Voet D., Voet Ju.G. Biochemistry

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3CMP

trans Golgi

Polypeptide

3UDP

871871

CHAPTER 23

Other Pathways of

Carbohydrate

Metabolism

1 Gluconeogenesis

A. The Gluconeogenesis Pathway

B. Regulation of Gluconeogenesis

C. The Cori Cycle

2 The Glyoxylate Cycle

3 Biosynthesis of Oligosaccharides and Glycoproteins

A. Lactose Synthesis

B. Glycoprotein Synthesis

4 The Pentose Phosphate Pathway

A. Oxidative Reactions of NADPH Production

B. Isomerization and Epimerization of Ribulose-5-Phosphate

C. Carbon–Carbon Bond Cleavage and Formation Reactions

D. Control of the Pentose Phosphate Pathway

E. Glucose-6-Phosphate Dehydrogenase Deficiency

Heretofore, we have dealt with many aspects of carbohy-

drate metabolism. We have seen how the free energy of

glucose oxidation is sequestered in ATP through glycolysis,

the citric acid cycle, and oxidative phosphorylation. We

have also studied the mechanism by which glucose is stored

as glycogen for future use and how glycogen metabolism is

controlled in response to the needs of the organism. In this

chapter, we examine several other carbohydrate metabo-

lism pathways of importance:

1. Gluconeogenesis, through which noncarbohydrate

precursors such as lactate, pyruvate, glycerol, and amino

acids are converted to glucose.

2. The glyoxylate cycle, through which plants convert

acetyl-CoA to glucose.

3. Oligosaccharide and glycoprotein biosynthesis,

through which oligosaccharides are synthesized and added

to specific amino acid residues of proteins.

4. The pentose phosphate pathway, an alternate path-

way of glucose degradation, which generates NADPH, the

source of reducing equivalents in reductive biosynthesis,

and ribose-5-phosphate, the sugar precursor of the nucleic

acids.

This chapter completes our study of carbohydrate metabo-

lism in animals; photosynthesis, which occurs only in plants

and certain bacteria, is the subject of Chapter 24.

1 GLUCONEOGENESIS

Glucose occupies a central role in metabolism, both as a

fuel and as a precursor of essential structural carbohy-

drates and other biomolecules. The brain and red blood

cells are almost completely dependent on glucose as an en-

ergy source. Yet the liver’s capacity to store glycogen is

only sufficient to supply the brain with glucose for about

half a day under fasting or starvation conditions. Thus,

when fasting, most of the body’s glucose needs must be met

by gluconeogenesis (literally, new glucose synthesis), the

biosynthesis of glucose from noncarbohydrate precursors.

Indeed, isotopic labeling studies determining the source of

glucose in the blood during a fast showed that gluconeogen-

esis is responsible for 64% of total glucose production over

the first 22 hours of the fast and accounts for almost all the

glucose production by 46 hours.Thus, gluconeogenesis pro-

vides a substantial fraction of the glucose produced in fast-

ing humans, even after a few hours’ fast. Gluconeogenesis

occurs in liver and, to a smaller extent, in kidney.

The noncarbohydrate precursors that can be converted

to glucose include the glycolysis products lactate and pyru-

vate,citric acid cycle intermediates, and the carbon skeletons

of most amino acids. First, however, all these substances

must be converted to oxaloacetate, the starting material for

gluconeogenesis (Fig. 23-1). The only amino acids that can-

not be converted to oxaloacetate in animals are leucine

and lysine because their breakdown yields only acetyl-

CoA (Section 26-3F). There is no pathway in animals for

the net conversion of acetyl-CoA to oxaloacetate. Likewise,

fatty acids cannot serve as glucose precursors in animals

because most fatty acids are degraded completely to

acetyl-CoA (Section 25-2C). Unlike animals, however,

plants do contain a pathway for the conversion of acetyl-

CoA to oxaloacetate, the glyoxylate cycle (Section 23-2), so

that fatty acids can serve as a plant cell’s only carbon

source. Glycerol, a triacylglycerol breakdown product, is

converted to glucose via synthesis of the glycolytic inter-

mediate dihydroxyacetone phosphate, as described in

Section 25-1.

JWCL281_c23_871-900.qxd 6/8/10 9:43 AM Page 871

A. The Gluconeogenesis Pathway

Gluconeogenesis utilizes glycolytic enzymes. Yet three of

these enzymes, hexokinase, phosphofructokinase (PFK),

and pyruvate kinase, catalyze reactions with large negative

free energy changes in the direction of glycolysis. These

reactions must therefore be replaced in gluconeogenesis

by reactions that make glucose synthesis thermodynami-

cally favorable. Here, as in glycogen metabolism (Section

18-1D), we see the recurrent theme that biosynthetic and

degradative pathways differ in at least one reaction.This not

only permits both directions to be thermodynamically fa-

vorable under the same physiological conditions but allows

the pathways to be independently controlled so that one di-

rection can be activated while the other is inhibited.

a. Pyruvate Is Converted to Oxaloacetate before

Conversion to Phosphoenolpyruvate

The formation of phosphoenolpyruvate (PEP) from

pyruvate, the reverse of the pyruvate kinase reaction, is en-

dergonic and therefore requires free energy input. This is

accomplished by first converting the pyruvate to oxaloac-

etate. Oxaloacetate is a “high-energy” intermediate whose

exergonic decarboxylation provides the free energy neces-

sary for PEP synthesis. The process requires the participa-

tion of two enzymes (Fig. 23-2):

1. Pyruvate carboxylase catalyzes the ATP-driven for-

mation of oxaloacetate from pyruvate and .

2. PEP carboxykinase (PEPCK) converts oxaloacetate

HCO



872 Chapter 23. Other Pathways of Carbohydrate Metabolism

Figure 23-1 Pathways converting lactate, pyruvate, and citric

acid cycle intermediates to oxaloacetate. The carbon skeletons of

CCH

–

CHCH C

–

C SCoA

–

Oxaloacetate

Acetyl-CoA

Citrate

Isocitrate

Succinyl-CoA

α-Ketoglutarate

Isoleucine

Methionine

Valine

Succinate

Phenylalanine

Tyrosine

Fumarate

Malate

Arginine

Glutamate

Glutamine

Histidine

Proline

Aspartate

Asparagine

Pyruvate

Alanine

Cysteine

Glycine

Serine

Threonine

Tryptophan

Lactate

Glucose

gluconeogenesis

CITRIC

ACID

CYCLE

CCC

all amino acids but leucine and lysine may be, at least in part,

converted to oxaloacetate and thus to glucose by these reactions.

JWCL281_c23_871-900.qxd 3/24/10 11:33 AM Page 872

Oxaloacetate

C CH

–

O O

–

Phosphoenol-

pyruvate (PEP)

GDP + CO

GTP

–

Pyruvate

–

PEPCK

ADP + P

HCO

–

+ ATP

pyruvate

carboxylase

to PEP in a reaction that uses GTP as a phosphorylating

agent.

b. Pyruvate Carboxylase Has a Biotin

Prosthetic Group

Pyruvate carboxylase, discovered in 1959 by Merton

Utter, is a tetrameric protein of identical ⬃130-kD sub-

units, each of which has a biotin prosthetic group. Biotin

(Fig. 23-3a) functions as a CO

carrier by acquiring a car-

boxyl substituent at its ureido group (Fig. 23-3b). Biotin is

covalently bound to the enzyme by an amide linkage be-

tween the carboxyl group of its valerate side chain and the

ε-amino group of an enzyme Lys residue to form a biocytin

(alternatively, biotinyllysine) residue (Fig. 23-3b). The

biotin ring system is therefore at the end of a 16-Å-long

flexible arm, much like that of the lipoic acid prosthetic

group in the pyruvate dehydrogenase multienzyme com-

plex (Section 21-2Ac).

Biotin, which was first identified in 1935 as a growth fac-

tor in yeast, is an essential human nutrient. Its nutritional

deficiency is rare, however, because it occurs in many foods

and is synthesized by intestinal bacteria. Human biotin de-

ficiency almost always results from the consumption of

large quantities of raw eggs. This is because egg whites

contain a protein, avidin, that binds biotin so tightly (disso-

ciation constant, K ⫽ 10

⫺15

M) as to prevent its intestinal

absorption (cooked eggs do not cause this problem be-

cause cooking denatures avidin).The presence of avidin in

eggs is thought to inhibit the growth of microorganisms in

this highly nutritious environment. The avidin homolog

streptavidin, which is secreted by Streptomyces avidinii, is

used as a linking agent in numerous biotechnological appli-

cations (e.g., Section 22-3Ce) because of its particularly

high affinity for biotin.

c. The Pyruvate Carboxylase Reaction

The pyruvate carboxylase reaction occurs in two phases

(Fig. 23-4):

Phase I Biotin is carboxylated at its N1 atom by bicar-

bonate ion in a three-step reaction in which the hydrolysis

of ATP to ADP ⫹ P

functions, via the intermediate forma-

tion of carboxyphosphate, to dehydrate bicarbonate. This

yields free CO

, which has sufficient free energy to carboxy-

late biotin.The resulting carboxyl group is activated relative

to bicarbonate (⌬G°¿ for its cleavage is ⫺19.7 kJ ⴢ mol

⫺1

)

and can therefore be transferred without further free

energy input.

Phase II The activated carboxyl group is transferred

from carboxybiotin to pyruvate in a three-step reaction to

form oxaloacetate.

These two reaction phases occur on different active sites of

the same enzyme.

d. Acetyl-CoA Regulates Pyruvate Carboxylase

Oxaloacetate synthesis is an anaplerotic (filling up) reac-

tion that increases citric acid cycle activity (Section 21-5b).

Accumulation of the citric acid cycle substrate acetyl-CoA

is therefore indicative of the need for more oxaloacetate.

Section 23-1. Gluconeogenesis 873

Figure 23-3 Biotin and carboxybiotinyl–enzyme.

(a) Biotin consists of an imidazoline ring that is cis-fused to a

tetrahydrothiophene ring bearing a valerate side chain.The

chirality at each of its three asymmetric centers is indicated.

Positions 1, 2, and 3 constitute a ureido group. (b) In

carboxybiotinyl–enzyme, N1 of the biotin ureido group is the

carboxylation site. Biotin is covalently attached to carboxylases

by an amide linkage between its valeryl carboxyl group and the

ε-amino group of an enzyme Lys side chain to form biocytin.

Figure 23-2 Conversion of pyruvate to oxaloacetate and then to phosphoenolpyruvate. The

enzymes involved are (1) pyruvate carboxylase and (2) PEP carboxykinase (PEPCK).

Carboxybiotinyl–enzyme

COO

–

(CH

)

Lys residue

(CH

)

C NH

–

(b)

Biotin

Valerate side chain

(a)

CHH

JWCL281_c23_871-900.qxd 7/2/10 12:01 PM Page 873

–

OOC O

Adenosine

Biotinyl–enzyme

Carboxybiotinyl–enzyme

Pyruvate enolate

Oxaloacetate

ATP Carboxyphosphate

–

HO P

–

ADP

Pyruvate

Biotinyl–enzyme

(CH

)

NH E(CH

)

(CH

)

NH E(CH

)

NHN

–

OOC O

–

OOC O

–

OOC O

–

NHN

–

Phase II

Phase I

–

Indeed, acetyl-CoA is a powerful allosteric activator of

pyruvate carboxylase; the enzyme is all but inactive with-

out bound acetyl-CoA. If, however, the citric acid cycle is in-

hibited (by ATP and NADH, whose presence in high con-

centrations indicates a satisfied demand for oxidative

phosphorylation; Section 21-4), oxaloacetate instead under-

goes gluconeogenesis.

e. X-Ray Structure of Pyruvate Carboxylase

Suggests a Mechanism for Carboxyl Group Transfer

The X-ray structure of pyruvate carboxylase from the

soil bacterium Rhizobium etli in complex with ATPS and

ethyl-CoA (acetyl-CoA with an ethyl group in place of its

acetyl group), determined by Ivan Rayment, reveals that

each subunit of this homotetrameric, 1154-residue protein

consists of four domains (Fig. 23-5a): a biotin carboxylation

(BC) domain (residues 1–465) that carries out Phase I of

the pyruvate carboxylase reaction (Fig. 23-4); an allosteric

domain (residues 466–510 and 1001–1073) that binds

acetyl-CoA; a carboxyltransferase (CT) domain (residues

511–1000) that catalyzes Phase II of the pyruvate carboxy-

lase reaction (Fig. 23-4); and a domain named the biotin

carboxyl carrier protein (BCCP; residues 1074–1154) to

which the enzyme’s biotin prosthetic group is covalently

linked via Lys 1119 (Fig. 23-3).The active site of the BC do-

main is marked by its bound ATPS, the CT domain is

mainly an / barrel (viewed from the side in Fig. 23-5a)

whose active site is located in the mouth of the barrel and

is marked by its bound Zn

2

ion, and the allosteric domain

binds the ethyl-CoA, which like acetyl-CoA, activates the

enzyme.

The biotin prosthetic group on the BCCP domain is dis-

ordered and hence not visible. Nevertheless it is clear that

the ⬃80-Å distance between a subunit’s active sites is too

874 Chapter 23. Other Pathways of Carbohydrate Metabolism

Figure 23-4 Two-phase reaction mechanism of pyruvate

carboxylase. Phase I is a three-step reaction in which

carboxyphosphate is formed from bicarbonate and ATP, followed

by the generation of CO

on the enzyme, which then

carboxylates biotin. Phase II is a three-step reaction in which

is produced at the active site via the elimination of the

biotinyl enzyme, which accepts a proton from pyruvate to

generate pyruvate enolate. This, in turn, nucleophilically attacks

the CO

, yielding oxaloacetate. [After Knowles, J.R., Annu. Rev.

Biochem. 58, 217 (1989).]

JWCL281_c23_871-900.qxd 3/24/10 11:33 AM Page 874

Section 23-1. Gluconeogenesis 875

Figure 23-5 X-ray structure of R. etli pyruvate carboxylase.

(a) Ribbon diagram in which the BC and CT domains are each

colored in rainbow order from their N-termini (blue) to their

C-termini (red), the allosteric domain is light green, and the

BCCP domain is pink.The ATP␥S bound at the active site of the

BC domain and the ethyl-CoA bound to the allosteric domain

(with only its nucleotide portion visible) are drawn in space-

filling form with ATP␥S C green, ethyl-CoA C cyan, N blue, O

red, and P orange.The Zn

2⫹

ion bound at the CT domain active

site is represented by a purple sphere. (b) Surface representation

of the tetramer viewed along its 2-fold axis with the two active

subunits closest to the viewer.The BC domain is purple, the

allosteric domain is light green, the CT domain is yellow, and the

BCCP domain is red. For clarity, one of the subunits is outlined

in black.The distance between the ATP␥S in the BC active site

and the Zn

2⫹

ion in the CT active site is 65 Å. (c) View relative to

Part b by a 180° rotation about the vertical axis.The top pair of

subunits have undergone a conformational change relative to

the top pair in Part b such that the ATP␥S–Zn

2⫹

distance

between neighboring subunits is 80 Å. In addition, the BCCP

domains in the top pair here are disordered. (d) Model of the

tetramer indicating how the BCCP domain transfers a carboxyl

group between the BC domain on the same subunit and the CT

domain of its neighboring subunit.The view and domain colors

are the same as in Part b. [Part a based on an X-ray structure by

and Parts b, c, and d courtesy of Ivan Rayment, University of

Wisconsin, Madison,Wisconsin. PDBid 2QF7.]

(a)

(b)

(c)

(d)

JWCL281_c23_871-900.qxd 6/16/10 1:54 PM Page 875

large to be bridged by the 16-Å-long carboxybiotinyl arm

(Fig. 23-3b). However, the BCCP domain is attached to the

enzyme by a flexible polypeptide linker that is 34 Å long,

much like that linking the lipoyl domain(s) to each dihy-

drolipoyl transacetylase (E2) subunit of the pyruvate dehy-

drogenase complex (Section 21-2Ae). Even so, it would re-

quire a dramatic movement of the entire BCCP domain to

transfer substrate between the two active sites of a single

subunit. How, then, does a BCCP domain translocate its

carboxybiotin group between the active sites of a BC do-

main and a CT domain?

Pyruvate carboxylase’s homotetrameric structure is re-

quired for its enzymatic activity; isolated subunits are cat-

alytically inactive. However, the tetramer has only 2-fold

symmetry because the top pair of subunits in Fig. 23-5b

differ in conformation from the top pair in Fig. 23-5c by a

40° rotation and a 40-Å translocation of the BC domain

relative to the CT domain of the same subunit. Indeed,

the BCCP domains in the top pair of subunits in Fig. 23-5c

are disordered, probably because the allosteric domains

on these subunits do not bind ethyl-CoA. Consequently,

the distance between active sites from adjacent subunits is

65 Å for the top pair in Fig. 23-5b, whereas it is 80 Å for

the top pair in Fig. 23-5c. This suggests the model drawn

in Fig. 23-5d in which each BCCP domain at the top of

Fig. 23-5b shuttles CO

in the form of carboxybiotin from

the active site of the BC domain on the same subunit to

the CT domain on the adjacent subunit, whereas the

other two subunits are inactive. This is an unusual exam-

ple of allosteric activation coupled with negative cooper-

ativity. It may permit pyruvate carboxylase to carry out

efficient catalysis in association with other metabolic

enzymes.

The foregoing model is supported by experiments

involving two mutant forms of pyruvate carboxylase:

K1119Q, which eliminates the biotinylation of the BC do-

main; and K718Q, which impairs the Phase II reaction.

Tetramers of each of these mutant subunits exhibited 0.1%

and 4% of the wild-type enzymatic activity, respectively.

However, mixed tetramers exhibit 20% activity, thus indi-

cating the formation of neighboring pairs of functional BC

and CT domains.

f. PEP Carboxykinase

PEPCK, a monomeric ⬃630-residue enzyme, catalyzes

the GTP-driven decarboxylation of oxaloacetate to form

PEP and GDP (Fig. 23-6). Note that the CO

that carboxy-

lates pyruvate to yield oxaloacetate is eliminated in the for-

mation of PEP. Oxaloacetate may therefore be considered

to be “activated” pyruvate, with CO

and biotin facilitating

the activation at the expense of ATP hydrolysis. Acetyl-

CoA is similarly activated for fatty acid biosynthesis

through such a carboxylation decarboxylation process

(forming malonyl-CoA; Section 25-4B). In general, -keto

acids may be considered “high-energy” compounds because

of the high free energy of decarboxylation of the -carboxyl

group. The enolates they generate are used to form

carbon–carbon bonds in fatty acid biosynthesis or phospho-

enolpyruvate here in gluconeogenesis.

g. Gluconeogenesis Requires Metabolite Transport

between Mitochondria and Cytosol

The generation of oxaloacetate from pyruvate or citric

acid cycle intermediates occurs only in the mitochondrion,

whereas the enzymes that convert PEP to glucose are cy-

tosolic. The cellular location of PEPCK varies with the

species. In mouse and rat liver it is located almost exclu-

sively in the cytosol, in pigeon and rabbit liver it is mito-

chondrial, and in guinea pig and humans it is more or less

equally distributed between both compartments. In order

for gluconeogenesis to occur, either oxaloacetate must

leave the mitochondrion for conversion to PEP or the PEP

formed there must enter the cytosol.

PEP is transported across the mitochondrial membrane

by specific membrane transport proteins. There is, how-

ever, no such transport system for oxaloacetate. It must

first be converted either to aspartate (Fig. 23-7, Route 1) or

to malate (Fig. 23-7, Route 2), for which mitochondrial

transport systems exist (Section 22-1B).The difference be-

tween these two routes involves the transport of NADH

reducing equivalents. The malate dehydrogenase route

(Route 2) results in the transport of reducing equivalents

from the mitochondrion to the cytosol, since it utilizes mi-

tochondrial NADH and produces cytosolic NADH.The as-

partate aminotransferase route (Route 1) does not involve

NADH. Cytosolic NADH is required for gluconeogenesis

so, under most conditions, the route through malate is a ne-

cessity. If the gluconeogenic precursor is lactate, however

(Section 23-1C), its oxidation to pyruvate generates cytoso-

lic NADH, so that either transport route may then be used.

Of course, as we have seen, during oxidative metabolism

876 Chapter 23. Other Pathways of Carbohydrate Metabolism

CCC

–

CCC

–

OPGuanosine

–

Phosphoenolpyruvate

(PEP)

Pyruvate enolateOxaloacetate

GTP

GDP

Figure 23-6 The PEPCK mechanism. Decarboxylation of

oxaloacetate (a -keto acid) forms a resonance-stabilized

enolate anion whose oxygen atom attacks the  phosphoryl

group of GTP forming PEP and GDP.

JWCL281_c23_871-900.qxd 6/8/10 9:43 AM Page 876

the two routes may also alternate (with Route 2 reversed)

to form the malate–aspartate shuttle, which transports

NADH reducing equivalents into the mitochondrion (Sec-

tion 22-1Bc).

In the liver, where the urea cycle occurs (Section 26-2),

a third route, a modification of Route 1, may be followed

for transporting oxaloacetate into the cytosol. The aspar-

tate that enters the cytosol by Route 1 may be converted to

fumarate as part of the urea cycle (Fig. 26-8), instead of be-

ing transaminated. Fumarate is then hydrated to malate

and dehydrogenated to oxaloacetate by cytosolic equiva-

lents of citric acid cycle enzymes.This third route generates

cytosolic NADH in the same way as does Route 2.

h. Hydrolytic Reactions Bypass PFK and Hexokinase

The opposing pathways of gluconeogenesis and glycolysis

utilize many of the same enzymes (Fig. 23-8). However, the

free energy change is highly unfavorable in the gluconeogenic

direction at two other points in the pathway in addition to

the pyruvate kinase reaction: the PFK reaction and the

hexokinase reaction. At these points, instead of generat-

ing ATP by reversing the glycolytic reactions, FBP and G6P

are hydrolyzed, releasing P

in exergonic processes cat-

alyzed by fructose-1,6-bisphosphatase (FBPase) and

glucose-6-phosphatase, respectively. Glucose-6-phosphatase

is unique to liver and kidney, permitting them to supply

glucose to other tissues.

Because of the presence of separate gluconeogenic en-

zymes at the three irreversible steps in the glycolytic conver-

sion of glucose to pyruvate, both glycolysis and gluconeo-

genesis are rendered thermodynamically favorable. This is

accomplished at the expense of the free energy of hydrol-

ysis of two molecules each of ATP and GTP per molecule

of glucose synthesized by gluconeogenesis in addition to

that which would be consumed by the direct reversal of

glycolysis.

Section 23-1. Gluconeogenesis 877

COO

–

COO

–

HHO

COO

–

COO

–

COO

–

COO

–

COO

–

COO

–

HHO

COO

–

COO

–

COO

–

COO

–

Gluconeogenesis

Cytosol Mitochondrion

NAD

MalateMalate

Oxaloacetate

Route

AspartateAspartate

Oxaloacetate

NADH + H

malate

dehydrogenase

Amino acid

α-Keto acid

aspartate

aminotransferase

PEPPEP

Amino acid

α-Keto acid

aspartate

aminotransferase

NAD

NADH + H

malate

dehydrogenase

Inner

mitochondrial

membrane

Figure 23-7 Transport of PEP and oxaloacetate from the

mitochondrion to the cytosol. PEP is directly transported

between these compartments. Oxaloacetate, however, must first

be converted to either aspartate through the action of aspartate

aminotransferase (Route 1) or to malate by malate

dehydrogenase (Route 2). Route 2 involves the mitochondrial

oxidation of NADH followed by the cytosolic reduction of

NAD



and therefore also transfers NADH reducing equivalents

from the mitochondrion to the cytosol.

See the Animated

Figures

JWCL281_c23_871-900.qxd 3/24/10 11:33 AM Page 877

NADH + H

NAD

+ P

Glucose

Glucose-6-phosphate

glucose-6-phos-

phatase

(–5.1)

hexokinase (32.9)

Fructose-6-phosphate

Fructose-1,6-bisphosphate

fructose bisphos-

phatase

(–8.6)

ATP

ADP

phosphofructokinase (24.5)

ATP

ADP

triose phosphate

isomerase

Glyceraldehyde-3-

phosphate

Dihydroxyacetone

phosphate

+ NAD

glyceraldehyde-3-phos-

phate dehydrogenase

1,3-Bisphosphoglycerate

ATP

ADP

phosphoglycerate

kinase

phosphoglucose isomerase (1.1)

aldolase (12.1)

3-Phosphoglycerate

phosphoglycerate mutase (1.3)

2-Phosphoglycerate

enolase (1.2)

ATP

ADP

Phosphoenolpyruvate

pyruvate kinase

(26.4)

ATP

ADP

Pyruvate

Oxaloacetate

GTP

+ GDP

PEPCK

+ ADP

ATP

+ CO

pyruvate

carboxylase

(4.1)

(–22.6)

Glycolysis:

Gluconeogenesis:

Overall:

Such free energy losses in a cyclic process are thermody-

namically inescapable.They are the price that must be paid

to maintain independent regulation of the two pathways.

B. Regulation of Gluconeogenesis

If both glycolysis and gluconeogenesis were to proceed in

an uncontrolled manner, the net effect would be a futile

cycle wastefully hydrolyzing ATP and GTP. This does not

occur. Rather, these pathways are reciprocally regulated so

as to meet the needs of the organism. In the fed state, when

the blood glucose level is high, the liver is geared toward

fuel conservation: Glycogen is synthesized and the gly-

colytic pathway and pyruvate dehydrogenase are acti-

vated, breaking down glucose to acetyl-CoA for fatty acid

biosynthesis and fat storage. In the fasted state, however,

the liver maintains the blood glucose level both by glyco-

gen breakdown and by reversing the flux through glycol-

ysis toward gluconeogenesis [using mainly protein degra-

dation products via the glucose–alanine cycle (Section

26-1Ad) and glycerol from triacylglycerol hydrolysis

(Section 25-1e)].

a. Glycolysis and Gluconeogenesis Are Controlled

by Allosteric Interactions and Covalent Modifications

The rate and direction of glycolysis and gluconeogenesis

are controlled at the points in these pathways where the for-

ward and reverse directions can be independently regulated:

the reactions catalyzed by (1) hexokinase/glucose-6-

phosphatase, (2) PFK/FBPase,and (3) pyruvate kinase/pyru-

vate carboxylase–PEPCK (Fig. 23-8). Table 23-1 lists these

regulatory enzymes and their regulators. The dominant

mechanisms are allosteric interactions and cAMP-dependent

covalent modifications (phosphorylation/dephosphoryla-

tion; Section 18-3). cAMP-dependent covalent modification

renders this system sensitive to control by glucagon and

other hormones that alter cAMP levels.

One of the most important allosteric effectors involved

in the regulation of glycolysis and gluconeogenesis is

fructose-2,6-bisphosphate (F2,6P), which activates PFK

and inhibits FBPase (Section 18-3F). The concentration of

F2,6P is controlled by its rates of synthesis and breakdown

by phosphofructokinase-2 (PFK-2) and fructose bisphos-

phatase-2 (FBPase-2), respectively. Control of the activi-

ties of PFK-2 and FBPase-2 is therefore an important

aspect of gluconeogenic regulation even though these

2ATP ⫹ 2GTP ⫹ 4H

2ADP ⫹ 2GDP ⫹ 4P

glucose ⫹ 2NAD

⫹

⫹ 4ADP ⫹ 2GDP ⫹ 6P

2 Pyruvate ⫹ 2NADH ⫹ 4H

⫹

⫹ 4ATP ⫹ 2GTP ⫹ 6H

2 pyruvate ⫹ 2NADH ⫹ 4H

⫹

⫹ 2ATP ⫹ 2H

Glucose ⫹ 2NAD

⫹

⫹ 2ADP ⫹ 2P

enzymes do not catalyze reactions of the pathway. PFK-2

and FBPase-2 activities, which occur on separate domains

of the same bifunctional enzyme, are subject to allosteric

878 Chapter 23. Other Pathways of Carbohydrate Metabolism

Figure 23-8 Pathways of gluconeogenesis and glycolysis. The

three numbered steps, which are catalyzed by different enzymes

in gluconeogenesis, have red arrows. The ⌬G’s for the reactions

in the direction of gluconeogenesis under physiological

conditions in liver are given in parentheses in kJ ⴢ mol

⫺1

.[⌬G’s

obtained from Newsholme, E.A. and Leech, A.R., Biochemistry

for the Medical Sciences, p. 448, Wiley (1983).]

See the

Animated Figures

JWCL281_c23_871-900.qxd 7/2/10 12:02 PM Page 878

regulation as well as control by covalent modifications

(Table 23-1). Low levels of blood glucose result in hor-

monal activation of gluconeogenesis through regulation of

[F2,6P] (Fig. 23-9).

Activation of gluconeogenesis in liver also involves in-

hibition of glycolysis at the level of pyruvate kinase. Liver

pyruvate kinase is inhibited both allosterically by alanine

(a pyruvate precursor; Section 26-1Ad) and by phosphory-

lation. Glycogen breakdown, in contrast, is stimulated by

phosphorylation (Section 18-3C). Both pathways then flow

toward G6P, which is converted to glucose for export to

muscle and brain. Muscle pyruvate kinase, an isozyme of

the liver enzyme, is not subject to these controls. Indeed,

such controls would be counterproductive in muscle since

this tissue lacks glucose-6-phosphatase and thus the ability

to synthesize glucose via gluconeogenesis.

b. PEPCK Concentration Is Transcriptionally

Controlled

PEPCK is the enzyme that catalyzes the first commit-

ted reaction of gluconeogenesis. It is therefore of interest

(Table 23-1) that PEPCK’s activity is controlled solely

through the transcriptional regulation of the gene encod-

ing it (transcriptional regulation is outlined in Section 5-

4Aa and discussed in detail in Sections 31-3 and 34-3). In

particular, the transcription of the PEPCK gene is stimu-

lated by glucagon, glucocorticoids, and thyroid hormones,

and is inhibited by insulin. For instance, the cAMP that is

produced in response to stimulation of the liver by

glucagon, in addition to its initiation of phosphorylation

cascades (Section 18-3), induces the transcription of the

PEPCK gene. Richard Hanson has shown that this occurs

because the PEPCK gene promoter (a control region that

precedes the transcriptional initiation site of genes en-

coding proteins; Section 5-4Aa) contains a specific DNA

sequence called the cAMP response element (CRE) that

is bound by a transcription factor named CRE binding

protein (CREB), but only when CREB is also binding

cAMP (recall that a transcription factor is a protein that

binds to a specific segment of its target promoter and, in

doing so, activates RNA polymerase to initiate the tran-

scription of the associated gene; Section 5-4Aa). How-

ever, the PEPCK gene promoter contains numerous

other binding sites for specific transcription factors.

Among them are the thyroid hormone response element

(TRE), which is bound by thyroid hormone receptor in

complex with thyroid hormone (Section 19-1D), and the

glucocorticoid hormone response element (GRE), which

is bound by the glucocorticoid receptor in complex with a

glucocorticoid hormone (Sections 19-1G and 34-3Bn). In

contrast, PEPCK gene transcription is strongly repressed

by protein factors phosphorylated by the PI3K signaling

cascade (Section 19-4D) initiated by the binding of in-

sulin to the insulin receptor (these protein factors may re-

press transcription by interfering with the binding of the

above transcription factors; the mechanism of insulin sig-

naling is discussed in Sections 19-3Ac,19-3Cg, and 19-4F).

The rate of PEPCK mRNA production is determined by

the integration of these various interactions and hence of

the signals that caused them.

Section 23-1. Gluconeogenesis 879

Table 23-1 Regulators of Gluconeogenic Enzyme Activity

Allosteric Allosteric Enzyme Protein

Enzyme Inhibitors Activators Phosphorylation Synthesis

PFK ATP, citrate AMP, F2,6P

FBPase AMP, F2,6P

Pyruvate kinase Alanine F1,6P Inactivates

Pyruvate carboxylase Acetyl-CoA

PEPCK Stimulated by glucagon, thyroid hormone,

and glucocorticoids, and inhibited by insulin

PFK-2 Citrate AMP, F6P, P

Inactivates

FBPase-2 F6P Glycerol-3-P Activates

Low blood [glucose]

Increased glucagon secretion

Increased [cAMP]

Increased enzyme phosphorylation

Activation of FBPase-2 and inactivation of PFK-2

Decreased [F2,6P]

Inhibition of PFK and activation of FBPase

Increased gluconeogenesis

Figure 23-9 Hormonal regulation of [F2,6P]. This process

activates gluconeogenesis in liver in response to low blood

[glucose].

JWCL281_c23_871-900.qxd 10/19/10 11:35 AM Page 879

C. The Cori Cycle

Muscle contraction is powered by hydrolysis of ATP,

which is then regenerated through oxidative phosphoryla-

tion in the mitochondria of slow-twitch (red) muscle fibers

and by glycolysis yielding lactate in fast-twitch (white)

muscle fibers. Slow-twitch fibers also produce lactate

when ATP demand exceeds oxidative flux. The lactate is

transferred, via the bloodstream, to the liver, where it is re-

converted to pyruvate by lactate dehydrogenase and then

to glucose by gluconeogenesis. Thus, through the interme-

diacy of the bloodstream, liver and muscle participate in a

metabolic cycle known as the Cori cycle (Fig. 23-10) in

honor of Carl and Gerty Cori, who first described it.This is

the same ATP-consuming glycolysis/gluconeogenesis “fu-

tile cycle” we discussed above. Here, however, instead of

occurring in the same cell, the two pathways occur in dif-

ferent organs. Liver ATP is used to resynthesize glucose

from lactate produced in muscle. The resynthesized glu-

cose is returned to the muscle, where it is stored as glyco-

gen and used, on demand, to generate ATP for muscle

contraction. The ATP utilized by the liver for this process

is regenerated by oxidative phosphorylation. After vigor-

ous exertion, it often takes at least 30 min for all of the lac-

tate so produced to be converted to glycogen and the oxy-

gen consumption rate to return to its resting level, a

phenomenon known as oxygen debt.

2 THE GLYOXYLATE CYCLE

Plants, but not animals, possess enzymes that mediate the

net conversion of acetyl-CoA to succinate, which is then

converted, via malate,to oxaloacetate.This is accomplished

via the glyoxylate cycle (Fig. 23-11), a pathway involving

enzymes of the glyoxysome (a membranous plant or-

ganelle; Section 1-2Ad). The glyoxylate cycle involves five

enzymes, three of which also participate in the citric acid cy-

cle: citrate synthase, aconitase, and malate dehydrogenase.

The two other enzymes, isocitrate lyase and malate syn-

thase, are unique to the cycle.

The glyoxalate cycle consists of five reactions (Fig. 23-11):

Reactions 1 and 2. Glyoxysomal oxaloacetate is con-

densed with acetyl-CoA to form citrate, which is isomer-

ized to isocitrate as in the citric acid cycle. Since the gly-

oxysome contains no aconitase, Reaction 2 presumably

takes place in the cytosol.

Reaction 3. Glyoxysomal isocitrate lyase cleaves the

isocitrate to succinate and glyoxylate (hence the cycle’s

name).

Reaction 4. Malate synthase, a glyoxysomal enzyme,

condenses glyoxylate with a second molecule of acetyl-

CoA to form malate.

Reaction 5. Glyoxysomal malate dehydrogenase cat-

alyzes the oxidation of malate to oxaloacetate by NAD



thereby completing the cycle.

The glyoxylate cycle therefore results in the net conversion

of two acetyl-CoA to succinate instead of to four molecules

of CO

, as would occur in the citric acid cycle.The succinate

produced in Reaction 3 is transported to the mitochon-

drion, where it enters the citric acid cycle and is converted

to malate, which has two alternative fates: (1) It can be con-

verted to oxaloacetate in the mitochondrion, continuing

the citric acid cycle and thereby making the glyoxylate

pathway an anaplerotic process (Section 21-5b); or (2) it

can be transported to the cytosol, where it is converted to

oxaloacetate for entry into gluconeogenesis.

The overall reaction of the glyoxylate cycle can be con-

sidered to be the formation of oxaloacetate from two mol-

ecules of acetyl-CoA.

Isocitrate lyase and malate synthase, the only enzymes of

the glyoxylate pathway unique to plants, enable germinat-

ing seeds to convert their stored triacylglycerols, through

acetyl-CoA, to glucose. It had long been assumed that this

was a requirement of germination. However, a mutant of

Arabidopsis thaliana (an oilseed plant) lacking isocitrate

lyase, and hence unable to convert lipids to carbohydrate,

nevertheless germinated. This process was only inhibited

when the mutant plants were subjected to low light condi-

tions. It therefore appears that the glyoxylate cycle’s im-

portance in seedling growth is its anaplerotic function in

providing 4-carbon units to the citric acid cycle, which can

then oxidize the triacylglycerol-derived acetyl-CoA.

3 BIOSYNTHESIS OF

OLIGOSACCHARIDES AND

GLYCOPROTEINS

Oligosaccharides consist of monosaccharide units joined

together by glycosidic bonds (linkages between C1, the

anomeric carbon, of one unit and an OH group of a second

oxaloacetate  2CoA  2NADH  FADH

 2H



2 Acetyl-CoA  2NAD



 FAD

880 Chapter 23. Other Pathways of Carbohydrate Metabolism

Figure 23-10 The Cori cycle. Lactate produced by muscle

glycolysis is transported by the bloodstream to the liver, where it

is converted to glucose by gluconeogenesis.The bloodstream

carries the glucose back to the muscles, where it may be stored as

glycogen.

See the Animated Figures

Liver Blood Muscle

Glucose

Lactate Lactate

Glucose Glycogen

ADP + GDP

ATP ATP

ADP

+ GTP

gluconeogenesis

glycogenolysis

and glycolysis

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