-15). The enzymethat catalyzes
this reaction,FBPase-l,is stronglyinhibited (allosteri-
cally) by AMP;when the cell'ssupply of AIP is low (cor-
respondingto high [AMP]),the ATP-requiringsynthesis
of glucoseslows.
fFructose6-phosphateJ Thustheseopposingstepsin the glycolyticandgluco-
neogenicpathways-PFK- 1 andFBPase- 1-are regulated
(b) in a coordinatedand reciprocalmanner.In general,when
sufflcient concentrationsof acetyl-CoA or citrate (the
AJP AMP,ADP
\\
Gluconeogenesis
Fructose6- + ATP Fructose 1,6- + ADP
phosphate
tt
bisphosphate
1
citLte fJuctose 2,6-
bisphosphate

(c)

ATP is not only a substratefor PFK-1but alsoan

end product of the glycolytic pathway.When high
cellular [ATP] signals that ATP is being produced
faster than it is being consumed,ATP inhibits PFK-1
t
Glycolysis
by binding to an allosteric site and lowering the affin- FIGURE 15-15 Regulation of fructose1,6-bisphosphatase(FBPase-l)
ity of the enzymefor its substratefructose 6-phos- and phosphofructokinase-l (PFK-I). The importantrole of fructose
phate (Fig. 15-f4). ADP and AMP,which increase 2,6-bisphosphate
in the regulation
of thissubstrate
cycleis detailedin
in concentrationas consumption of ATP outpaces subsequentfigures.
 t eedg u l a t ioofG
1 5 . 3C o o r d i n aR Itt'j
n l y c o l yasni sdG l u c o n e o g e n e s i s

product of acetyl-CoAcondensationwith oxaloacetate) The rapid hormonalregulationof glycolysisand glu-

are present,or when a hrghproportionof the cell'sadeny- coneogenesisis mediated by fructose 2,6-bisphos-
late is in the form of ATP, gluconeogenesisis favored. phate, an allostericeffector for the enzlirnesPFK-1 and
Whenthe levelof AMPincreases,it promotesglycolysisby FBPase-l:
stimulatingPFK-I (and, as we shall see in Section15.5, o o
promotes glycogen degradationby activating glycogen -o-T-o-fH, o-P-o-
phosphorylase). I
o- o-
Fruttose
2,6-Sisphosphate
lsa Potent Regulator
Allosteric
CH2OH
CIfPfK-1
andtBPase-1
OHH
The specialrole of liver in maintaininga constantblood
Fructose2,6-bisphosphate
glucoselevel requiresadditionalregulatorymechanisms
to coordinate glucose production and consumption. When fructose 2,6-bisphosphate binds to its allosteric
When the blood glucoselevel decreases,the hormone site on PFK-I, it increasesthe enzyme'saffinity for
glucagon signalsthe liver to produce and releasemore its substrate fructose 6-phosphateand reduces its
glucoseand to stop consumingit for its own needs,One affinity for the allosteric inhibitors ATP and citrate
sourceofglucoseis glycogenstoredin the liver; another (Fig. 15-f6). At the physiologicalconcentrationsof
sourceis gluconeogenesis, usingpyruvate,lactate,glyc- its substrates,ATP and fructose6-phosphate, and of its
erol, or certain amino acids as starting material. When other positive and negative effectors (ATP, AMR
blood glucoseis high, insulin signalsthe liver to use glu- citrate), PFK-I is virtually inactive in the absence of
coseas a fuel and as a precursorfor the synthesisand fructose 2,6-bisphosphate. Fructose 2,6-bisphosphate
storageof glycogenand triacylglycerol. has the opposite effect on FBPase-l: it reduces its

100 100

t6u
> 80
N
960 h 60
:E 't

t40
i

a
Ftn

0
0 0.050.1 0.2 0.4 0.7 1.0 2.0
(mu)
lFructose6-phosphatel lFructose 1,6-bisphosphate]krlrr)
(a) (b)
Gluconeogenesis

I
J
Glycolysis
(c)

FIGURE l5-16 Roleof fructose2,5-bisphosphate in regulationof gly- ThusF26BPactivates PFK-1by increasing itsapparent affinityfor fruc-
colysisand gluconeogenesis. Fructose 2,6-bisphosphate(F268P)has tose6-phosphate (seeFig. 15-14b\. (b) FBPase-1 activity is inhibited
oppositeeffectson the enzymaticactivitiesof phosphofructokinase-1 by aslittleas1 pu F26BP and is stronSly inhibited by 25 1t'u.Intheab-
(PFK-1,a glycolyticenzyme)andfructose1,6-bisphosphatase (FBPase- senceof this inhibitor(bluecurve) the K65 for fructose 1,6-bisphos-
1, a gluconeogenic (a)
enzyme). PFK-I activityin the absenceof phateis 5 pu, but in the presence ol 25 p'uF26BP(redcurve)the K65
F26BP(bluecurve)is half-maximal whentheconcentrationof fructose is >70 1tu.Fructose 2,6-bisphosphate alsomakesFBPase-1 moresen-
6-phosphate is 2 mv (thatis, K6s = 2 mv). When 0.13p,uF26BPis sitiveto inhibitionby anotherallosteric regulator, AMP (c) Summary of
present(redcurve),the K6r for fructose6-phosphateis only 0.08 mv. by F26BP.
regulation
iroul P r i n c i p loefsM e t a b oRl i ce g u l a t i o n

\\ | I | //
------l-
\ f-:

trzoepr :-l Ei'":;!,

Stimulates glycolysis,
l;"
inhibits gh--- - - --- - --'- | (rnacrrve, |
\

glucagon
(+ tcAMPl)

(a)
J

'/
l I 1 t t
(b)

FIGURIl5-17 Regulationof fructose2,6-bisphosphate level.(a) The (PFK-2)and itsbreakdownby fructose2,6-bisphosphatase(FBPase-2).

cellularconcentration
of the regulator (F26BP)
fructose2,6-bisphosphate (b) Bothenzymeactivitiesarepartof the samepolypeptidechain,and
is determinedby the ratesof its synthesisby phosphofructokinase-2 theyarereciprocallyregulated by insulinand glucagon.

affinity for its substrate(Fig. t5-16c), thereby slowing Xylulose

gluconeogenesis.
The cellularconcentrationof the allostericregu-
lator fructose2,6-bisphosphate is set by the relative
rates of its formation and breakdown (F'iS.
15-l7a). It is formed by phosphorylationof fruc-
tose 6-phosphate,cata\yzedby phosphofructoki-
nase-2 (PFK-2), and is broken down by fructose
2,G-bisphosphatase(FBPase-2). (Note that these
enzymesare distinct from PFK-I and FBPase-l,
which catalyze the formation and breakdown, re-
spectively,of fructose 1,6-bisphosphate.) PFK-2 and
FBPase-2are two separateenzymaticactivitiesof a
single, bifunctional protein. The balance of these
two activities in the liver, which determines the cel-
lular level of fructose 2,6-bisphosphate, is regulated
by glucagonand insulin (Fig. 15-17b).
As we sawin Chapter12 (p. 431), glucagonstim-
ulatesthe adenylylcyclaseof liver to synthesiz e 3' ,b'-
cyclic AMP (cAMP) from ATP. Cyclic AMP then
activates cAMP-dependentprotein kinase, which
transfers a phosphorylgroup from ATP to the bifunc-
tional protein PFK-2/FBPase-2. Phosphorylationof
this protein enhancesits FBPase-2 activity and in-
hibits its PFK-2 activity. Glucagonthereby lowers the
cellularlevel of fructose2,6-bisphosphate, inhibiting
glycolysisand stimulatinggluconeogenesis. The re-
sulting production of more glucose enablesthe liver
to replenishblood glucosein responseto glucagon.
Insulin has the opposite effect, stimulating the activ-
ity of a phosphoprotein phosphatasethat catalyzes
removal of the phosphoryl group from the bifunc-
tional protein PFK-2/FBPase-2,activating its pFK-2
activity,increasingthe level of fructose2,6-bisphos-
phate, stimulatingglycolysis,and inhibiting gluconeo-
genesis.
5-Phosphate
lsa KeyRegulator
of
(arbohydrate
andFatMetabolism
Another regulatory mechanismalso acts by controlling
the level of fructose2,6-bisphosphate. In the mammalian
Iiver,xylulose5-phosphate(seep. 560),a productofthe
pentose phosphatepathway (hexose monophosphate
pathway), mediates the increasein glycolysisthat fol-
lows ingestion of a high-carbohydratemeal. The xylu-
lose5-phosphateconcentrationrisesasglucoseentering
the liver is convertedto glucoseO-phosphate and en-
ters both the glycolytic and pentosephosphatepath-
ways. Xylulose 5-phosphateactivatesphosphoprotein
phosphatase2A (PP2A; Fig. 15-18), which dephos-
phorylates the bifunctional PFK-2/FBPase-2enzyme
(Fig. 15-17). DephosphorylationactivatesPFK-2 and
inhibitsFBPase-2,and the resultingrise in fructose2,6-
bisphosphateconcentrationstimulatesglycolysisand
inhibits gluconeogenesis.The increased glycolysis
boosts the production of acetyl-CoA,while the in-
creasedflow of hexosethrough the pentosephosphate
pathway generatesNADPH. Acetyl-CoA and NADPH
are the starting materialsfor fatty acid synthesis,which
has long been known to increasedramaticallyin re-
sponseto intake of a high-carbohydrate meal.Xylulose
5-phosphatealso increasesthe syrrthesisof all the en-
zymes required for fatty acid synthesis,meeting the
prediction from metaboliccontrol analysis.We return to
this effectin our discussionof the integrationof carbo-
hydrateand lipid metabolismin Chapter23.
TheGlycolytic
Enzyrne
Pyruvate
Kinase
ls
Allosterirally
Inhibited
byATP
At least three isozymesof pyruvate kinaseare found in
vertebrates,differing in their tissue distribution and
 G l y c o l yasni sdG l u c o n e o g e n leCsti {s
t eedg u l a t ioofn
1 5 . 3C o o r d i n aR

(a)

Catalytic
subunit

2Mn2*

Scaffold,/
A subunit

their responseto modulators.High concentrationsof

ATP, acetyl-CoA, and long-chain fatty acids (signs
of abundant energy supply) allosterically inhibit all
isozymesof pyruvate kinase (FiS. 15-19). The liver
isozyme (L form), but not the muscle isozyme (M
form), is subjectto further regulationby phosphoryla-
tion. When low blood glucosecausesglucagonrelease,
cAMP-dependentprotein kinasephosphorylatesthe L
isozymeof pyruvate kinase,inactivatingit. This slows
the use of glucoseas a fuel in liver, sparingit for export
to the brain and other organs In muscle,the effect of
increased[cAMP]is quite different.In responseto epi-
nephrine, cAMP activates glycogen breakdown and
glycolysisand providesthe fuel neededfor the flght-or-
flioht roqnnnqo
Holoenzyme 1 Holoenzyme 2
FIGURI15-18 Structureand action of phosphoproteinphosphatase
2A (PP2A).(a) Thecatalyticsubunithastwo Mn2* ionsin its active
site,positionedcloseto the substraterecognitionsurfaceformedby the
interfacebetweenthe catalyticsubunitand the regulatorysubunit
(PDBlD 2NPP).Microcystin-LR, shownherein red, is a specificin-
hibitorof PP2A The catalyticand regulatorysubunitsrestin a scaffold
(theA subunit)that positions them relativeto eachotherand shapes
the substraterecognitionsite.(b) PP2Arecognizesseveraltargetpro-
providedby the regulatory
teins,itsspecificity subunit.Eachof several
regulatory subunitsfits the scaffoldcontainingthe catalyticsubunit,
and eachregulatory subunitcreatesits uniquesubstrate-binding site.

Liver only All glycolytic tissues, including liver

glucagon
I
I F16BP------
+ 1l
A-DP C ATP | | 6 steps
It
PEP

Pyruvate
l ltransamination
i
|| i
J)
A l a n i n e- - - - - /

FIGURE 15-19 Regulation of pyruvatekinase.

Theenzymeis allosteri-
callyinhibitedby ATP,acetyl-CoA, fattyacids(allsigns
and long-chain
of an abundantenergysupply),and the accumulation of fructose1,6-
bisphosphate triggersits activation.
Accumulation of alanine,which
can be synthesized from pyruvatein one step,allostericallyinhibits
pyruvatekinase,slowingthe productionof pyruvate by glycolysis.
The
liverisozyme(Lform)isalsoregulated hormonally.
Clucagonactivates
cAMP-dependent proteinkinase(PKA;seeFig.15-35),which phos-
phorylates the pyruvatekinaseL isozyme,inactivating it. When the
glucagonleveldrops,a proteinphosphatase (PP)dephosphorylates
pyruvatekinase,activating it. Thismechanism preventsthe liverfrom
consuming glucoseby glycolysis when bloodglucose is low; instead,
the liverexportsglucose.The muscleisozyme (M form) is not affected
by thisphosphorylation mechanism
f_l
1 5 9 0] P r i n c i p l0ef sM e t a b 0R
l i ce g u l a t i o n

TheGluconeogeni(
C0nversion to Phosphoenol allowing conversionof excesspyruvate to oxaloacetate
ofPyruvate
Pyruvate
lsUnder
Multiple
TypesofRegulation (and,eventually,glucose).
In the pathway leading from pyruvate to glucose,the
fust control point determinesthe fate of pyruvate in the
mitochondrion:its conversioneither to acetyl-CoA(by
the pyruvate dehydrogenasecomplex) to fuel the citric
acid cycle (Chapter16) or to oxaloacetate(by pyruvate
carboxylase)to start the processof gluconeogenesis
(Fig. f 5-20). When fatty acidsare readilyavailableas
fuels, their breakdown in liver mitochondria yields
acetyl-CoA,a signalthat further oxidation of glucosefor
fuel is not necessary. Acetyl-CoAis a positiveallosteric
modulator of pyruvate carboxylaseand a negativemod-
ulator of pyruvate dehydrogenase,through stimulation
of a protein kinasethat inactivatesthe dehydrogenase.
When the cell'senergyneedsare being met, oxidative
phosphorylationslows, NADH rises relative to NAD+
and inhibits the citric acid cycle,and acetyl-CoAaccu-
mulates.The increasedconcentrationof acetyl-CoAin-
hibits the pyruvate dehydrogenasecomplex,slowingthe
formation of acetyl-CoAfrom pyr"uvate,and stimulates
gluconeogenesisby activating pyruvate carboxylase,
Glucose
tI
I scriptionfactors.
S1";""""g"""ri. ]
II
Oxaloacetate
Oxaloacetateformed in this way is converted to
phosphoenolpy'ruvate(PEP) in the reaction catalyzed
by PEP carboxykinase(Fig. 15-11). In mammals,the
regulation of this key enz}rmeoccurs primarily at the
level of its synthesisand breakdown,in responseto di-
etary and hormonal signals.Fasting or high glucagon
levels act through cAMP to increase the rate of tran-
scription and to stabilizethe mRNA. Insulin, or high
blood glucose,has the oppositeeffects.We discussthis
transcriptional regulation in more detail below. Gener-
ally triggeredby a signalfrom outsidethe cell (diet, hor-
mones), these changestake place on a time scale of
minutesto hours.
Transcriptional
Regulation
ofGlycolysis
and
Gluconeogenesis
Changes
theNumberof
Enzyme
Molecules
Most of the regulatory actions discussedthus far are
mediated by fast, quickly reversiblemechanisms:al-
losteric effects, covalentalteration (phosphorylation)
of the enz;rme,or binding of a regulatory protein. An-
other set of regulatoryprocessesinvolveschangesin
the number of molecules of an enzyme in the cell,
through changesin the balanceof enzyme synthesis
and breakdovm,and our discussionnow turns to regu-
lation of transcription through signal-activatedtran-
In Chapter 72 we encounterednuclear receptors
and transcription factorsin the context of insulin signal-
ing. Insulin actsthrough its receptorin the plasmamem-
brane to turn on at leasttwo distrnct signalingpathways,
each involving activation of a protein kinase.The MAP

---,o Ora.r
","I
carbox) tuse
kinaseERK, for example,phosphorylatesthe transcrip-
tion factorsSRFand EIkl (seeFig. 12-15),which then
I stimulate the synthesis of enzymesneeded for cell
Pyruvate

--'I '""'iii;il.", growth and division. Protein kinaseB (PKB; also called
Akt) phosphorylatesanother set of transcription factors
l.* Acetyl-CoA
(PDX1,for example),and these stimulate the syrrthesis
of enz;.'rnesthat metabolizecarbohydratesand the fats
II formed and storedfollowing excesscarbohydrateintake
I in the diet. In pancreaticp cells, PDX1 also stimulates
+ the slnthesis of insulin itself.
More than 150 genesare transcriptionallyregulated
Citric acid cycle
by insulin; humanshave at least sevengeneraltypes of
I insulin responseelements,eachrecognizedbya subset
I
I of transcription factors activated by insulin under vari-
+
Energy
ousconditions.Insulin stimulatesthe transcriptionof the
genesthat encodehexokinasesII and IV, PFK-1, pyru-
FIGURE 15-20 Two alternativefates for pyruvate.pyruvatecan be vate kinase,and PFK-2lFBPase-2(all involvedin glycol-
converted to glucoseandglycogenvia gluconeogenesisor oxidizedto ysis and its regulation); several enzyrnesof fatty acid
acetyl-CoAfor energyproduction.Thefirstenzymein eachpath is reg- synthesis;and glucose6-phosphatedehydrogenaseand
ulatedallosterically;
acetyl-CoA,producedeitherby fatty acid oxida- 6-phosphogluconate dehydrogenase,enzpnes of the
tion or by the pyruvatedehydrogenase complex,stimulatespyruvate pentose phosphatepathway that generatethe NADPH
carboxylase and inhibitspyruvate
dehydrogenase. required for fatty acid synthesis.Insulin also slows the
 1 5 . 3( o o r d i n a tR
eed g u l a t ioof n
G l y c o l yasni sdG l u c o n e o g e n [etsnits l

Changein geneexpression Pathway

Increased expression
HexokinaseII Glycolysis
HexokinaseIV Glycolysis
1 (PFK-1)
Phosphofructokinase- Glycolysis
Pyruvatekinase Glycolysis
PFK-2/FBPase-2 Regulationof glycolysis/gluconeogenesis
Glucose6-phosphatedehydrogenase Pentosephosphatepathway (NADPH)
6-Phosphogluconate
dehydrogenase Pentosephosphatepathway (NADPH)
Py'ruvatedehydrogenase Fatty acid synthesis
Acetyl-CoAcarboxylase Fatty acid synthesis
Malic enzyme Fatty acid synthesis(NADPH)
ATP-citratelyase Fatty acid slmthesis(providesacetyl-CoA)
Fatty acid synthasecompiex Fatty acid s5.'nthesis
Stearoyl-CoAdehydrogenase Fatty acid desaturation
Acyl-CoA-glyceroltransferases Tfiacylglycerolsynthesis
Decreased expression
PEP carboxykinase Giuconeogenesis
Glucose6-phosphatase(cataly'ticsubunit) Glucosereleaseto blood

expressionof the genesfor two enz).rnes of gluconeoge-

nesis:PEP carboxykinaseand glucose 6-phosphatase
(Table15-5) Glucose Plasma
One transcription factor important to carbohydrate J GLUT2 membrane
rrl,
metabolismis ChREBP (carbohydrate response ele- 'll
ment binding protein; Fig. 15-2f ), whichis expressed I Cytosol
primarily in liver, adiposetissue,and kidney.It servesto Glucose
coordinatethe synthesisof enzl'rnesneededfor carbohy-
drate and fat s;,nthesis ChREBPin its iractive state is
phosphorylated,and is locatedin the cy'tosol When the Glucose
6-phosphate
phosphoprotein phosphatase PP2A(Fig. I 5-18) removes J
a phosphorylgroup from ChREBP,the transcriptionfac-
tor can enter the nucleus.Here,nuclearPP2Aremoves .@@
anotherphosphorylgroup,and ChREBPnow joins with a _ Xylulose dhREBF)
b-pnospnale
partnerprotein,Mlx, andturns on the synthesisof several
enzynes: pyruvate kinase, fatty acid s;mthase, and
acetyl-CoAcarboxylase,the first enzyrnein the path to
fatty acid syarthesis (Fig. 15-21).

FIGURE 15-21 Mechanismof gene regulationby the transcription Nucleus

factorChREBP. WhenChREBP in the cytosolof a hepatocyte is phos-
phorylatedon a Seranda Thrresidue, it cannotenterthe nucleusDe-
phosphorylation of @Ser by protein phosphatase PP2A allows
ChREBP to enterthe nucleus,wherea seconddephosphorylation, of
@Thr, activatesChREBPso that it can associate with its partner
protein,Mlx. ChREBP-Mlx now bindsto the carbohydrate response
element(ChoRE) in the promoterand stimulates transcription PP2Ais
activated
allosterically by xyluloseS-phosphate, an intermediate in the
pentosephosphatepathway.
 592', Principles
ofMetabolic
Regulation
Controllingthe activity of PP2A-and thus, r.rltimately,
the synthesisof this groupof metabolicen4,'rnes-is xylu-
lose5-phosphate,an intermediatenot of glycolysisor glu-
coneogenesisbut of the pentose phosphate pathway.
Whenblood$ucose concentrationis high, $ucose enters
the liver and is phosphorylatedby hexokinaseM The gu-
cose 6-phosphatethus formed can enter either the gly-
cobtic pathwayor the pentosephosphatepathway.Ifthe
latter,two initial oxidationsproducexylulose5-phosphate,
which servesasa signalthat the glucose-utilizugpathways
arewell-suppliedwith substrate.It accomplishes
this by al-
lostericallyactivatingPP2A,which then dephosphorylates
ChREBRallowingthe transcriptionfactorto tum on the ex-
pressionof genesfor enz;..rnes of glycolysisand fat ryrrthe-
sis(Fig. l5-2I) . Glycolysisyieldspyruvate,and conversion
of pyuvate to acetyl-CoAprovidesthe starting material
for fatty acid syrthesis:acetyl-CoAcarboxylaseconverts
acetyl-CoAto malonyl-CoA, the first committedintermedi-
ate in the path to fatty acids.The fatty acid s1r-rthasecom-
plex producesfatty acidsfor export to adiposetissueand
storageastriacylglycerols(Chapter21). In this way,excess
dretarycarbohydrateis storedasfat.
Another transcriptionfactor in the liver, SREBP-
lc, a member of the family of sterol response ele-
ment binding proteins (see Fig. 2l-43), turns on the
syrrthesisof pyruvatekinase,hexokinaseId lipoprotein
lipase,acetyl-CoAcarboxylase,and the fatty acid syn-
thase complexthat will convert acetyl-CoA(produced
from pyruvate)into fatty acidsfor storagein adipocytes.
The symthesis
of SREBC-1cis stimulatedby insulinand
depressedby glucagon.SREBP-1calso suppressesthe
expressionof severalgluconeogenicenzymes:glucose
6-phosphatase, PEP carboxykinase, and FBPase- 1
The transcriptionfactor CREB (cyclic AMp re-
sponse element binding protein) turns on the syr-r-
thesisof glucose6-phosphatase and PEP carboxykinase
in responseto the increase in [cAMP] triggered by
glucagon.In contrast,insulin-stimulatedinactiuati,on
of other transcription factors turns off severalgluco-
neogenicenzyrnesin the liver:PEP carboxykinase, fruc-
tose 1,6-bisphosphatase, the glucose 6-phosphate
transporterof the endoplasmicreticulum, and glucose
6-phosphatase. For example,FOXOf (forkhead box
other) stimulatesthe synthesisof gluconeogenicen-
zymesand suppressesthe syrrthesisof the enzyrnesof
glycolysis,the pentosephosphatepathway,and triacyl-
glycerol synthesis(Fig. 15-22) In its unphosphory-
latedform,FOXOl actsasa nucleartranscriptionfactor.
In responseto insulin,FOXO1leavesthe nucieusand in
the cytosolis phosphorylatedby PKB,then taggedwith
ubiquitin and degradedby the proteasome.Glucagon
preventsthis phosphorylationby PKB, and FOXO1re-
mainsactivein the nucleus.
Complicatedthough the processesoutlined above
may seem,regulationof the genesencodingenz}rmes
carbohydrateand fat metabolismis proving far more
complex and more subtle than we have shor,r,n here.
Plasma
,/,membrane
Cytosol
It-i'i i i.- /\
Ly'n
<-?a
J cp
I Proteasomy'
Y
Ubiquitin,,'t
\ PKBI
(;;;;i'
>/,- 7
','**o,'"
o
Pi_.,!"- -l,ts'P
".'--j-f o_
DNA
mRNA
carboxykinase
Glucose6-phosphatase
FIGURE 15-22 Mechanismof gene regulationby the transcription
factorFOXO1.Insulinactivates the signalingcascade shownin Figure
12-.1 6, leadingto activation
of proteinkinaseB (PKB).FOXOI in the
cytosolis phosphorylated by PKB,and the phosphorylated transcrip-
tion factoris taggedby the attachmentof ubiquitinfor degradationby
proteasomes. FOXOl thatremainsunphosphorylated or isdephospho-
rylatedcan enterthe nucleus,bind to a response element,and trigger
transcription of theassociatedgenes.Insulinthereforehastheeffectof
turningoflthe expression of thesegenes,which includePEPcarboxy-
kinaseand glucose6-phosphatase
Multiple transcription factors can act on the same gene
promoter; multiple protein kinases and phosphatases
can activate or inactivate these transcription factors;
and a variety of protein accessory factors modulate the
action of the transcription factors. This complexity is
apparent, for example, in the gene encoding PEP car-
boxykinase, a very well-studied case of transcriptional
control. Its promoter region (Fig. 15-23) has 15 or
more response elements that are recognized by at least
a dozen known transcription factors, with more likely to
be discovered The transcription factors act in combina-
tion on this promoter region, and on hundreds of other
gene promoters, to flne-tune the levels of hundreds of
metabolic enzymes, coordinating their activity in the
metabolism of carbohydrates and fats. The critical im-
portance of transcription factors in metabolic regulation
is made clear by observing the effects of mutations in
their genes. For example, at least flve different types of
of maturity-onset diabetes of the young (MODYI are asso-
ciated with mutations in speciflc transcription factors
(Box 15-3).
 15.3(oordinated
Regulation andGluconeogenesis
ofGlycolysis I sol I

HNF-4cY Fos/Jun ATFB CREB C/EBP

COUP-TF
RAR SREBP-1
H NF-36
TSR J +**t- -t mRNA
J CiXBP Nfi
I
,\//.;:

Tlanscription factors Response elements and regulatory

FOXO1 forkhead box other 1 binding sites in promoter
PPAR72 peroxisomeproliferator-activated receptor,y2 dAF2 distal accessoryfactor2
HNF-38 hepatic nuclear factor-3B dAFl distal accessoryfactor 1
SREBP-I sterol regulatory element binding protein-l SRE sterol regu.latory element
HNF-4cy hepatic nuclear factor-4o Atr'1 accessoryfactor 1
COUP-TF chicken ovalbumin upstream AF2 accessoryfaclor2
promoter-transcription factor GRE glucocorticoid regulatory
RAR retinoic acid receptor element
GR glucocorticoid receptor TRE thynoid hormone regulatory
TsR thyroid hormone receptor element
C/EBP CAAT/enhancebindingprotein CRE cAMP regulatory element
HNF-I hepatic nuclear factor-l
NF1 nuclear factor 1
ATFS activating transcription factor 3
CREB cAMP regulatory element binding protein
NFrB nuclear factor rB
TBP TATA-box binding protein
Med. mediator
TFIIH transcription factor IIH

FIGURE 15-23 The PEPcarboxykinase promoterregion,showingthe whichcan reflecttheavailability

factors, of nutrients, bloodglucoselevel,
complexityof regulatoryinputto thisgene.Thisdiagramshowsthetran- andotherfactorsthatgo intomakingup the cell'sneedfor thisenzymeat
scriptionfactors(smallericons,boundto the DNA)knownto regulate the this particulartime. Pl, P2, P3l, P3ll,and P4 are proteinbindingsites
transcriptionof the PEPcarboxykinase gene.The extentto which this identifiedby DNaseI footprinting (seeBox26-1).TheTATA box istheas-
geneis expressed dependson the combinedinputaffectingall of these semblypoint for the RNA polymerase ll (Polll) transcription
complex.

The term "diabetes"describesa variety of medicalcon-
ditionsthat havein commonan excessiveproductionof
urine. In Box 11-2 we describeddiabetesinsipidus,in
which defectivewater reabsorptionin the kidney results
from a mutation in the gene for aquaporin. "Diabetes
mellitus" refers speciflcallyto diseasein which the abil-
ity to metabolizeglucoseis defective,due either to the
failureof the pancreasto produceinsulinor to tissuere-
sistanceto the actionsof insulin.
There are two common t5,pesof diabetesmellitus.
Tlrpe 1, also called insulin-dependentdiabetesmellitus
(IDDM),is causedby autoimmuneattackon the insulin-
producing B cells of the pancreas.Individuals with
IDDM must take insulin by injection or inhalation to
compensatefor their missingB cells.IDDM developsin
childhoodor in the teen years;an older name for the
diseaseis juvenile diabetes.Tlrpe 2, also called non-
insulin-dependentdiabetesmellitus (NIDDM), typically
developsin adultsover 40 yearsold. It is far more com-
mon than IDDM,and its occurrenceh the populationis
strongly correlated with obesity.The current epidemic
of obesityin the more developedcountriesbrings with it
the promise of an epidemic of NIDDM, providing a
strongincentiveto understandthe relationshipbetween
obesityand the onsetof NIDDM at the geneticand bio-
chemicallevels.After completingour look at the metab-
olism of fats and proteins in later chapters,we will
return (in Chapter 23) to the discussionof diabetes,
which has a broad effect on metabolism:of carbohy-
drates,fats, and proteins.
Here we consideranother type of diabetesin which
carbohydrateand fat metabolismis deranged:mature
onset diabetesof the young (MODY), in which genetic
mutation affects a transcription factor important in car-
rying the insulin signalinto the nucleus,or affectsan en-
4nne that respondsto insulin. In MODY2,a mutation in
the hexokinaseIV (glucokinase)gene affects the liver
and pancreas,tissuesin which this is the main isoform of
hexokinase.The $ucokinase of pancreaticB cells func-
tions as a glucosesensor.Normally,when bloodglucose
(conti,nued on nert pa'ge)
rEr-l P r i n c i p loefsM e t a b oRl i ce g u l a t i o n
rises,so doesthe glucoselevel in B cells,and because
glucokinasehas a relativelyhigh K* for glucose,its ac-
tivity increaseswith risingbloodglucoseIevels.Metabo-
Iism of the glucose6-phosphateformedin this reaction
raisesthe ATP level in B cells,and this triggersinsulin
releaseby the mechanismshown in Figure 23-28. In
healthyindir,rduals, bloodglucoseconcentrationsof -b
mu trigger this insulin release.But individualswrth inac-
tivating mutations in both copies of the glucokinase
gene havevery high thresholdsfor rnsulinrelease,and
consequently,from birth, they have severe hyper-
glycemia-permanent neonataldiabetes.In individuals
with one mutated and one normal copy of the glucoki-
nasegene,the glucosethresholdfor insulinreleaserises
to about 7 mu. As a result theseindividualshaveblood
glucoselevels only slightly abovenormal: they generally
have only mild hyperglycemiaand no syrnptoms.This
S U M M A R1Y5 . 3 C o o r d i n a t eRd e g u l a t i o n
o f G l y c o l y s iasn d
Gluconeogenesis
r Gluconeogenesisand glycolysis share seven
enzlmes, catalyzing the freely reversible reactions
of the pathways. For the other three steps, the
forward and reverse reactions are catalyzed by
different enzymes, and these are the points of
regulation of the two pathways.
r Hexokinase IV (glucokinase) has kinetic properties
related to its special role in the liver: releasing
glucose to the blood when blood glucose is low,
and taking up and metabolizing glucose when blood
glucose is high.
r PFK-I is allosterically inhibited by ATP and citrate.
In most mammalian tissues, including liver, fructose
2,6-bisphosphateis an allosteric activator of this
enz).rne.
Pyruvate kinase is allosterically inhibited by
ATP, and the liver isozyme also is inhibited r.ry
cAMP-dependent phosphorylation.
Gluconeogenesisis regulated at the level of
pyruvate carboxylase (which is activated by
acetyl-CoA) and FBPase-l (which is inhibited by
fructose 2,6-bisphosphateand AMP)
To limit substrate cycling between glycolysis and
gluconeogenesis,the two pathways are under
reciprocal allosteric control, mainly achieved by
the opposing effects of fructose 2,6-bisphosphate
on PFK-1 and FBPase-1.
Glucagon or epinephrine decreases[fructose
2,O-bisphosphatel,by raising [cAMP] and bringing
condition(MODY2)is generallydiscoveredby accident
duringroutine bloodglucoseanalysis.
There are at least flve other types of MODI each
the result of an inactivating mutation in one or another
of the transcriptionfactors essentialto the normal de-
velopmentand function of pancreaticF cells.Individu-
als with these mutations have varying degrees of
reducedinsulin production and the associateddefects
in blood glucosehomeostasis.In MODY1and MODY3,
the defectsare severeenoughto producethe long-term
complications associatedwith IDDM and NIDDM-
cardiovascular problems,kidney failure,and blindness.
MODY4,5, and 6 are lesssevereformsof the disease.Al-
together,MODYdisordersrepresenta smallpercentage
of NIDDM cases.Also very rare are individualswith mu-
tations in the insulin gene itself; they have defects in
insulin signalingof varying severity.
about phosphorylationof the bifunctional enzyrne
PFK-2/FBPase-2. Insulinincreases[fructose
2,6-bisphosphatelby activatinga phosphoprotein
phosphatasethat dephosphorylatesand thus
activatesPFK-2.
Xylnlose5-phosphate,an intermediateof the pentose
phosphatepathway,activatesphosphoprotein
phosphatasePP2A,which dephosphorylates several
target proteins,includingPFK-2/FBPase-2,tilting the
balancetoward glucoseuptake,glycogenslnthesis,
and lipid s;,nthesisin the liver.
TranscriptionfactorsincludingChREBRCREB,
SREBP,and FOXO1act in the nucleusto regulate
the expressionof specificgenescodingfor enz;rmes
of the glycolytic and gluconeogenicpathways.
Insulin and glucagonact antagonisticallyin
activatingthese transcription factors, thus turning
on and off largenumbersof genes.
15.4 TheMetabolism
of Glycogen
in Animals
Our discussionof metabolic regulation, using carbohy-
drate metabolismas the primary example,now turns to
the synthesisand breakdownofglycogen.In this section
we focuson the metabolicpathways;in Section15.5we
turn to the regulatorymechamsms.
In organismsfrom bacteriato plants to vertebrates,
excess$ucose is convertedto poly.rnericforms for stor-
age-$ycogen in vertebratesand many microorganisms,
starchin plants.In vertebrates,glycogenis for.rndprimarily
in the liver andskeletalmuscle;it mayrepresentup to 10%
of the weightof liver and lo/oto 2o/oof the weightof muscle.
If this much glucosewere dissolvedin the cytosolof a he-
patocyte,its concentrationwould be about 0.4 nl, enough

tlGURt15-24 Glycogengranulesin a hepatocyte.Clycogen,a stor-
ageform of carbohydrate, appearsaselectron-denseparticles,oftenin
aggregatesor rosettes.In hepatocytesglycogenis closelyassociated
with tubulesof thesmoothendoolasmic reticulum.
Manvmitochondria
arealsoevidentin thismicrograph.
to dominate the osmotic properties of the cell. When
storedas a large pol),mer($ycogen),however,the same
massof $ucosehasa concentrationof only 0.01pM.Glyco-
gen is storedin large cltosolic granules.The elementary
particleof glycogen,the B-particle,is about21 nm in diam-
eter and consistsof up to 55,000glucoseresidueswith
about2,000nonreducingends.TWentyto 40 ofthese par-
ticles clustertogetherto form a-rosettes,easilyseenwith
the microscopein tissue samplesfrom well-fed animals
(Fig,. 15-24) but essentiallyabsenta^ftera 24-hourfast.
The glycogenin muscle is there to provide a quick
sourceof energyfor either aerobicor anaerobicmetab-
olism.Muscleglycogencanbe exhaustedin lessthan an
hour during vigorousactivity. Liver glycogenservesas a
reservoirof glucosefor other tissueswhen dietaryglu-
coseis not available(betweenmealsor during a fast);
Nonreducing end
6cH2oH cH2oH
--l * scrr
'I
", p rhor vlast
J
Nonreducing end
6cH2oH
Glucose 1-phosphate
inAnimals
ofGlycogen
15.4TheMetabolism | 595i
this is especiallyimportant for the neurons of the brain,
which cannot use fatty acids as fuel. Liver glycogencan
be depleted in 12 to 24 hours. In humans, the total
amountof energystoredas glycogenis far lessthan the
amount stored as fat (triacylglycerol) (see Table 23-5),
but fats cannotbe convertedto glucosein mammalsand
cannotbe catabolizedanaerobically.
Glycogengranulesare complexaggregatesof glyco-
gen and the enz5'rnes that synthesizeit and degradeit, as
well as the machineryfor regulatingthese enz5.'rnes. The
generalmechanismsfor storing and mobiLizingglycogen
are the samein muscleand liver' but the erzSrmesdiffer
in subtle yet important ways that reflect the different
rolesof glycogenin the two tissues.Glycogenis alsoob-
tained in the diet and brokendorvnin the gut, and this in-
volvesa separateset of hydrolytic enzymesthat convert
glycogento free glucose.(Dietary starchis hydrolyzedin
a similar way.) We begin our discussionwith the break-
dou'n of glycogento glucose1-phosphate(glycogenoly-
sis), then turn to synthesisofglycogen(glycogenesis)'
by
lsCatalyzed
Breakdown
Glycogen
Phosphorylase
Glycogen
In skeletalmuscleand liver,the glucoseunits of the outer
branches of glycogen enter the glycoly'tic pathway
through the action of three enzymes:glycogenphospho-
rylase, glycogendebranchingenzyrne,and phospho$u-
comutase.Glycogenphosphorylasecatalyzesthe reaction
in wluch an (a1-+4) glycosidiclinkagebetweentwo glu-
cose residuesat a nonreducingend of $ycogen under-
goes attack by inorganic phosphate (P), removing the
terminal glucoseresidue as c-D-glucose l-phosphate
(Fig. 15-25). This phosphorolgszsreactionis different
from the hAd,rolAsi'sof glycosidicbondsby amylasedur-
ing intestinaldegradationof dietary glycogenand starch.
In phosphorolysis,some of the energy of the glycosidic
Glycogen chain
(glucose)"
FIGURE 15-25 Removalof a glucoseresidue
from the nonreducing end of a glycogen
O- chain by glycogen phosphorylase'This
processis repetitive;the enzyme removes
successive glucoseresiduesuntil it reaches
Glycogen shortened
by one residue the fourth glucoseunit from a branch point
(glucose)", t ( s e eF i 8 . 1 5 - 2 6 ) .
ft
1596 Principles
ofMetabolic
Regulation

bond is preservedin the formationof the phosphateester, Nonreducing

glucosel-phosphate(seeSection14.2). ends

Pyridoxalphosphateis an essentialcofactorin the

glycogenphosphorylasereaction;its phosphategroup
actsasa generalacid catalyst,promotingattackby p1on
the glycosidic bond. (This is an unusual role for pyri-
Glycogen
doxalphosphate;its more typical role is as a cofactorin
aminoacid metabolism;seeFig. 18-6.)
gLycogen
Glycogenphosphoryiaseacts repetitively on the lrhosphorvlase
nonreducingendsofglycogenbranchesuntil it reaches
a point four glucoseresiduesaway from an (a1-+6)
branch point (see Fig. 7-14), where its action stops. o
Further degradationby glycogenphosphorylase can oc- o
cur only after the debranching enzyme, formally Glucose 1-phosphate
molecules
known as oligo (a1--+6) to (a1-+4) glucan-trans-
ferase, catalyzestwo successive reactionsthat transfer tr unsler trstr
irctr\ lt\. 0t
branches(Fig. l5-26). Oncethesebranchesarerrans- c k r l r r l u r c hI n g
etn zvul('
ferred and the glucosylresidue at C-6 is hydrolyzed,
glycogenphosphorylase activity can continue.

I -Phosphate
Glucose (anEnter
Glycolysis
or,inLiver,
Replenish
Blood
Glucose {a1l )61
glu(l0slttLlse
Glucosel-phosphate,the end product of the glycogen at t il itr, ol
tlebr trnchilg
phosphorylase reaction,is convertedto glucose6-phos- ctlzvllle
ffi cln.or"
phate by phosphoglucomutase, which catalyzesthe
reversiblereaction
Glucose l-phosphate =+ glucose G-phosphate

Initiallyphosphorylated
u"o:3il|;i,ii:#JnT:l-*'
at a Serresidue,the enz)ryne do-
natesa phosphorylgroup to C-6 of the substrate,then phosphorylaseaction
acceptsa phosphoryigroup from C-1 (t'ig. t5-27). FIGURI15-26 Gtycogenbreakdownnear an (at-+6) branch point.
The glucose6-phosphateformed from glycogenin Following
sequential
removalof terminalglucoseresidues
by glycogen
skeletalmusclecan enter glycolysisand serveas an en- (seeFig.I 5-25),glucoseresidues
phosphorylase neara brancharere-
ergy source to support muscle contraction. In liver, movedin a two-stepprocess that requires a bifunctional
debranching
glycogenbreakdownservesa different purpose:to re- enzyme.First,the transferaseactivityof the enzymeshiftsa block of
Ieaseglucoseinto the blood when the blood glucose threeglucoseresidues from the branchto a nearbynonreducing end,
level drops,as it doesbetweenmeals.This requiresthe to which they are reattachedin (a1--+4)linkageThe singleglucose
enzyrneglucose6-phosphatase, presentin liver and kid- residueremainingat the branchpoint,in (a1--+6) linkage,is then re-
ney but not in other tissues.The enzymeis an integral leasedas freeglucoseby the debranching enzyme,s (a1--+6)glucosi-
membraneprotein of the endoplasmicreticulum, pre- daseactivity.
Theglucoseresidues areshownin shorthand form,which
dicted to containnine transmembranehelices,with its omitsthe-H, --{H, and-{H2OH groupsfromthe pyranose rings.
active site on the Iumenalside of the ER. Glucose6-
phosphateformedin the cytosolis transportedinto the
Becausemuscleand adiposetissuelack glucose6-
ER lumen by a speciflctransporter(T1) (Fig. fE-28) phosphatase,they cannot convert the glucose6-phos-
and hydrolyzedat the lumenai surfaceby the glucose6- phate formed by glycogenbreakdownto glucose,and
phosphatase. The resultingPi and glucoseare thought thesetissuesthereforedo not contributeAlucoseto the
to be carried back into the cytosol by two different blood.
transporters(T2 and T3), and the glucoseleavesthe he-
patoc).tevia the plasmamembranetransporter,GLUT2.
Notice that by havingthe activesite of glucose6-phos-
The5ugar
Nucleotide
UDP-Glucose
Donates
Glucose
phataseinsidethe ER lumen,the cell separatesthis re- forGlycogen
Synthesis
actionfrom the processof glycolysis,which takesplace Many of the reactionsin which hexosesare transformed
in the cy'tosoland would be aborted by the action of or polymerizedinvolve sugax nucleotides, compounds
glucose6-phosphatase. Genetic defectsin either glu- in which the anomericcarbon of a sugaris activatedby
cose6-phosphatase or T1 lead to seriousderangement attachment to a nucleotide through a phosphate ester
of glycogenmetaboiism,resulting in type Ia glycogen Iinkage.Sugarnucleotidesarethe substratesfor polymer-
storagedisease(Box 15-4). izationof monosaccharide s into disaccharides,glycogen,
 lt'l
G l y c o giennA n i m a l s
1 5 . 4T h eM e t a b o l i os fm

FIGURE 15-27 Reactioncatalyzedby phosphoglucomutase.

Phosphoglucomutase The reactionbeginswith the enzymephosphorylated on a Ser
'r/rt residue.In step @, the enzymedonatesits phosphorylgroup
\
to glucose1-phosphate,
(green) producingglucose1,6-bisphos-

\{a HHO
phate.In step@, the phosphorylgroupat C-1 of glucose1,6-
bisphosphate (red)istransferred
backto the enzyme,re-forming
Glucose 1-phosphate thephosphoenzyme andproducingglucose6-phosphate.

HHO
Glucose1,6-bisphosphate

o
-o_P_o_
I
o-H
t\
V
HO

HHO
Glucose 6-phosphate

FIGURE l5-28 Hydrolysisof glu-

Cytosol
cose 6-phosphateby glucose6-
Glucose
G6P G-phosphatase phosphatase of the ER,Thecatalytic
G6P siteof glucose6-phosphatase faces
transporter the lumenof the ER.A glucose6-
(T1)
phosphate(C6P)transporter(T1)
caniesthe substrate from the cy-
tosolto the lumen,and the prod-
ucts glucoseand P; passto the
(T2
cytosolon specifictransporters
ER
lumen and T3) Clucoseleavesthe cell
via the CLUT2 transporter in the
plasmamembrane.
Increased
blood glucose
concentration

o-Glucosyl group

starch, cellulose, and more com-

plex extracellular polysaccharides.
Uridine
They are also key intermediates in o
the production of the aminohex-
oses and deoxyhexoses found in
some of these polysaccharides,
o
and in the synthesis of vitamin C
(l-ascorbic acid). The role of
sugar nucleotides in the biosyn-
thesis of glycogen and many other
carbohydrate derivatives was dis- OH OH
covered in 1953 by the Argentine UDP-glucose
Luis Leloir, 1906-1987 biochemist Luis Leloir. (a sugar nucleotide)
[rrd P r i n c i p loef sM e t a b oR
l i ce g u l a t i o n
Much of what is written in present-daybiochemistrytext-
books about the metabolismof glycogenwas discovered
betweenabout1925and 1950by the remarkablehusband
and wife team of Carl F. Cori and Gerty T. Cori. Both
trained in medicrneir Europe at the end of World War I
(shecompletedpremedicalstudiesandmedicalschoolin
oneyear!).Theyleft Europetogetherin 1922to establish
researchlaboratoriesin the United States,first for rune
years in Buffalo, New York, at what is now the Roswell
Park MemorialInstitute, then from 1931until the end of
their lives at WashingtonUniversityin St. Louis.
In their early physiologicaistudies of the origin
and fate of glycogen in animal muscle, the Coris
T h e C o r i s i n C e r t y C o r i ' s l a b o r a t o r y ,a r o u n d 1 9 4 7
The suitability of sugar nucleotides for biosynthetic
reactions stems from several properties:
1. Their formation is metabolically irreversible,
contributing to the irreversibility of the slnthetic
pathways in which they are intermediates. The
condensation of a nucleoside triphosphate with a
hexose 1-phosphate to form a sugar nucleotide
has a small positive free-energy change,but the
reaction releasesPP1,which is rapidly hydrolyzed
by rnorganic pytophosphatase (Fig. lE-Zg),
in a reaction that is strongly exergonic
(AG'' : - 19.2 kJ/mol). This keeps the cellular
concentration of PP1low, ensuring that the
demonstratedthe conversionof glycogento lactate in
tissues,movementof lactate in the blood to the liver,
and, in the liver, reconversionof lactateto glycogen-
a pathwaythat cameto be known asthe Cori cycle (see
Fig. 23-20). Pursuing these observationsat the bio-
chemicallevel, they showedthat glycogenwas mobi-
Iized in a phosphorolysisreaction catalyzedby the
enzyme they discovered,glycogen phosphorylase.
They identified the product of this reaction (the "Cori
ester") as glucose l-phosphate and showed that it
could be reincorporatedinto glycogenin the reverse
reaction.Althoughthis did not prove to be the reaction
by which glycogenis synthesizedin cells, it was the
first in vitro demonstrationof the synthesisof a macro-
moleculefrom simple monomericsubunits,and it in-
spired others to search for polymerizing enzymes.
Arthur Kornberg, discoverer of the first DNA poly-
merase,saidof his experiencein the Coris'lab, "Glyco-
gen phosphorylase,not basepairing, was what led me
to DNA polymerase."
ffi Gerty Cori becameinterestedin human genetic
II diseasesin which too much glycogenis stored
in the liver. She was able to identify the biochemical
defect in severalof these diseasesand to show that
the diseasescould be diagnosedby assaysof the en-
zymesof glycogenmetabolismin small samplesof tis-
sue obtainedby biopsy.Table I summarizeswhat we
now know about 13 geneticdiseasesof this sort. I
Carl and Gerty Cori sharedthe Nobel Prize in Phys-
iologyor Medicinein1947with BernardoHoussayof Ar-
gentina, who was cited for his studies of hormonal
regulationof carbohydratemetabolism.The Cori labora-
toriesin St. Louisbecamean internationalcenterof bio-
chemicalresearchin the 1940sand 1950s,and at least
six scientistswho trained with the CorisbecameNobel
laureates:Arthur Kornberg (for DNA synthesis,1959),
SeveroOchoa (for RNA synthesis,1959), Luis Leloir
(for the role of sugarnucleotidesin polysaccharidesyn-
thesis, 1970), Earl Sutherland (for the discovery of
actual free-energy change in the cell is favorable.
In effect, rapid removal of the product, driven by
the large, negative free-energy change of PP,
hydrolysis, pulls the synthetic reaction forward,
a conunon strategy in biological poll'rnerization
reactions.
2. Although the chemical transformations of sugar
nucleotides do not involve the atoms of the
nucleotide itself, the nucleotide moiety has many
groups that can undergo noncovalent interactions
with enzyrnes; the additional free energy of binding
can contribute signiflcantly to catalytic activity
(Chapter 6; see also p.297).
 1 5 . 4T h eM e t a b o l iosfm
G l y c o gi enA
n n i m a l s[rrol

cAMP in the regulation of carbohydrate metabolism, 1974),and Edwin Krebs (for the discoveryof phospho-
1971),Christiande Duve (for subcellular fractionation, rylasekinase,1991).

Primary organ
Tlpe (name) Enrymeaffected affected Symptoms
I}pe o Glycogenslnthase Liver Low bloodglucose,high
ketonebodies,earlydeath
Tlpe Ia (von Gierke's) Glucose6-phosphatase Liver Enlargedliver, kidney failure
$pe Ib Microsomalglucose Liver As in Ia; alsohigh
6-phosphatetranslocase susceptibiiityto bacterial
infections
Tlpe Ic MicrosomalP1 Liver As in Ia
transporter
lVpe II (Pompe's) Lysosomalglucosidase Skeletaland Infantile form: death by age2;
cardiacmuscle juveniie form: muscle defects
(myopathy); aduit form: as
in musculardystrophy
Tlpe IIIa (Cori'sor Forbes's) Debranchingenz,'rne Liver, skeletal Enlargedliver in infants;
and cardiac myopathy
muscle
Dpe IIIb Liver debranching Liver Enlargedliver in infants
enzlme (muscle
enzlme normal)
Dpe IV (Andersen's) Branchingenzyrne Liver, skeletal Enlargedliver and spieen,
muscle myoglobinin urine
15pe V (McArdie's) Musclephosphorylase Skeletalmuscle Exercise-inducedcrampsand
pain; myoglobinin urine
Tpe VI (Hers's) Liver phosphorylase Liver Enlargedliver
Tlpe VII (Tarui's) MusclePFK-I Muscle, As in type t also hemoMic
anrfhrnn\f
!r,t !r!r vvJ
ac
vvv anenua
T},pe\4b, VIII, or IX Phosphorylasekinase Liver, Ieukocy'tes, Enlargedliver
muscle
T\rpeXI (Fanconi-Bickel) Glucosetransporter Liver Failure to thrive, enlarged
(GLUT2) liver, rickets, kidney
dysfunction

3. Like phosphate,the nucleotidyigroup (UMP or
AMP,for example)is an excellentleavinggroup,
facilitating nucleophilicattack by activatingthe
sugarcarbonto which it is attached.
4. By "tagging"somehexoseswith nucleotidylgroups,
cellscan set them asidein a pool for one purpose
(glycogensynthesis,for example),separatefrom
hexosephosphatesdestinedfor anotherpurpose
(suchasglycolysis).
Glycogensynthesistakes place in virtually all ani-
mal tissuesbut is especiallyprominentin the liver and
skeletal muscies.The starting point for synthesisof
glycogenis glucose6-phosphate. As we haveseen,this
can be derived from free glucose in a reaction cat-
alyzedby the isozymes hexokinase I and hexokinase II
in muscle and hexokinase IV (glucokinase) in liver:
o-Glucose+ ATP -----+l-glucose 6-phosphate+ ADP
However, some ingested glucose takes a more round-
about path to glycogen. It is first taken up by ery4hro-
c1'tes and converted to lactate glycolytically; the lactate
is then taken up by the liver and converted to glucose
6-phosphateby gluconeogenesis.
To initiate glycogen synthesis, the glucose 6-phos-
phate is converted to glucose l-phosphate in the phos-
phoglucomutasereaction:
Glucose6-phosphate+ glucosel-phosphate
 P r i n c i p l eo sf M e t a b o l R
i ce g u l a t i o n
L600_l

FIGURE 15-29 Formationof a sugarnucleotide.A con-

densationreactionoccursbetweena nucleosidetriphos- o
I | \ll
phate (NTP) and a sugar phosphate.The negatively
@o-r P-O- + o-P* o f q, f-or ntu"."-[s"!q
chargedoxygenon the sugarphosphateservesasa nucle- I I vl
o ooo-
ophile, attackingthe a phosphateof the nucleoside
Sugar phosphate
triphosphate and displacingpyrophosphate. The reaction
is pulledin the forwarddirectionby the hydrolysis
by i norganicpyrophosphatase
of PP;
*o"-.'.u.
I
rrorhosrhorvlas[

oo oo
llll
o-P--o-P-o r o-f -o-f -o-lTtnoseJ s.*
Suear
tl
oo oo-
Pyrophosphate(PP1) Sugar nucleotide
(NDP-sugar)
inors
pyrophospha "I
"J
o
-O-P-OH
2
- oI
Phosphate(P)

Net reaction: Sugar phosphate + NTP -----+ NDP-sugar -t 2Pi

The product of this reactionis convertedto UDP-glucose glucoseformation,becausepyrophosphateis rapidly hy-

by the action of UDP-glucose pyrophosphorylase, in a drolyzedby inorganicpyrophosphatase(Fig. 15-29).
key step of glycogenbiosymthesis: UDP-glucoseis the immediate donor of glucose
residues in the reaction catalyzedby glycogen sJrn-
Glucosel-phosphate+ UTP -----+UDP-glucose
* PPi
thase, which promotes the transfer of the glucose
Noticethat this enz),rneis namedfor the reversereaction; residue from UDP-glucoseto a nonreducingend of a
in the cell, the reactionproceedsh the direction of UDP- branchedglycogenmolecule(Fig. 15-30). The overall

6cH2oH FIGURE l5-30 Glycogensynthesis. A glycogenchain is elongatedby

glycogensynthase. Theenzymetransfers the glucoseresidueof UDP-
glucoseto the nonreducingend of a glycogenbranch(seeFig.7-14) to
makea new tal -+4) linkage.

o-P
I
o o-cH2

UDP-glucose

\./
HOH
Nonreducing end of
a glycogen chain
glycogcn
synthase
with n residues
(n> 4)

New nonreducing
end

Elongated glycogen
withz+lresidues
 ofGlycogen
15.4TheMetabolism inAnimals
latf

.iCI"JCI"f I "f ;L"f [ "fI.

Nonreducing (aI---+4)
end

Nonred.ucing,[i"{I"f)-"_c)-
(a1--+6) Branch
^
-r\, pornt

Nonreducing -Z\- crycogA.Sg

end cofq"'t'r

FIGURE 15-31 Branchsynthesisin glycogen.The glycogen-branchingenzyme(alsocalledamylo (1+4)

to (1-+6) transglycosylase, forms a new branch point during glycogen
or glycosyl-(4-+6){ransferase)
synthesis.

equilibrium of the path from glucose 6-phosphateto glucoseresidues,each derived from UDP-glucose;the
glycogenlengthenedby one glucoseunit greatly favors reactions are catalyzedby the chain-extendingactivity
synthesisof glycogen. of glycogenin.At this point, glycogensynthasetakes
Glycogensynthasecannotmakethe (a1-+6) bonds over, further extending the glycogenchain. Glycogenin
found at the branch points of glycogen; these are remains buried within the B-particle, covalently at-
formed by the glycogen-branchingenzyme,also called tachedto the singlereducingend of the glycogenmol-
amylo (l-+4) to (1-+6) transglyeosylase, or glyco- ecule(Fig. 15-33b).
syl-(4-+6)-transferase.The glycogen-branchingen-
zyme catalyzestransfer of a terminal fragment of 6 or 7
glucoseresiduesfrom the nonreducingend of a glyco-
gen branch having at least 11 residuesto the C-6 hy-
droxyl group of a glucoseresidue at a more interior
position of the same or another glycogenchain, thus
creatinga new branch (Fig. 154f ). Further glucose
residuesmay be addedto the new branch by glycogen
slmthase.The biologicaleffect of branchingis to make
the glycogenmoleculemore solubleand to increasethe
number of nonreducingends.This increasesthe num-
ber of sites accessibleto glycogenphosphorylaseand
glycogensSmthase, both of which act only at nonreduc-
ing ends.

Glycogenin
Primes
thelnitialSugar
Residues
inGlycogen
Glycogen synthase cannot initiate a new glycogen
chain de novo. It requires a primer, usually a pre-
formed (a1-+4) polyglucosechain or branch havingat
Ieasteight glucoseresidues.So,how is anew glycogen
moleculeinitiated?The intriguing protein glycogenin
(Fig. 15-32) is both the primer on which new chains
are assembledand the enzy'rnethat catalyzesthef as-
sembly.The first step in the synthesisof a new glyco-
gen moleculeis the transfer of a glucoseresiduefrom
UDP-glucoseto the hydroxyl group of TVrt" of glyco-
genin, catalyzed by the protein's intrinsic glucosyl-
transferaseactivity (Fig. 15-33). The nascentchain
is extendedby the sequentialaddition of sevenmore
FIGURE 15-32 Glycogeninstructure.(PDB1D 1LL2)Muscleglyco-
genin(M, 37,000)formsdimersin solution.Humanshavea second
isoformin liver,glycogenin-2. Thesubstrate, UDP-glucose (shownasa
red ball-and-stickstructure), is bound to a Rossmann fold near the
from the Tyrlea residues
amino terminus and is some distance
(turquoise)-15 A from theTyrin the samemonomer,12 A from theTyr
in the dimericpartner.EachUDP-glucose is boundthroughits phos-
phatesto a Mn2* ion (green)that is essentialto catalysis.Mn2* is be-
lievedto functionasan electron-pairacceptor(Lewisacid)to stabilize
the leavinggroup, UDP.The glycosidicbond in the producthas the
sameconfiguration aboutthe C-1 of glucoseasthe substrate UDP-glu-
cose,suggesting thatthe transferof glucosefrom UDP toTyrreaoccurs
in two steps.The firststepis probablya nucleophilicattackby Aspr62
(orange),forming a temporaryintermediatewith invertedconfigura-
tion. A secondnucleophilicattackbyTyrreathen restoresthe starting
configuration.
Iuul P r i n c i p loef sM e t a b o R
l i ce g u l a t i 0 n

(a) (b)

Glycogenin

UDP-glucose -o-P-o-P-o-
llr
d o--@-lE;EIl

UDP-glucose

cH2oH

G glycogenin third tier

I primer fourth tier

outer tier
secono[rer (unbrancned)

FIGURE 15-33 Glycogeninand the structureofthe glycogenparticle.

(a)Clycogenin catalyzes two distinctreactions.
Initialattackby the hy-
droxylgroupolTyrleaon C-l of the glucosylmoietyof UDP-glucose
resultsin a glucosylatedTyrresidue.TheC-1 of anotherUDP-glucose
moleculeis now attackedby the C-4 hydroxylgroupof the terminal
glucose,and this sequencerepeatsto form a nascentglycogenmole-
culeof eightglucoseresidues attached by (a1-+4)glycosidiclinkages.
(b) Structureof the glycogenparticle.Startingat a centralglycogenin
molecule,glycogenchains(12 to.l4 residues) extendin tiers.Inner
chainshavetwo (a1--+6) branches each.Chainsin theoutertierareun-
Repeatssix times branched. Thereare 12 tiersin a matureglycogenparticle(only5 are
shownhere),consisting of about55,000glucoseresidues in a mole-
culeof about21 nm diameterandM. -1 x 1O7.

SUMMAR
1Y5 . 4 T h eM e t a b o l i somf
G l y c o g ei n A n i m a l s
r Glycogenis storedin muscleand liver as large
particles.Containedwithin the particlesare the
enzyrnesthat metabolizeglycogen,aswell as
regulatory enzyrnes.
r Glycogenphosphorylase catalyzesphosphorol;,tic
cleavageat the nonreducingends of glycogen
chains,producingglucose1-phosphate.
The
debranchingenzyrnetransfersbranchesonto main
chainsand releasesthe residueat the (a1-+6)
branchas free giucose.
r Phosphoglucomutaseinterconvertsglucose
1-phosphateand glucose6-phosphate.Glucose
6-phosphatecan enter glycolysisor, in liver,
can be convertedto free glucoseby glucose
6-phosphatase in the endoplasmicreticulum,
then releasedto replenishblood glucose.
r The sugarnucleotideUDP-glucose donatesglucose
residuesto the nonreducingend of glycogenin
the reaction catalyzedby glycogensynthase.A
separatebranchingenzyrneproducesthe (a1-+6)
linkagesat branch points.
r New glycogenparticles begin with the autocataly'tic
formation of a glycosidicbond betweenthe glucose
of UDP-glucose and a Tlr residuein the protein
glycogenin,followed by addition of severalglucose
residuesto form a primer that can be actedon by
glycogens;.'rLthase.
15.5Coordinated
Regulation
ofGlycogen
Synthesis
andBreakdown
As we haveseen,the mobilizationof storedglycogenis
brought about by glycogenphosphorylase,which de-
gradesglycogento glucosel-phosphate (Fig. 15-25).

Glycogenphosphorylaseprovides an especiallyinstruc-
tive case of erzyme regulation. It was one of the first
knovm examplesof an allostericallyregulated enzyrne
and the first enz;.rneshownto be controlledby reversible
phosphorylation.It was alsoone of the first ailostericen-
zyrnesfor which the detailed three-dimensionalstruc-
tures of the active and inactive forms were revealedby
x-ray crystallographicstudies. Glycogenphosphorylase
is also another illustration of how isoz}.'rnesplay their
tissue-speciicroles.
Glycogen
Phosphorylase
lsRegulated
Allosterically
andHormonally
In the late 1930s, Carl and
Gerty Cori (Box 15-4) discov-
ered that the glycogenphos-
phorylase of skeletal muscle
exists in two interconvertible
forms: glycogen phosphory-
lase a, which is cataly'tically
active, and glyeogen phos-
phorylase b, which is less ac-
tive (Fig. f5+4). Subsequent
studies by Earl Sutherland
EarlW. Sutherland,
Jr., showed that phosphorylaseb
1915-1974 predominatesin resting mus-
cle, but during vigorousmus-
cular actMty epinephrinetriggers phosphorylationof a
speci-flcSer residuein phosphorylase b, converthg it to
its more active form, phosphorylasea. (Note that glyco-
gen phosphorylaseis often referred to simply as
phosphorylase-so honored becauseit was the first
phosphoryIaseto be discovered;the shortenedname
has persistedin commonusageand in the literature.)
The enzyme (phosphorylaseb kinase) responsi-
ble for activatingphosphorylaseby transferring a phos-
phoryl group to its Ser residue is itself activated by
Ser14 OH OH
side
chain QHz 9Hz
Phosphorylaseb
(lessactive)
p h o s p h o rr l a s c o phosphorvlasc6
phosphatasc klnase
t,t)1,
o / ,o
cr-Iz 9H,
Phosphorylaseo
(active)
ofGlycogen
Regulation
15.5Coordinated andBreakdown
Synthesis I 60
epinephrineor glucagonthrough a seriesof stepsshovm
in Figure 15-35. Sutherland discoveredthe second
messengercAMP,which increasesin concentrationin
responseto stimulation by epinephrine (in muscle) or
glucagon(in liver). Elevated [cAMP] initiates an en-
zJ'rnecascade, in which a catalyst activatesa catalyst,
which activatesa catalyst(seeSection12.1).Suchcas-
cades allow for Iarge amph-flcationof the initial signal
(seepink boxesin Fig. 15-35). The rise in [cAMP]acti-
vates cAMP-dependent protein kinase,also calledpro-
tein kinase A (PKA). PKA then phosphorylatesand
activatesphosphorylase b kinase, which catalyzesthe
phosphorylationof Ser residuesin eachof the two iden-
tical subunits of glycogen phosphorylase,activating it
and thus stimulating glycogen breakdown. In muscle,
this provides fuel for glycolysisto sustain muscle con-
traction for the flght-or-flight responsesignaledby epi-
nephrine.In liver, glycogenbreakdowncountersthe low
blood glucosesignaledby glucagon,releasingglucose.
These different roles are reflected in subtle differences
in the regulatory mechanismsin muscle and liver. The
glycogen phosphorylasesof liver and muscle are
isozl'rnes,encoded by different genes and differing in
their regulatoryproperties.
In muscle,superimposedon the regulationof phos-
phorylaseby covalentmodification are two allosteric
control mechanisms(FiS. 15-35).Ca'*, the signalfor
muscle contraction, binds to and activates phosphory-
Iaseb kinase,promotingconversionof phosphorylase b
to the active a form. Ca2+binds to phosphorylaseb
kinase through its 6 subunit, which is calmodulin (see
Fig. 12-11).AMP,which accumulatesin vigorouslycon-
tracting muscle as a result of ATP breakdown,binds to
and activatesphosphorylase,speedingthe releaseof
glucosel-phosphatefrom glycogen.When ATP levels
are adequate,ATP blocks the allostericsite to which
AMP binds, inactivatingphosphorylase.
Whenthe musclereturns to rest, a secondenzlirne,
phosphorylase a phosphatase, also called phospho-
protein phosphatase I (PPl), removesthe phospho-
Serl4
side ryl groups from phosphorylaseo, converting it to the
chain Iessactive form, phosphorylaseb.
Like the enzyme of muscle, the glycogen phos-
phorylase of liver is regulated hormonally (by phos-
phorylation/dephosphorylation)and allosterically.The
dephosphorylatedform is essentiallyinactive.Whenthe
glucagon
-. (liver)
blood glucoselevel is too low, glucagon(acting through
the cascademechanismshownin Fig. 15-35) activates
epinephrine, FIGURE 15-34 Regulationof muscleglycogenphosphorylase by cova-
--f tc^'-l,
ttAMPl
(muscle) lent modification.In the moreactiveform of the enzyme/phosphory-
lasea, Serla residues,one on each subunit,are phosphorylated'
Phosphorylase a is convertedto the lessactiveform, phosphorylaseb,
by enzymaticlossof thesephosphorylgroups,catalyzedby phospho-
rylasea phosphatase (alsoknown as phosphoprotein phosphatase 1,
PPi). Phosphorylase b can be reconverted (reactivated)
to phosphory-
lasea by the actionof phosphorylase b kinase,(SeealsoFig.6-36 on
glycogen phosphorylase regulation.)
L604l Principles
0f Metab0lic
Regulation

FIGURE 15-35 Cascademechanismof epinephrineand -.,.

t.-. -.. - -.-- - - _ t- - _
Epinephrine i*moleculesl
---"-*--*-r G gon
glucagonaction. By binding to specificsurfacereceptors, Myocyte | Hepatocyte
either epinephrineacting on a myocyte(left)or glucagon ruIl l
I
actingon a hepatocyte(right)activatesa CTP-bindingpro- I

tein G,o (seeFig.12-4).ActiveC.o triggersa risein [cAMP], tl

I
activatingPKA.Thissetsoff a cascadeof phosphorylations;
PKAactivatesphosphorylase b kinase,which then activates I
I
glycogenphosphorylase. Suchcascadeseffecta largeam-
plificationof the initialsignal;the figuresin pink boxesare
probablylow estimatesof the actualincreasein numberof ATP Cyclic AMP
moleculesat eachstageof the cascade. Theresultingbreak- izii;i:t:@l
down of glycogenprovidesglucose,which in the myocyte
can supplyATP(via glycolysis)for musclecontractionand
Inactive PKA Active PKA
in the hepatocyteis releasedinto the blood to counterthe
low blood glucose.
Active
phosphorylase b
--- kinase
; : ^ ^ molegules
, ; ,
iluujr I
Inactive Active
glycogen glycogen
phosphorylaseb phosphorylase o
-----l
I f--' -^ -' ^ -"--

IIAMP] t 11,000s molecules j

r. :--.-"......"....."
I

Glycogen Glucose1-phosphate

.t
Glucose

.1,
Blood glucose

phosphorylaseb kinase, which h turn converts phos-
phorylaseb to its active o form, initiating the releaseof
glucoseinto the blood.Whenblood glucoselevelsreturn
to normal, glucose enters hepatocytesand binds to an
hhibitory allosteric site on phosphorylased. This bind-
ing alsoproducesa conformationalchangethat exposes
the phosphorylatedSer residues to PPl, which cat-
alyzestheir dephosphorylationand inactivatesthe phos-
phorylase(Fig. f5-36). The allostericsite for glucose
allows liver glycogen phosphorylaseto act as its own
glucosesensorand to respondappropriatelyto changes
in bloodglucose.

o O Insulin
I Ir
QHz c

phosphorylase o
phosphatase
(PP1)

sites empty

FIGURE 15-35 Glycogenphosphorylase of liver as a glucosesensor. phosphatase convertsphosphorylase a to phosphorylase

b, sharplyre-
Clucosebindingto an allosteric
siteof the phosphorylase a isozymeol ducingthe activityof phosphorylase
and slowingglycogenbreakdown
liverinducesa conformationalchangethatexposesits phosphorylated in responseto highbloodglucose.lnsulinalsoactsindirectlyto stim-
Serresiduesto the actionof phosphorylase a phosphatase (ppl). This ulatePPl and slow glycogenbreakdown.
 t eedg u l a t ioofn
1 5 . 5C o o r d i n aR e ynn t h e a
G l y c o gS s insdB r e a k d 0 w[ n
ttt]

Glycogen
Synthase
lsAlso byPhosphorylation which addsphosphorylgroupsto tluee Ser residuesnear
Regulated
andDephnsphorylation the carboxylterminusof glycogens),nthase,
stronglyinac-
tivating it. The action of GSK3is hierarchical;it carmot
Like glycogenphosphorylase,glycogen synthasecan phosphorylateglycogensynthaseuntil anotherprotein ki-
exist in phosphorylatedand dephosphorylatedforms nase,casein kinase II (CKII), hasfirst phosphorylated
(Fig. 15-:37).Its activeform, glycogen s5,'nthasea, is the glycogens),Trthase on a nearbyresidue,an eventcalled
unphosphorylated.Phosphorylationof the hydroxyl priming (FiS. 15-38a).
side chainsof severalSer residuesof both subunitscon- In liver,conversionofglycogensynthaseb to the ac-
verts giycogen synthase a to glycogen synthase b, tive form is promoted by PP1, which is bound to the
which is iractive unless its allosteric activator,glucose glycogenparticle.PPl removesthe phosphorylgroups
6-phosphate,is present.Glycogensyrrthaseis remarkable
for its ability to be phosphorylatedon variousresiduesby
at least 11 different protein kinases The most rmportant
I
regulatorykinaseis glycogen s5mtlnse kinase 3 (GSK3), I
I

3ADP 3AIPlo"
Phosphoserines (}SK3
FIGURE 15-37 Effectsof GSK3on glycogensynthase activity.C lycogen near carboxyl
synthase a, the activeform, hasthreeSerresiduesnearitscarboxylter- terminus

minus,whicharephosphorylated by glycogen synthase kinase3 (CSK3)

Thisconvertsglycogensynthase to the inactive(b)form CSK3actionre- I

quiresprior phosphorylation (priming)by caseinkinase(CKll) lnsulin

triggersactivationof glycogensynthaseb by blockingthe activityof
.12-1
CSK3(seethe pathwayfor this actionin Fig. 6) and activating a
phosphoprotein (PP1
phosphatase in muscle,anotherphosphatase rn lnactive

liver).In muscle,epinephrine activates PKA,whichphosphorylates the

glycogen-targetrng proteinCv (seeFig.15-40)on a sitethatcausesdis-
sociationof PPl from glycogen Clucose6-phosphate favorsdephos-
phorylationof glycogensynthase by bindingto it and promotinga
conformationthat is a good substrate for PP.l. Clucosealso promotes
dephosphorylation; thebindingof glucose to glycogen phosphorylase a
forcesa conformational changethatfavorsdephosphorylation to glyco-
genphosphorylase b, thusrelievingitsinhibition of PP.l(seeFig 15-39)

Active site

Priming site

ATP
o- phosphorylated
-o-P:o kinase II
H i
o o
lycogen
synthase

Ser residues
phosphorylatedin
(a) glycogensynthase (b) Pseudosubstrate
Pseudosubstrate

tIGURE 15-38 Primingof GSK3phosphorylation of glycogensynthase.
(a) Clycogensynthase
kinase3 firstassociates (glyco-
with its substrate
gen synthase) by interactionbetweenthreepositivelychargedresidues
1Arge6,Arg180,Lys2os)anda phosphoserine residueat position+4 in the
substrate(Fororientation,theSerorThrresidue to be phosphorylated in
the substrateis assignedthe index0 Residues on the amino{erminal
sideof thisresidue arenumbered -1 , -2, andsoforth;residues on the
carboxyl{erminal sidearenumbered +1, +2, andsoforth)Thisassoci-
ationalignstheactivesiteof theenzymewith a Serresidue at position0,
which it phosphorylates Thiscreatesa new primingsite,and the en-
zymemovesdown the proteinto phosphorylate the Serresidueat posi-
tion -4, and then the Serat -8. (b) CSK3 has a Serresiduenearits
amino terminusthat can be phosphorylated by PKA or PKB(seeFig.
15-39) Thisproducesa "pseudosubstrate" regionin CSK3thatfoldsinto
theprimingsiteandmakestheactivesiteinaccessible to anotherprotein
substrate, inhibitingCSK3until the primingphosphoryl groupof its
pseudosubstrate regionis removedby PP1. Otherproteinsthataresub-
stratesfor CSK3alsohavea primingsiteat position+4, which mustbe
phosphorylated by anotherproteinkinasebeforeCSK3canacton them
(SeealsoFigs6-37 and12J2b on glycogensynthase regulation.)
 f l
606 Principles
ofMetabolic
Regulation
from the three Ser residuesphosphorylatedby GSK3.
Glucose6-phosphatebindsto an allostericsite on glyco-
gen synthaseb, making the enz}.'rnea better substrate
for dephosphorylationby PPI and causingits activation.
By analogywith glycogenphosphorylase, which acts as
a glucosesensor,glycogenslmthasecan be regardedas
a glucose 6-phosphatesensor.In muscle, a different
phosphatasemay have the role played by PPl in liver,
activatingglycogensynthaseby dephosphorylatingit.
Glycogen
Synthase
Kinase
3Mediates
5ome
ofthe
Artions
ofInsulin
As we saw in Chapter 12, oneway in which insulin trig-
gers intracellular changesis by activating a protein ki-
nase [PKB) that in turn phosphorylatesand inactivates
GSK3(Fig. 15-39; seealsoFig. 12-16).Phosphoryla-
tion of a Ser residuenear the aminoterminus of GSK3
convertsthat regionof the proteinto a pseudosubstrate,
which folds into the site at which the priming phospho-
rylated Ser residuenormallybinds (Fig. 15-38b). This
prevents GSK3 from binding the priming site of a real
substrate,thereby inactivating the enzymeand tipping
the balancein favor of dephosphorylationof glycogen
synthaseby PPl. Glycogenphosphorylasecan also af-
fect the phosphorylationof glycogensynthase:active
glycogenphosphorylasedirectly inhibits PPl, prevent-
ing it from activatingglycogens;mthase(Fig. 15-37).
Although flrst discoveredin its role in glycogenme-
tabolism (hence the name glycogenslmthasekinase),
GSK3 clearly has a much broader role than the regula-
tion of glycogen synthase.It mediates signalingby in-
sulin and other growth factors and nutrients, and it acts
in the specificationof cell fates during embryonicdevel-
opment.Amongits targetsare cytoskeletalproteinsand
proteins essential for mRNA and protein synthesis.
These targets, Iike glycogensynthase,must first un-
FIGURE 15-39 The path from insulinto GSK3and
glycogensynthase.Insulinbindingto its receptor
activatesa tyrosineprotein kinasein the receptor,
which phosphorylates insulinreceptorsubstrate-1
(lRS-1). The phosphotyrosine in this proteinis then
b o u n d b y p h o s p h a t i d y l i n o s i3t o- kl i n a s e( p t - 3 K ) ,
which convertsphosphatidylinosirol
phate(PlP2)in the membrane to phosphatidylinosi-
tol 3,4,5-trisphosphate (PlP3).A protein kinase
(PDK-I)that is activatedwhen boundto plp, acti-
vatesa secondproteinkinase(pKB),which phos-
phorylates glycogensynthase kinase3 (CSK3)in its
pseudosubstrate region,inactivating it by the mech-
anismsshownin Figure15-38b.The inactivation
CSK3allowsphosphoprotein phosphatase
dephosphorylate and thus activateglycogensyn-
thase In thisway,insulinstimulates glycogensynthe-
sis (SeeFig.12-16for moredetails on insulinaction.)
dergo a priming phosphorylationby another protein ki-
nasebeforethey canbe phosphorylated by GSK3.
Phosphoprotein 1ls(entral
Phosphatase to
Glyrogen
Metabolism
A single enzyme,PPl, can removephosphorylgroups
from all three of the enzymesphosphorylatedin re-
sponseto glucagon (liver) and epinephrrne (liver and
muscle): phosphorylasekinase, glycogenphosphory-
lase,and glycogensyrrthase.Insulin stimulatesglycogen
synthesisby activating PP1 and by inactivating GSK3.
Phosphoproteinphosphatase1 doesnot exist free in
the cytosol,but is tightlybourd to its targetproteinsby one
of a family of glycogen-taxgeting proteins that bind
$ycogen and each of the tlree enzymes,$ycogen phos-
phorylase,phosphorylasekinase,and $ycogen synthase
(Fig. 15-40). PPI is itsel-fsubject to covalentand al-
lostericregulation:it is inactivatedwhen phosphorylatedby
PKA and is allostericallyactivatedby gucose6-phosphate.
(arbohydrate
Allosteric
dndll0rm0nal
5ignals
Coordinate
Metabolism
Globally
Having looked at the mechanismsthat regulate individ-
ual enz;.'rnes,
we can now consider the overall shifts in
carbohydratemetabolismthat occur in the well-fed
state,during fasting,and in the flght-or-flightresponse-
signaledby insulin, glucagon,and epinephrine,respec-
tively. We need to contrasttwo casesin which regulation
servesdifferent ends:(1) the role of hepatocytesin sup-
plying glucoseto the blood,and (2) the selfishuse of car-
bohydratefuelsby nonhepatictissues,typiied by skeletal
muscle(myoc1'tes),to supporttheir own activities.
After ingestionof a carbohydrate-richmeal,the ele-
vation of blood glucose triggers insulin release
(FiS. 15-41, top). In a hepatocy'te,insulin hastwo imme-
Insulin
4,S-bisphos-
of
1 (PP.l )ro
 1 5 . 5C o o r d i n aR G l y c o g5eynn t h e sainsdB r e a k d o w[na t l
t eedg u l a t ioof n

FIGUREl5-40 Glycogen-targetingprotein G,u. The

glycogentargeting proteinCv isoneof a familyof proteins
that bind otherproteins(includingPP1)to glycogenpafti-
cles.Cu can be phosphorylated at two differentsitesin re-
sponseto insulinor epinephrine. @ Insulin-stimulated
phosphorylation of Cv site1 activatesPPl, whichdephos-
phorylates phosphorylase kinase,glycogenphosphorylase,
and glycogensynthase(not shown). @ Epinephrine-
stimulated phosphorylation of Cv site 2 causesdissoci-
ation of PP.lfrom the glycogenparticle,preventingits
accessto glycogenphosphorylase and glycogensyn-
thase.PKA also phosphorylates a protein(inhibitor1)
that,when phosphorylated, inhibitsPP.l By thesemeans,
insulin inhibits glycogenbreakdownand stimulates
glycogensynthesis, and epinephrine (or glucagonin the
liver)hasthe oppositeeffects.

High blood
glucose

llnsulin GLUT2

,,,
f Insulin-sensitive lPKB Synthesis of
"
protein kinase hexokinase II,
PFK-I, pyruvate ,
kinase ,u
,IPPl ,l.GSK-3 ' t lclucosel6"i6"

JPhosphorylase t Glycogen
kinase synthase
'f Glycogen
phosphorylase

lGlycogen
breakdown

tGlycogen
breakdown
+I
t Glycogen
phosphorylase

tI
I
I
l
tPhosphorylase
kinase

FIGURE 15-41 Regulationof carbohydratemetabolism

in the liver.Arrowsindicatecausalrelationshios
between
the changesthey connect.Forexample,an arrowfrom JA
to 18 meansthata decrease in A causesan increase
in B.
Pink arrowsconnecteventsthat resultfrom high blood
glucose;blue arrowsconnecteventsthat resultfrom lol.
bloodglucose.
fI
608 Principles
ofMetabolic
Regulation

diate effects:it inactivatesGSK3,actingthrough the cas- Epinephrine

II
cadeshownin Figure 15-39,and activatesa protein phos- Glucagon
phatase,perhaps PPl. These two actions fully activate
glycogens;mthase.PPl also inactivatesglycogenphos-
phorylasea and phosphorylasekinaseby dephosphory-
+
Liver Muscle

lating both, effectively stopping glycogen breakdown.

Glucoseentersthe hepatocy[ethrough the high-capacity
transporter GLUT2,alwayspresent in the plasmamem-
brane,and the elevatedintracellularglucoseIeadsto drs-
sociationof hexokinaseIV (glucokinase)from its nuclear
regulatoryprotein(Fig. 15-13).Hexokinase IVentersthe
c;4osoland phosphorylatesglucose,stimulatingglycoly-
sis and supplyingthe precursor for glycogensy'nthesis.
Undertheseconditions,hepatocytesuse the excessglu-
cosein the bloodto sy'nthesize glycogen,up to the limit of
about 10%of the total weight of the liver.
Betweenmeals,or during an extendedfast, the drop
in blood glucosetriggers the releaseof glucagon,which,
acting through the cascadeshown in Figure 15-35,
activatesPKA. PKA mediatesall the effects of glucagon
(Fig. 15-41, bottom). It phosphorylatesphosphorylase
kinase,activatingit and leadng to the activationof $yco-
gen phosphorylase.It phosphorylatesglycogens;mthase,
inactivatingit and blocking glycogensyrrthesisIt phos-
phorylatesPFK-2/FBPase-2, leadingto a drop in the con-
cen- tration of the regulatorfructose2,6-bisphosphate,
which hasthe effect of inactivatingthe glycolyticenzyrne
PFK-I and activatingthe gluconeogenicenzyrneFBPase-
1.And it phosphorylatesand inactivatesthe glycolltic en-
zyrnep),'ruvatekinase.Under these conditions,the liver
producesglucose6-phosphateby glycogenbreakdown
and by gluconeogenesis, and it stopsusing$ucose to fuel
glycolysisor make $ycogen, maximizingthe amount of
glucoseit canreleaseto the blood.Thisreleaseofglucose
is possibleonly in liver and kidney,becauseother tissues
lack glucose6-phosphatase(Fig. 15-28).
The physiologyof skeletalmuscle differs from that
of liver in three ways important to our discussionof
metabolicregulation(Fig. 15-42): (1) muscleusesits
stored glycogenonly for its own needs; (2) as it goes
from rest to vigorous contraction,muscle undergoes
very large changes in its demand for ATP, which is
supportedby glycolysis;(3) musclelacks the enz}..rnatic
machineryfor gluconeogenesis. The regulationof carbo-
hydratemetabolismin musclereflectsthesedifferences
from liver. First, myocy[eslack receptorsfor glucagon.
Second,the muscleisozymeof pyruvatekinaseis not
phosphorylatedby PKA, so glycolysisis not turned off
when [cAMP]is high. In fact, c.\IVP,increosesthe rate of
glycolysisin muscle, probably by activatingglycogen
phosphorylase. When epinephrineis releasedinto the
blood in a flght-or-flight situation, PKA is activated by
the rise in [cAMP] and phosphorylatesand activates
glycogenphosphorylase kinase.The resultingphospho-
rylation and activation of glycogen phosphorylasere-
sults in faster glycogenbreakdown.Epinephrine is not
releasedunder low-stress conditions,but with each
Glycogen Glycogen
t tGlycogenolysisf I
Y Y
Blood - Glucose Glucose
glucose - 6-phosphate 6-phosphate
A {Glycolysist I
I lGluconeogenesis V
Pyruvate Pyruvate
FIGURE 15-42 Differencein the regulationof carbohydratemetabo-
lism in liver and muscle.In liver,eitherglucagon(indicatinglow
bloodglucose) or epinephrine(signaling
the needto fightor flee)has
the effectof maximizingthe outputof glucoseinto the bloodstream.
ln muscle,epinephrine increasesglycogenbreakdown andglycolysis,
which togetherprovidefuel to producethe ATPneededfor muscle
contraction
neuronal stimulation of muscle contraction, cytosolic
rises briefly and activates phosphorylase kinase
[Ca'*]
through its calmodulinsubunit
Elevatedinsulin triggersincreasedglycogensynthe-
sisin myocytesby activatingPPi and inactivatingGSK3.
Unlike hepatocytes,myocyteshavea reserveof GLUT4
sequesteredin rntracellularvesicles.Insultntriggerstheir
movement to the plasma membrane (see Fig. 12-16),
wherethey allowincreasedglucoseuptake.In response
to insulin, therefore, myocytes help to lower blood
glucose by increasingtheir rates of glucose uptake,
glycogens;mthesis,and glycolysis.
(arbohydrate
and[ipidMetabolism
Arelntegrated
by
Hormonal
andAllosteric
Mechanisms
As complexas the reguiationof carbohydratemetabo-
lism is, it is far from the whole story of fuel metabolism.
The metabolism of fats and fatty acids is very closely
tied to that of carbohydratesHormonalsignalssuch as
insulin and changesin diet or exerciseare equally im-
portant in regulating fat metabolismand integrating it
with that of carbohydrates.We return to this overall
metabolic integration h mammalsin Chapter 23, after
first consideringthe metabolicpathwaysfor fats and
aminoacids(Chapters17and 18).The message we wish
to convey here is that metabolic pathwaysare overlaid
wrth complex regulatory controls that are exquisitely
sensitiveto changesin metaboliccircumstances. These
mechanismsact to adjust the flow of metabolites
through various metabolic pathways,as needed by the
cell and organism,and to do so without causingmajor
changesin the concentrationsof intermediatesshared
with other pathways
 .fI
F u r t h eRre a d i n g 6 0 9 ]

SUMMAR
1Y5 . 5 C o o r d i n a t eRde g u l a t i o n fructose2,6-bisphosphatase UDP-glucose
(FBPase-2) 588 pyrophosphorylase 600
o f G l y c o g eSny n l h e s i s amylo (1-+4) to (1-+6)
carbohydrateresponse
a n dB r e a k d o w n elementbindingprotein trans$ycosylase 601
r Glycogenphosphorylaseis activatedin responseto (chREBP) 591 glycogenin 601
glucagonor epinephrine,which raise [cAMP]and ctornl raqnnnqp glycogen
activatePKA. PKA phosphorylatesand activates alemont hindino phosphorylaseo 603
phosphorylasekinase,which convertsglycogen protein (SREBP) 592 glycogen
phosphorylaseb to its activeo form. Phosphoprotein cyclic AMP response phosphorylaseb 603
phosphatase1 (PPl) reversesthe phosphorylation elementbinding enzyme cascade 603
of glycogenphosphorylaseo, inactivatingit. Glucose protein (CREB) 592 phosphorylaseb kinase 603
of glycogenphosphorylase
binds to the liver isozSrme forkheadbox other phosphoprotein
a, favoringits dephosphorylationand inactivation. (FOXO1) 592 phosphatase1 (PPI) 603
glycogenolysis 595 glycogens5mthasea 605
r Glycogensynthaseo is inactivatedby
glycogenesis 595 glycogenslrrthaseb 605
phosphorylationcatalyzedby GSK3.Insulin
glucose1-phosphate 595 glycogensynthasekinase3
blocksGSK3.PP1,which is activatedby insulin,
debranchingenzlrne 596 (GSK3) 605
reversesthe inhibition by dephosphorylating
oligo (a1-+6) to (a1-+4) caseinkinaseII (CKn) 605
glycogensynthaseb.
glucantransferase 596 priming 605
r Insulinincreasesglucoseuptakeinto myocytesand phosphoglucomutase596 glycogen-targeting
adipoc;,tesby triggering movementof the glucose sugarnucleotides 596 proteins 606
transporterGLUT4to the plasmamembrane. freeze-clamping 611
r Insulin stimulatesthe synthesisof hexokinases
II and IV, PFK-I, pyruvate kinase,and several
enzyrnesinvolvedin lipid synthesis.Insulin Further
Reading
stimulatesglycogensynthesisin muscleand liver.
Regulation of Metabolic Pathways
r In liver, glucagonstimulatesglycogenbreakdov,n
Desvergne, 8., Michalik, L., & Wahli, W. (2006) T[anscriptional
and gluconeogenesis while blockingglycolysis, regulationof metabolism.Physi,ol Reu. 86, 465-514
thereby sparingglucosefor export to the brain Advancedand comprehensivereview.
and other tissues. Gibson, D. & Harris, R.A, (2001)Metabolic Regulati'on i'n
MammaLs,Taylor & Francis,New York.
r In muscle,epinephrinestimulatesglycogen
Excellent,readableaccountof metabolicregulation.
breakdownand glycolysis,providing ATP to
Storey, K.B, (ed.). (2004)Functinnal Metabolism: Regulati'on
supportcontraction. and Adaptati,on, Wley-Liss,Inc., Hoboken,NJ.
Excelient discussionof the principles of metabolicregulation,
signaltransduction,transcriptionalcontrol, and energymetabolismin
KeyTerms health and disease.

Tenns i,n bold are defined i,n th,eglossarg
glucose6-phosphate569
flux 57\
homeostasis 57I
eellular
differentiation 571
transcriptionfactor 57I
response element 571
turnover 572
transcriptome 572
proteome 572
metabolome 573
metabolicregulation 574
metabolic control 574
mass action
ratio, @ 574
adenylatekinase 576
AMP-activatedprotein
kinase (AMPK) 576
Analysis of Metabolic Control
Fell, D.A. (1992) Metaboliccontrol analysis:a surveyofits theoreti
flux control cal and experimentaldevelopmentBi'ochem.J 286, 313-330
coefflcient,C 578 Clearstatementof the principiesof metaboliccontrol analysis.
flux,J 578 Fell, D.A, (1997) Und,erstand'i'ngthe Control oJMetaboli'sm,Port-
elasticity land Press,Ltd , London
coefflcient,e 580 An excellent,clear expositionof metabolicregulation,from the
If you read only one treat-
point of view of metaboliccontrol ana"lysis.
responsecoefflcient,-B 581
ment on metaboliccontrol analysis,this shouldbe it.
gluconeogenesis 582
Heinrich, R. & Rapoport, T.A' (1974) A linear steady-statetreat-
futile cycle 583 ment of enzjrmaticchains:generalproperties,controi and effector
substratecycle 583 strength Eur J. Bi,ochem 42,89-95.
hexokinaseII 583 Early statementof principlesof metabolc control analysis.See
hexokinaseI 583 alsothe paperby Kacser& Burns,Iisted below
hexokinaseIV 584 Jeffrey, F.M.H., Rqiagopal, A., Maloy, C.R.' & Sherry' A.D.
(1991) r3C-NMR:a simpleyet comprehensivemethod for analysisof
GLUTz 584
intermediarymetabolism.Ttends Bi'ochem Sci'. 16, 5-10.
glucagon 587
Brief, intermediatelevel review.
fructose
Kacse4 H. & Burns, J.A' (1973) The control of flux. Sgm'p Soc
2,6-bisphosphate587 Erp 8i,o1.32,65-104.
phosphofructokinase-2 A classicpaper in the field. Seealsothe paper by Heinrich &
(PFK-2) 588 Rapoport,listed above
 10 l P r i n c i p l eo sf M e t a b o l R
i ce g u l a t i o n
Kacser, H., Burns, J.A., & Fell, D.A. (1995) The control of flux:
21 yearson.Bi,ochemSoc Tlans.z8, 341-366.
Schilling, C,H., Schuster,S., Palsson, B.O., & Heinrich, R.
(1999) Metabolicpathwayanalysis:basic conceptsand scientiflcap-
plicationsin the post-genomicera.Biotech:nol Prog 15, 296-303.
Short, advanceddiscussionof theoreticaltreatmentsthat attempt
to find ways of manipulatingmetabolismto optimizethe formation of
metabolicproducts.
Schuster, S., Fell, D.A., & Dandekar, T. (2000) A generaldefini-
tion of metabolicpathwaysuseful for systematicorganizationand
analysisof complexmetabolicnetworks Nat Bi,otechnol.18,
326-332
An interestingand provocativeanalysisof the interplay between
the pentosephosphatepathwayand glycolysis,from a theoretical
standpoint.
Westerhoff, H,V., Hofmey'r, J.-H.S., & Kholodenko, B.N. (1994)
Getting to the inside of cells using metaboliccontrol analysis.Bzo-
phgs Chem 60,273-283.
Coordinated Regulation of Glycolysis and
Gluconeogenesis
Armoni, M., Harel, C., & Karnieli, E. (2007) T[anscriptional
regulationof the GLUT4gene:from PPAR-gamma and FOXOI to
FFA and inflammation.Tfends Endocrinol Metab 18, 100-107.
Barthel, A,, Schmoll, D., & Unterman, T.G. (2005) FoxO proteins
in insulin action and metabolism.Tiends Endocrino| Metab 16,
183-189.
Intermediate-levelrevrewof the transcriptionfactor'seffectson
carbohydratemetabolism.
Brady, M.J., Pessin, J,8,, & Saltiel, A.B. (1999)Spatialcompart-
mentalizationin the regulationof giucosemetabolismby insulin.
Ttends Endocri,nol Metab 10, 408-413.
Intermediate-1evelreview.
Carling, D. (2004) The AMP-activatedprotein kinasecascade-a
unifying systemfor energycontrol. Tlends Bi,ochem Sci,.29, 78-24
intermediatelevel review of AMPK and its role in energy
metabolism
de la Iglesia, N., Mukhtar, M., Seoane, J., Guinovart, J.J., &
Agius, L. (2000) The role of the regulatoryprotein of glucokinasein
the glucosesensorymechanismof the hepatocyte.J. Bi,ol Chem,
275, 10,597-10,603
Report of the experimentaldeterminationof the flux control
coefflcientsfor glucokinaseand the glucokinaseregulatoryprotein
in hepatocytes
Dean, L. & McEntyre, J. (2004) The GenetzcLandscape oJ
Dza,betes,NationalCenterfor BiotechnologyInformation,
www.ncbi.nim.nih. gov/books/bvfcgi?rid=diabetes.
An excellent,highly readable,downloadablebook (free)
It includesan introduction to diabetes,a history of studiesof dia-
betes,and chapterson the geneticfactorsin IDDM, NIDDM, and
MODY
Desverne,8., Michalik, L., & Wahli, W (2006) Tianscriptional
regulationof metabolism Phgsi,ol Reu 86,465-514
Extensive,advancedreview of transcriptionfactors,including
those that regulatecarbohydrateand fat metabolism
Hardie, D.G. (2007) AMP-activatedprotein kinaseas a drug target
Annu Reu PhaymacoL Tori,col.47, 185-210
Advancedreview,with emphasison the possiblerole of this
enz).rnein t5,peII diabetes.
Herma.n, M.A. & Kahn, B.B. (2006) Glucosetransport and sensing
rn the maintenanceof glucosehomeostasisand metabolicharmony.
J. Clin Inuest 116,1767-1775.
Beautifullyillustrated,intermediate-levelreview.
Hers, H.G. & Van Schaftingen, E. (1982)Fructose2,6-bisphos-
phate 2 yearsa"fterits discovery.Briochem J. 2O6,1-12.
Classicdescriptionof this regulatorymolecuieand its role rn
regulatingcarbohydratemetabolism
Hue, L. & Rider, M.H. (1987)Roleof fructose2,6-bisphosphate in
the control of glycolysisin mammaliantiss;rtesB'inchem.J. 245,
3t3-324
Jorgensen, S.8., Richter, E.A., & IVojtaszewski, J.F.P, (2006)
Role of AMPK in skeletalmusclemetabolicregulationand adaptation
in relationto exercise.J. PhEsioI 674,17-3I
Kahn,8.B., Alquier, T., Carling, D., & Hardie, D.G. (2005)
AMP-activatedprotein kinase:ancient energygaugeprovidesclues
to modern understandingof metabolism.CeLL Metab. L,15-25
Well-illustrated,intermediate-levelreview.
Long, Y.C. & Zierath, J.R. (2006) AMP-activatedprotein kinase
signalingin metabolicregulation.J. Cli,n Inuest l16, 1776-1783.
Advanced,short review of AMPK role in metaboiism,including
data on knockout mice
Nordlie, R.C., Foste4 J.D., & Lange, A.J. (1999)Regulationof
glucoseproduction by the hver.Annu Reu Nutr. f 9, 379-406.
Advancedreview.
Okar, D.A., Manzano, A., Navarro-Sabate, A., Riera, L.,
Bartrons, R., & Lange, A.J. (2001)PFK-2/TBPase-2: makerand
breaker of the essentialbiofactor fructose-2,6-bisphosphaleT?ends
B'inchem Scz.26, 30-35.
Pilkis, S.J. & Granner, D.K. (1992) Molecularphysiologyof the
regulationof hepaticgluconeogenesisand glycolysis.Annu Reu
Physiol.64, 885-909.
Postic, C., Dentin, R., Denechaud,P.-D.,& Girard, J. (2007)
ChREBRa transcriptionalregulatorof glucoseand lipid metabolism.
Annu Reu Nutr 27,179-192.
Advancedreview of the role of transcriptionfactor ChREBPin
carbohydratemetabolism.
Schirmer, T. & Evans, P.R. (1990) Structural basisof the allosteric
behaviorof phosphofructokinase.
Nafzre 343, 740-l4b
Towle, H.C. (2005) Glucoseas a regulator of eukaryoticgenetran-
scription Ttends Endocrinol Metab 16, 489494
Intermedlatelevel review.
Towler, M.C. & Hardie, D.G. (2007) AMP-activatedprotein kinase
in metaboliccontrol and insulin signaling.Ci,rc. Res.100, 328-341.
van Shaftingen, E. & Gerin, I. (2002) The glucose-6-phosphatase
sysLem.
Bi,ochem J. 362, 513-532.
Veech, R.L. (2003) A humble hexosemonophosphatepathway
metaboliteregulatesshort- and long-term control of lipogenesis.
Proc Natl Acad Sci, US,A100,5578-5580
Short review of the work from K. Uyeda'slaboratoryon the role
of xylulose5-phosphatein carbohydrateand fat metabolism;Uyeda's
papersare cited in this rer,rew.
Yamada, K. & Noguchi, T. (i999) Nutrient and hormonairegula-
tion of pyruvatekinasegeneexpression.
Bzochem J. 337, 1-11.
The Metabolism of Glycogen in Animals
Whelan, WJ. (i976) On the origin of primer for glycogensynthesis
Tlends Biochem Sci L, 13-75.
Intermediatereview of the discovery,propertiesand role of
glycogenin
Coordinated Regulation of Glycogen Synthesis
and Breakdown
Aiston, S., Hampson, L., Gomez-Foix, A.M., Guinovart, J.J., &
Agius, L. (2001) Hepatlcglycogensynthestsis highly sensitiveto
phosphorylaseactivity: evidencefrom metaboliccontrol analysis
J. Btol. Chem 276,23,858-23,866.
Jope, R.S. & Johnson, G.V.W.(2004) The glamourand gloom of
glycogenslrrthasekinase-3 Ttends B,iochem Sci, 29, 95-702
Intermediatelevel,well-illustratedreview
 Problems
[utt

Problems 3. Effect of O2 Supply on Glycolytic Rates The regulated

steps ofglycolysis in intact cells can be identifled by studying
l. Measurement of Intracellular Metabolite Concentra- the catabolism of glucose in whole tissues or organs. For ex-
tions Measuring the concentratlons of metabolic intermedlates ample, the glucose consumption by heart muscle can be mea-
in a living cell presents great experimental difflculties-usually a sured by artiflcially circulating blood through an isolated intact
cell must be destroyed before metabolite concentrations can be heart and measurlng the concentration of glucose before and
measured Yet enz5,'rnes catalyze metabolic interconversions very after the blood passes through the heart If the circulating
rapidly, so a conunon problem associated with these t5,pes of blood ls deoxygenated, heart muscle consumes glucose at a
measurements is that the findings reflect not the physiological steady rate When oxygen is added to the blood, the rate of
concentrations of metabolites but lhe equilibrium concentra- glucose consumption drops dramatically, then ls maintained at
tions A reliable experimental technique requires all enzr,.,rne-cat- lhe new, lower rate Explain
alyzed reactions to be instantaneously stopped in the intact
4. Regulation of PFK-I The effect of ATP on the allosteric
tissue so that the metabolic intermediates do not undergo
enzyme PFK-1 is shown below. For a given concentration of
change. This objective is accomplished by rapidly compressing
fructose 6-phosphate, the PFK-1 activity increases with in-
the tlssue between large aluminum plates cooled with liquid m-
creasing concentrations of ATP, but a point is reached beyond
trogen (-190'C), a process called freeze-clamping. After
which increasing the concentration of ATP inhibits the enz;'rne.
freezing, which stops enzyne action ir-rstantly,the tissue is pow-
dered and the enzJ,lnesare inactivated by precipitation with per-
chloric acid. The precipitate is removed by centrifugation, and
[he clear supernatant extract ls analyzed for metabo]ites. To ca1- S80
culate intracellular concentrations, the intracellular volume is
S60
determined from the total water content of the tissue and a
measurement of the extracellular volume .t 40
The intracellular concentratlons of the substrates and
products of the phosphofructokinase-1 reaction ln isolated rat i20
heart tissue are given in the table below.
U
IATP]
Metabolite Concenhation(prvr)- (a) Explain how ATP can be both a substrateand an in-
Fructose6-phosphate 87.0 hibltor of PFK-1 How is the enzyrneregulatedby ATP?
Fructose1,6-bisphosphate 22.0 (b) In what ways is glycolysisregulatedby ATP levels?
ATP 11 , 4 0 0 (c) The inhibition of PFK-1by ATP is diminishedwhen the
ADP 1,320 ADP concentrationis high, as shown in the illustration. How
can this observationbe explained?
Source:FromWilliamson, J R (1965)Glycotytic
controlmechanismsl:
inhibitionof glycolysis
byacetate andpyruvate 5, Cellular Glucose Concentration The concentrationof
perfused
in the isolated, rat
heafi.J. Biol.Chem.240, 2308-2327.
glucosein human blood plasmais maintainedat about 5 mu
*Calculated as rrmol/mLof intracellular
water
The concentrationof free glucoseinside a myocyteis much
(a) CalculateQ, [fructose 1,O-bisphosphate][ADP]/[fruc- lower Why is the concentrationso low in the cell? What hap-
pens to glucoseafter entry into the cell?Glucoseis adminis-
tose 6-phosphatel[ATP], for the PFK-1reactionunderphysio-
tered intravenously as a food source in certain clinical
logicalconditions
(b) Givena AG'ofor the PFK-1reactionof -I4.2 situations.Giventhat the transformationof glucoseto glucose
kJ/mol,
6-phosphateconsumesATP,why not administer intravenous
calculatethe equilibrium constantfor this reaction.
glucose6-phosphate instead?
(c) Comparethe valuesof Q andKio. Is the physiological
reaction near or far from equilibrium?Explain What doesthis 6. Enz5'rneActivity and Physiological Function The Z^u*
experimentsuggestabout the role of PFK-1 as a regulatory of the glycogenphosphorylasefrom skeletal muscle is much
enzl'rne? greaterthan the 7-u" of the sameenzlme from [ver tissue.
(a) What is the physiologicalfunction of glycogenphos-
2. Are All Metabolic Reactions at Equilibrium?
phorylasein skeletalmuscle?In liver tissue?
(a) Phosphoenolpy'ruvate (PEP) is one of the two phos-
(b) Whv does the 7^u* of the muscle enzyrneneed to be
phoryl groupdonorsln the synthesisof ATP duringglycolysis.
greater than that of the liver enzyme?
In humanerythrocytes,the steady-state concentrationof ATP
ts 2.24 mM, that of ADP is 0 25 mu, and that of pyruvateis 7. Glycogen Phosphorylase Equilibrium Glycogenphos-
0 051mn. Calculatethe concentratlonof PEP at25"C, assum- phorylasecatalyzesthe removalof glucosefrom glycogen.The
ing that the pyruvatekinasereaction (see Fig 13-13) is at AG'ofor this reactionis 3 1 kJ/mol
equilibrium in the cell. (a) Caiculatethe ratio of [P1]to [glucose1-phosphate]
(b) The physiological concentrationof PEPin humanery- when the reaction is at equilibrium. (Hint: The removal of
throcyiesis 0 023mu. Comparethis with the valueobtainedin glucoseunits from glycogendoes not changethe glycogen
(a) Explain the slgniflcanceof this difference. concentration.)
 -
f
4 6 1 2 , P r i n c i p loefsM e t a b oR
l i ce g u l a t i o n
(b) The measuredratio [P1]/[glucose l-phosphate]in my-
ocytes under physiologicalconditions is more than 100:1.
What doesthis indicate about the direction of metaboliteflow
through the glycogenphosphorylasereaction in muscle?
(c) S&y are the equilibrium and physiologicalratios dif-
ferent?What is the possiblesigniflcanceof this difference?
8. Regulation of Glycogen Phosphorylase In muscle tis-
sue,the rate of conversionof giycogento glucose6-phosphate
is determinedby the ratio of phosphorylasea (active) to phos-
phorylaseb (lessactive).Determinewhat happensto the rate
of glycogenbreakdou'nif a muscle preparationcontaining
glycogenphosphorylaseis treated with (a) phosphorylaseki-
naseandATP;(b) PPl; (c) epinephrine.
9. Glycogen Breakdown in Babbit Muscle The intracellu-
lar use of glucoseand glycogenis tightly regulatedat four
points To comparethe regulationof glycoiysiswhen oxygenis
plentiful and when it is depleted,considerthe utilizationof
glucoseand glycogenby rabbit Ieg musclein two physiological
settings:a resting rabbit, with low ATP demands,and a rabbit
that sights its mortal enemy,the coyote, and dashesinto its
burrow. For each setting, determine the relative levels (high,
intermediate,or low) of AMP,ATP,citrate, and acetyl-CoAand
describe how these levels affect the flow of metabolites
through glycolysisby regulating speciic enz)'rnesIn periods
of stress,rabbit leg muscleproducesmuch of its ATPby anaer-
obic glycolysis(lactate fermentation) and very little by oxida-
tion of acetyl-CoAderivedfrom fat breakdown
10. Glycogen Breakdown in Migrating Birds Unlike the
rabbit with its short dash, migratory birds require energy for
extended periods of time. For example, ducks generally fly
severalthousandmiles during their annual migration. The
flight musclesof migratory birds have a high oxidative capac-
ity and obtain the necessaryATP through the oxidation of
acetyl-CoA(obtainedfrom fats) via the citric acid cycle Com-
pare the regulation of muscle glycolysisduring short-term in-
tense activity, as in the fleeing rabbit, and during extended
activity, as in the migrating duck. Why must the regulation in
these two settingsbe different?
11. Enzyme Defects in Carbohydrate Metabolism
Summariesof four clinical casestudiesfollow.For each
casedetermhe which enzyrneis defectiveand designatethe ap-
propriate treatment, from the lists provided at the end of the
problem.Justi-fyyour choices.Answer the questionscontahed
in each case study. ffou may need to refer to information in
Chapter14.)
Case A The patlent develops vomiting and diarrhea
shortly after milk ingestion A lactosetolerancetest is admin-
istered.(The patientingestsa standardamountof lactose,and
the glucoseand galactoseconcentrationsof blood plasmaare
measuredat intervals In individualswith normal carbohydrate
metabolism,the levelsincreaseto a maximum in about t hour,
then decline.)The patient'sblood glucoseand galactosecon-
centrationsdo not increaseduringthe test.Why do bloodglu-
coseand galactoseincreaseand then decreaseduringthe test
in healthy individuals?Why do they fail to rise in the patient?
CaseB The patient developsvomiting and diarrhea after
ingestion of milk. His blood is found to have a low concentra-
tion of glucosebut a much higher than normal concentration
of reducing sugars.The urine tests positivefor galactose.Why
is the concentrationofreducing sugarin the blood high?Why
doesgalactoseappearin the urine?
Case C The patient complainsof painful muscle cramps
when performing strenuousphysicalexercisebut hasno other
symptoms A muscle biopsy indicates a muscle glycogen
concentrationmuch higher than normal. Why does glycogen
accumulate?
CaseD The patient is lethargic,her liver is enlarged,and
a biopsy of the liver showslarge amounts of excessglycogen.
She also has a Iower than normal blood glucoselevel. What is
the reasonfor the low blood glucosein this patient?
DefectiueEnzyme
(a) MusclePFK-I
(b) Phosphomannose isomerase
(c) Galactose1-phosphateuridylyltransferase
(d) Liver glycogenphosphorylase
(e) Ttiose kinase
(f ) Lactasein intestinal mucosa
(g) Maltasein intestinai mucosa
(h) Muscledebranchingenzy'rne
Tteatmnnt
1. Jogging5 km eachday
2 Fat-free diet
3. Low-lactosediet
4. Avoidingstrenuousexercise
5. Largedosesof niacin (the precursorof NAD')
6. Frequent feedings (smaller portions) of a normal diet
12. Effects of Insufficient Insulin in a Person
with Diabetes A man with insulin-dependent dia-
betes is brought to the emergencyroom in a near-comatose
state.While vacationingin an isolatedplace,he lost his insulin
medicationand has not taken any insulin for two days.
(a) For each tissue listed below, is each pathway faster,
slower,or unchangedin this patient, comparedwith the nor-
mal level when he is getting appropriate amounts of insulin?
(b) For eachpathway,describeat least one control mech-
anismresponsiblefor the changeyou predict
?issue and Pathways
1. Adipose:fatty acid sSmthesis
2. Muscle: glycolysis;fatty acid synthesis;glycogen slm-
thesis
3 Liver: glycolysis;gluconeogenesis; glycogen synthesis;
fatty acid synthesis;pentosephosphatepathway
13. Blood Metabolites in Insulin Insufficiency
For the patient describedin Problem 12, predict the
levels of the following metaboiites in his blood bejore ireat-
ment in the emergency room, relative to levels maintained
during adequate insulin treatment: (a) glucose; (b) ketone
bodies; (c) free fatty acids.
14. Metabolic Effects of Mutant Enz5rrnes Predict and ex-
plain the effect on glycogen metabolism of each of the foliowing

defectscausedby mutation: (a) loss of the cAMP-bindingsite
on the regulatorysubunitof protein kinaseA (PKA); (b) lossof
the proteh phosphataseinhibitor (inhibitor 1 in Fig. 1b-40);
(c) overexpressionof phosphorylaseb kinasein iiver; (d) de-
fective glucagonreceptorsin liver
15. Hormonal Control of Metabolic Fuel Between your
eveningmeal and breakfast,your blood glucosedrops and
your liver becomesa net producerrather than consumerof
glucose.Describethe hormonalbasisfor this switch,and ex-
plain how the hormonalchangetriggersglucoseproduction by
the liver.
16. Altered Metabolism in Genetically Manipulated
Mice Researcherscan manipulate the genes of a mouse so
that a singlegenein a singletissueeither producesan inactive
proteln (a "knockout"mouse) or producesa protein that is
always (constitutively) active. What effects on metabolism
would you predict for mice with the following geneticchanges:
(a) knockout of glycogen debranching enzyrnein the liver;
(b) knockout of hexokinaseIV in liver; (c) knockout of
FBPase-2in liver; (d) constitutivelyactiveFBPase-2in liver;
(e) constitutively active AMPK in muscle; (f) constitutively
active ChREBPin liver?
DataAnalysis
Problem
17. Optimal Glycogen Structure Musclecells need rapid
accessto large amounts of glucoseduring hear,yexercise
This glucoseis storedin liver and skeletalmusclein pollrneric
form as particles of glycogen. The typical glycogen particle
contains about 55,000glucoseresidues (see Fig. 15-33b)
Mel6ndez-Hevia, Waddell,and Shelton(1993) exploredsome
theoreticalaspectsof the structureof glycogen,as described
in this problem.
(a) The cellular concentrationofglycogenin liver is about
0.01pu What cellularconcentrationof free glucosewouldbe
required to store an equivalentamount of glucose?Why would
this concentrationof free giucosepresenta problemfor the
cell?
Glucoseis releasedftom $ycogen by glycogenphosphory-
lase,an enzl'rnethat canremove$ucosemolecules,oneat a time,
from one end of a $ycogenchain.Glycogenchainsare branched
(seeFigs 15-26and 15-33b),and the degreeof branchmg-the
number of branchesper chain-has a powerful influence on the
rate at which $ycogenphosphorylase canrelease$ucose.
(b) WhV would a degree of branching that was too low
(i.e.,below an optimumlevel) reducethe rate of glucosere-
lease?(Hint: Considerthe extremecaseof no branchesin a
chainof 55,000glucoseresidues.)
Data
A n a l y sPi sr o b l e mlsa t f
(c) \VhVwould a degree of branching that was too high
also reduce the rate of glucose release?(Hint: Think of the
physicalconstraints)
Mel6ndez-Heviaand colleaguesdid a seriesof calculations
and found that two branchesper chain (see Fig. 15-33b) was
optimai for the constraints described above. This is what is
found in glycogenstored in muscleand liver.
To determine the optimum number of glucose residues
per chain,Mel6ndez-Hevia and coauthorsconsideredtwo key
parametersthat define the structure of a glycogenparticle:
I : the number of tiers of glucosechainsin a particle (the mol-
eculein Fig. 15-33bhas five tiers);9" : the number of glu-
coseresiduesin eachchain.They set out to find the valuesof
t and g. that would maximize three quantities: (1) the
amount of glucosestored in the particle (Gr) per unit vol-
ume; (2) the numberofunbranchedglucosechains(Cj per
unit volume (i.e., number of chains in the outermost tier,
readily accessibleto glycogenphosphorylase);and (3) the
amount of glucose available to phosphorylasein these un-
branchedchains(Gp1).
(d) Show thaLCo: 2t 1. This is the number of chains
availableto glycogenphosphorylasebefore the action of the
debranchingenzyme.
(e) Show that C1, the total number of chainsin the parti-
c l e , i s g i v e n b y C r : 2 ' - 1 T h u sG r : Q " ( C " i : g . ( 2 t- 1 ) ,
the total number of glucoseresiduesin the particle.
(f) Glycogenphosphorylasecannot remove glucosefrom
glycogen chains that are shorter than flve glucose residues.
Showthat Gpr : (Q. - 4)(2' t). This is the amountof glu-
cosereadily availableto glycogenphosphorylase.
(g) Basedon the sizeofa $ucose residueand the location
of branches,the thicknessof onetier of $ycogenis 0.129"nm *
0.35nm Showthat the volume of a particle, 7", is givenby the
,|
equationV": |nf(O.I2g" + 0.35)3nm3
o
Mel6ndez-Heviaand coauthorsthen determined the opti-
mum valuesof I andg"-those that gavethe maximumvalueof
a quality function, f, Ihat maximizes Gr, Co, and Gp1,while
GrC oGo.
minimizingVs:J : -;-.They found that the optimum
value ofg" is independeniof I
(h) Choosea value of f between 5 and 15 and find the op-
timum value of 9". How doesthis comparewith the g" found in
liver glycogen(seeFig. 15-33bX (Hint: Youmay find it useful
to usea spreadsheet program.)
Reference
E., Waddell,T.G., & Shelton, D.D. (1993)
Mel6ndez-Hevia,
Optimization of molecular design in the evolution of metabolism: the
glycogen molecrle. Biochem J. 295,477483.