559

CHAPTER 16

Introduction toMetabolism

1 Metabolic Pathways

2 Organic Reaction Mechanisms

A. Chemical LogicB. Group-Transfer ReactionsC. Oxidations and ReductionsD. Eliminations, Isomerizations, and RearrangementsE. Reactions That Make and Break Carbon–Carbon Bonds

3 Experimental Approaches to the Study of Metabolism

A. Metabolic Inhibitors, Growth Studies, and Biochemical Genetics

B. Isotopes in BiochemistryC. Isolated Organs, Cells, and Subcellular OrganellesD. Systems Biology

4 Thermodynamics of Phosphate Compounds

A. Phosphoryl-Transfer ReactionsB. Rationalizing the “Energy” in “High-Energy” CompoundsC. The Role of ATP

5 Oxidation–Reduction Reactions

A. The Nernst EquationB. Measurements of Redox PotentialsC. Concentration Cells

6 Thermodynamics of Life

A. Living Systems Cannot Be at EquilibriumB. Nonequilibrium Thermodynamics and the Steady StateC. Thermodynamics of Metabolic Control

Living organisms are not at equilibrium. Rather, they require

a continuous influx of free energy to maintain order in a uni-

verse bent on maximizing disorder. Metabolism is the over-

all process through which living systems acquire and utilize

the free energy they need to carry out their various func-

tions. They do so by coupling the exergonic reactions of nutri-ent oxidation to the endergonic processes required to main-tain the living state such as the performance of mechanical

work, the active transport of molecules against concentra-

tion gradients, and the biosynthesis of complex molecules.

How do living things acquire this necessary free energy?

And what is the nature of the energy coupling process?

Phototrophs (plants and certain bacteria; Section 1-1A)

acquire free energy from the sun through photosynthesis, a

process in which light energy powers the endergonic reac-

tion of CO2 and H2O to form carbohydrates and O2

(Chapter 24). Chemotrophs obtain their free energy by ox-

idizing organic compounds (carbohydrates, lipids, proteins)

obtained from other organisms, ultimately phototrophs.

This free energy is most often coupled to endergonic reac-tions through the intermediate synthesis of “high-energy”phosphate compounds such as adenosine triphosphate(ATP; Section 16-4). In addition to being completely oxi-dized, nutrients are broken down in a series of metabolicreactions to common intermediates that are used as precur-sors in the synthesis of other biological molecules.

A remarkable property of living systems is that, despite

the complexity of their internal processes, they maintain a

steady state. This is strikingly demonstrated by the observa-

tion that, over a 40-year time span, a normal human adult

consumes literally tons of nutrients and imbibes over 20,000 L

of water, but does so without significant weight change.This

steady state is maintained by a sophisticated set of metabolic

regulatory systems. In this introductory chapter to metabo-

lism, we outline the general characteristics of metabolic

pathways, study the main types of chemical reactions that

comprise these pathways, and consider the experimental

techniques that have been most useful in their elucidation.

We then discuss the free energy changes associated with re-

actions of phosphate compounds and oxidation–reduction

reactions. Finally we consider the thermodynamic nature

of biological processes, that is, what properties of life are

responsible for its self-sustaining character.

1 METABOLIC PATHWAYS

Metabolic pathways are series of consecutive enzymatic re-actions that produce specific products. Their reactants, inter-

mediates, and products are referred to as metabolites. Since

an organism utilizes many metabolites, it has many meta-

bolic pathways. Figure 16-1 shows a metabolic map for a

typical cell with many of its interconnected pathways. Each

reaction on the map is catalyzed by a distinct enzyme, of

which there are �4000 known. At first glance, this network

seems hopelessly complex. Yet, by focusing on its major

areas in the following chapters, for example, the main

pathways of glucose oxidation (the shaded areas of Fig.

16-1), we shall become familiar with its most important av-

enues and their interrelationships. Maps of metabolic path-

ways in a more readable form can be found on the Web

at http://www.expasy.org/cgi-bin/search-biochem-index,

http://www.iubmb-nicholson.org/, and http://www.genome.

ad.jp/kegg/metabolism.html.

JWCL281_c16_557-592.qxd 2/26/10 11:10 AM Page 559

560 Chapter 16. Introduction to Metabolism

Figure 16-1 Map of the major metabolic pathways in a typical cell. The main pathways of

glucose metabolism are shaded. [Designed by Donald Nicholson. Published by BDH Ltd., Poole

2, Dorset, England.]

JWCL281_c16_557-592.qxd 2/26/10 11:10 AM Page 560

The reaction pathways that comprise metabolism are

often divided into two categories:

1. Catabolism, or degradation, in which nutrients and

cell constituents are broken down exergonically to salvage

their components and/or to generate free energy.

2. Anabolism, or biosynthesis, in which biomolecules

are synthesized from simpler components.

The free energy released by catabolic processes is con-

served through the synthesis of ATP from ADP and phos-

phate or through the reduction of the coenzyme NADP� to

NADPH (Fig. 13-2). ATP and NADPH are the major free

energy sources for anabolic pathways (Fig. 16-2).

A striking characteristic of degradative metabolism is that

it converts large numbers of diverse substances (carbohy-drates, lipids, and proteins) to common intermediates. These

intermediates are then further metabolized in a central ox-

idative pathway that terminates in a few end products. Figure

16-3 outlines the breakdown of various foodstuffs, first to

their monomeric units, and then to the common intermedi-

ate, acetyl-coenzyme A (acetyl-CoA) (Fig. 21-2).

Biosynthesis carries out the opposite process. Relativelyfew metabolites, mainly pyruvate, acetyl-CoA, and the citricacid cycle intermediates, serve as starting materials for a hostof varied biosynthetic products. In the next several chapters

we discuss many degradative and biosynthetic pathways in

detail. For now, let us consider some general characteristics

of these processes.

Five principal characteristics of metabolic pathways

stem from their function of generating products for use by

the cell:

1. Metabolic pathways are irreversible. A highly exer-

gonic reaction (having a large negative free energy change)

is irreversible; that is, it goes to completion. If such a reaction

is part of a multistep pathway, it confers directionality on the

pathway; that is, it makes the entire pathway irreversible.

2. Catabolic and anabolic pathways must differ. If twometabolites are metabolically interconvertible, the pathway

from the first to the second must differ from the pathwayfrom the second back to the first:

This is because if metabolite 1 is converted to metabolite 2

by an exergonic process, the conversion of metabolite 2 to

metabolite 1 requires that free energy be supplied in order

to bring this otherwise endergonic process “back up the

hill.” Consequently, the two pathways must differ in at least

1 2A

Y X

Section 16-1. Metabolic Pathways 561

Figure 16-2 ATP and NADPH are the sources of free energyfor biosynthetic reactions. They are generated through the

degradation of complex metabolites.

Figure 16-3 Overview of catabolism. Complex metabolites

such as carbohydrates, proteins, and lipids are degraded first to

their monomeric units, chiefly glucose, amino acids, fatty acids,

and glycerol, and then to the common intermediate,

acetyl-coenzyme A (acetyl-CoA). The acetyl group is then

oxidized to CO2 via the citric acid cycle with the concomitant

reduction of NAD� and FAD. Reoxidation of these latter

coenzymes by O2 via the electron-transport chain and oxidative

phosphorylation yields H2O and ATP.

ATP

NADPH

NADP+

ADP + HPO2–4

Degradation

Simple products

Complex metabolites

Biosynthesis

CO2

H2O

O2

CO2

NH3CitricAcidCycle

Oxidativephosphorylation

Pyruvate

Acetyl-CoA

Glycolysis

GlucoseAmino acids Fatty acids & Glycerol

CarbohydratesProteins Lipids

FADNAD+

NAD+ADP

NADHATP

FADH2

FADH2

NADH

FADNAD+ NADH

ATP

ADP

JWCL281_c16_557-592.qxd 6/10/10 11:51 AM Page 561

one of their reaction steps. The existence of independent in-terconversion routes, as we shall see, is an important propertyof metabolic pathways because it allows independent controlof the two processes. If metabolite 2 is required by the cell, it

is necessary to “turn off” the pathway from 2 to 1 while

“turning on” the pathway from 1 to 2. Such independent

control would be impossible without different pathways.

3. Every metabolic pathway has a first committed step.Although metabolic pathways are irreversible, most of

their component reactions function close to equilibrium.

Early in each pathway, however, there is an irreversible

(exergonic) reaction that “commits” the intermediate it

produces to continue down the pathway.

4. All metabolic pathways are regulated. Metabolic

pathways are regulated by laws of supply and demand. In

order to exert control on the flux of metabolites through a

metabolic pathway, it is necessary to regulate its rate-limiting

step. The first committed step, being irreversible, functions

too slowly to permit its substrates and products to equili-

brate (if the reaction were at equilibrium, it would not be

irreversible). Since most of the other reactions in a path-

way function close to equilibrium, the first committed step

is often one of its rate-limiting steps. Most metabolic path-

ways are therefore controlled by regulating the enzymes

that catalyze their first committed step(s). This is an effi-

cient way to exert control because it prevents the unneces-

sary synthesis of metabolites further along the pathway

when they are not required. Specific aspects of such flux

control are discussed in Section 17-4C.

5. Metabolic pathways in eukaryotic cells occur in spe-cific cellular locations. The compartmentation of the eu-

karyotic cell allows different metabolic pathways to operate

in different locations, as is listed in Table 16-1 (these or-

ganelles are described in Section 1-2A). For example, ATP

is mainly generated in the mitochondrion but much of it is

utilized in the cytoplasm. The synthesis of metabolites in

specific membrane-bounded subcellular compartments

makes their transport between these compartments a vital

component of eukaryotic metabolism. Biological mem-

branes are selectively permeable to metabolites because of

the presence in membranes of specific transport proteins.The transport protein that facilitates the passage of ATP

through the mitochondrial membrane is discussed in

Section 20-4C, along with the characteristics of membrane

transport processes in general.The synthesis and utilization

of acetyl-CoA are also compartmentalized. This metabolic

intermediate is utilized in the cytosolic synthesis of fatty

acids but is synthesized in mitochondria. Yet there is no

transport protein for acetyl-CoA in the mitochondrial

membrane. How cells solve this fundamental problem is

discussed in Section 25-4D. In multicellular organisms, com-

partmentation is carried a step further to the level of tissues

and organs.The mammalian liver, for example, is largely re-

sponsible for the synthesis of glucose from noncarbohy-

drate precursors (gluconeogenesis; Section 23-1) so as to

maintain a relatively constant level of glucose in the circula-

tion, whereas adipose tissue is specialized for the storage

and mobilization of triacylglycerols. The metabolic interde-

pendence of the various organs is the subject of Chapter 27.

2 ORGANIC REACTION MECHANISMS

Almost all of the reactions that occur in metabolic path-

ways are enzymatically catalyzed organic reactions. Section

15-1 details the various mechanisms enzymes have at their

disposal for catalyzing reactions: acid–base catalysis, cova-

lent catalysis, metal ion catalysis, electrostatic catalysis,

proximity and orientation effects, and transition state bind-

ing. Few enzymes alter the chemical mechanisms of these

reactions, so much can be learned about enzymatic mecha-nisms from the study of nonenzymatic model reactions. We

therefore begin our study of metabolic reactions by outlin-

ing the types of reactions we shall encounter and the mech-

anisms by which they have been observed to proceed in

nonenzymatic systems.

Christopher Walsh has classified biochemical reactions

into four categories: (1) group-transfer reactions; (2) oxida-tions and reductions; (3) eliminations, isomerizations, and re-arrangements; and (4) reactions that make or break carbon–carbon bonds. Much is known about the mechanisms of

562 Chapter 16. Introduction to Metabolism

Table 16-1 Metabolic Functions of Eukaryotic Organelles

Organelle Function

Mitochondrion Citric acid cycle, electron transport and oxidative

phosphorylation, fatty acid oxidation, amino acid breakdown

Cytosol Glycolysis, pentose phosphate pathway, fatty acid

biosynthesis, many reactions of gluconeogenesis

Lysosomes Enzymatic digestion of cell components and ingested matter

Nucleus DNA replication and transcription, RNA processing

Golgi apparatus Post-translational processing of membrane and secretory

proteins; formation of plasma membrane and secretory vesicles

Rough endoplasmic reticulum Synthesis of membrane-bound and secretory proteins

Smooth endoplasmic reticulum Lipid and steroid biosynthesis

Peroxisomes (glyoxisomes in plants) Oxidative reactions catalyzed by amino acid oxidases and

catalase; glyoxylate cycle reactions in plants

JWCL281_c16_557-592.qxd 6/10/10 11:51 AM Page 562

these reactions and about the enzymes that catalyze them.

The discussions in the next several chapters focus on these

mechanisms as they apply to specific metabolic intercon-

versions. In this section we outline the four reaction cate-

gories and discuss how our knowledge of their reaction

mechanisms derives from the study of model organic reac-

tions.We begin by briefly reviewing the chemical logic used

in analyzing these reactions.

A. Chemical Logic

A covalent bond consists of an electron pair shared between

two atoms. In breaking such a bond, the electron pair can ei-

ther remain with one of the atoms (heterolytic bond cleav-age) or separate such that one electron accompanies each of

the atoms (homolytic bond cleavage) (Fig. 16-4). Homolytic

bond cleavage, which usually produces unstable radicals, oc-

curs mostly in oxidation–reduction reactions. Heterolytic

C¬H bond cleavage involves either carbanion and proton

(H�) formation or carbocation (carbonium ion) and hydride

ion (H�) formation. Since hydride ions are highly reactive

species and carbon atoms are slightly more electronegative

than hydrogen atoms, bond cleavage in which the electron

pair remains with the carbon atom is the predominant mode

of bond breaking in biochemical systems. Hydride ion

abstraction occurs only if the hydride is transferred directly

to an acceptor such as NAD� or NADP�.

Compounds participating in reactions involving het-

erolytic bond cleavage and bond formation are categorized

into two broad classes: electron rich and electron deficient.

Electron-rich compounds, which are called nucleophiles(nucleus lovers), are negatively charged or contain un-

shared electron pairs that easily form covalent bonds with

electron-deficient centers. Biologically important nucle-

ophilic groups include amino, hydroxyl, imidazole, and

sulfhydryl functions (Fig. 16-5a). The nucleophilic forms of

these groups are also their basic forms. Indeed, nucle-

ophilicity and basicity are closely related properties (Sec-

C¬H

tion 15-1Ba):A compound acts as a base when it forms a co-

valent bond with H�, whereas it acts as a nucleophile when

it forms a covalent bond with an electron-deficient center

other than H�, usually an electron-deficient carbon atom:

Electron-deficient compounds are called electrophiles(electron lovers). They may be positively charged, contain

an unfilled valence electron shell, or contain an electroneg-

ative atom.The most common electrophiles in biochemical

Nucleophilicreaction of anamine

Basic reactionof an amine R RNH2 H� H

H

H

N�

R CC O OH

H

R�

R�

R�

R�

N

�

R NH2 �

Section 16-2. Organic Reaction Mechanisms 563

Figure 16-4 Modes of bond breaking. Homolytic cleav-

age yields radicals, whereas heterolytic cleavage yields either (i)a carbanion and a proton or (ii) a carbocation and a hydride ion.

C¬H

Figure 16-5 Biologically important nucleophilic and electrophilic groups. (a) Nucleophiles are the conjugate bases

of weak acids such as the hydroxyl, sulfhydryl, amino, and

Homolytic:

Heterolytic:

Radicals

Carbanion Proton

Carbocation Hydrideion

C CC H�

C CH C�

H�

�

CC� H�

�

homolyticcleavage

(i)

(ii)

H

C H

OQ

S

(a) Nucleophiles

ROH

OQRSH

RNH 3

HN NH

R

�

R

OQRO

OQRS

ORNH2

HN N

�

�

S

S

Nucleophilicform

� H� Hydroxyl group

� H� Sulfhydryl group

� H� Amino group

� H� Imidazole group

(b) Electrophiles

H� Protons

Mn� Metal ions

R

C

R�

O Carbonyl carbon atom

R

C

R�

NH Cationic imine (Schiff base)�

�

imidazole groups. (b) Electrophiles contain an electron-deficient

atom (red).

JWCL281_c16_557-592.qxd 2/26/10 11:10 AM Page 563

systems are H�, metal ions, the carbon atoms of carbonyl

groups, and cationic imines (Fig. 16-5b).

Reactions are best understood if the electron pair re-

arrangements involved in going from reactants to products

can be traced. In illustrating these rearrangements we shall

use the curved arrow convention in which the movement of

an electron pair is symbolized by a curved arrow emanating

from the electron pair and pointing to the electron-

deficient center attracting the electron pair. For example,

imine formation, a biochemically important reaction

between an amine and an aldehyde or ketone, is represented:

NH2 OC

H+

R N

H R�R�

R�R�

C OHR+

Amine Aldehydeor

ketone

Carbinolamineintermediate

R�

R�

N

H

CR ++

H2O

Imine

OO OO

In the first reaction step, the amine’s unshared electron

pair adds to the electron-deficient carbonyl carbon atom

while one electron pair from its double bond trans-

fers to the oxygen atom. In the second step, the unshared

electron pair on the nitrogen atom adds to the electron-

deficient carbon atom with the elimination of water. At alltimes, the rules of chemical reason prevail: For example,

there are never five bonds to a carbon atom or two bonds

to a hydrogen atom.

B. Group-Transfer Reactions

The group transfers that occur in biochemical systems in-volve the transfer of an electrophilic group from one nucle-ophile to another:

They could equally well be called nucleophilic substitution

reactions. The most commonly transferred groups in bio-

chemical reactions are acyl groups, phosphoryl groups, and

glycosyl groups (Fig. 16-6):

Nucleophile Electrophile–nucleophile

Y YA AX X� �

C“O

564 Chapter 16. Introduction to Metabolism

Figure 16-6 Types of metabolic group-transfer reactions.(a) Acyl group transfer involves addition of a nucleophile (Y) to

the electrophilic carbon atom of an acyl compound to form a

tetrahedral intermediate. The original acyl carrier (X) is then

expelled to form a new acyl compound. (b) Phosphoryl group

transfer involves the in-line (with the leaving group) addition of

a nucleophile (Y) to the electrophilic phosphorus atom of a

tetrahedral phosphoryl group. This yields a trigonal bipyramidal

intermediate whose apical positions are occupied by the leaving

group (X) and the attacking group (Y). Elimination of the leaving

O

C XR

O

C Y X�

R

O�

C

Y

XR� �Y�

Tetrahedralintermediate

(a)

�

�

�

�

X

(c)

O

YO

Y

O

O O�double

displacement(SN1)

Resonance-stabilizedcarbocation (oxonium ion)single

displacement (SN2)

Y�

Y�

X�

X�

� �P

Y Y

X

P

O�O

�OO�

�OO�

X(b)

O

P

�O O�

Trigonalbipyramid

intermediate

Y�

X�

group to complete the transfer reaction results in the phosphoryl

group’s inversion of configuration. (c) Glycosyl group transfer

involves the substitution of one nucleophilic group for another at

C1 of a sugar ring. This reaction usually occurs via a double

displacement mechanism in which the elimination of the original

glycosyl carrier (X) is accompanied by the intermediate formation

of a resonance-stabilized carbocation (oxonuim ion) followed by

the addition of the adding nucleophile (Y). The reaction also

may occur via a single displacement mechanism in which Y directly

displaces X with inversion of configuration.

JWCL281_c16_557-592.qxd 2/26/10 11:10 AM Page 564

1. Acyl group transfer from one nucleophile to another

almost invariably involves the addition of a nucleophile to

the acyl carbonyl carbon atom so as to form a tetrahedral

intermediate (Fig. 16-6a). Peptide bond hydrolysis, as cat-

alyzed, for example, by chymotrypsin (Section 15-3C), is a

familiar example of such a reaction.

2. Phosphoryl group transfer proceeds via the in-line

addition of a nucleophile to a phosphoryl phosphorus atom

to yield a trigonal bipyramidal intermediate whose apexes

are occupied by the adding and leaving groups (Fig. 16-6b).

The overall reaction results in the tetrahedral phosphoryl

group’s inversion of configuration. Indeed, chiral phospho-

ryl compounds have been shown to undergo just such an

inversion. For example, Jeremy Knowles has synthesized

ATP made chiral at its �-phosphoryl group by isotopic sub-

stitution and demonstrated that this group is inverted on its

transfer to glucose in the reaction catalyzed by hexokinase(Fig. 16-7).

3. Glycosyl group transfer involves the substitution of

one nucleophilic group for another at C1 of a sugar ring

(Fig. 16-6c). This is the central carbon atom of an acetal.

Chemical models of acetal reactions generally proceed

via acid-catalyzed cleavage of the first bond to form a

resonance-stabilized carbocation at C1 (an oxonium ion).

The lysozyme-catalyzed hydrolysis of bacterial cell wall

polysaccharides (Section 15-2Bb) is such a reaction.

C. Oxidations and Reductions

Oxidation–reduction (redox) reactions involve the loss or

gain of electrons. The thermodynamics of these reactions

is discussed in Section 16-5. Many of the redox reactions

that occur in metabolic pathways involve bond

cleavage with the ultimate loss of two bonding electrons

by the carbon atom. These electrons are transferred to an

electron acceptor such as NAD� (Fig. 13-2). Whether

these reactions involve homolytic or heterolytic bond

cleavage has not always been rigorously established. In

most instances heterolytic cleavage is assumed when radi-

cal species are not observed. It is useful, however, to visu-

alize redox bond cleavage reactions as hydride

transfers as diagrammed below for the oxidation of an

alcohol by NAD�:

For aerobic organisms, the terminal acceptor for the

electron pairs removed from metabolites by their oxida-

tion is molecular oxygen (O2). Recall that this molecule is a

ground state diradical species whose unpaired electrons

have parallel spins. The rules of electron pairing (the Pauli

exclusion principle) therefore dictate that O2 can only ac-

cept unpaired electrons; that is, electrons must be trans-

ferred to O2 one at a time (in contrast to redox processes

in which electrons are transferred in pairs). Electrons that

are removed from metabolites as pairs must therefore be

passed to O2 via the electron-transport chain one at a

time. This is accomplished through the use of conjugated

coenzymes that have stable radical oxidation states and

can therefore undergo both 1e� and 2e� redox reactions.

One such coenzyme is flavin adenine dinucleotide (FAD;

Generalbase

Alcohol NAD�

Generalacid

Ketone NADH

B

B H H

H H

H

R

N

O

O

C

C

O CC

H�

R

R

�

� ��

� NH2

NH2

H

H

H

N

R

O

R

R

C¬H

C¬H

Section 16-2. Organic Reaction Mechanisms 565

Figure 16-7 The phosphoryl-transfer reaction catalyzed byhexokinase. During its transfer to the 6-OH group of glucose, the

�-phosphoryl group of ATP made chiral by isotopic substitution

undergoes inversion of configuration via a trigonal bipyramidal

intermediate.

�

�

P

Glucose

O ADP

ADP

ADP

P

O�O

O�

O�

O�

O�

O

O

P

H

H

HH

OH

OHOH

H

HO

O

HH

H

HHOH

OH

OH

HO

CH2OHO

16

16

16

18

18

18

17

17

O�17

γ

Glucose ATP

Trigonalbipyramidintermediate

H2C O

Glucose-6-phosphate

O�

JWCL281_c16_557-592.qxd 2/26/10 11:10 AM Page 565

Fig. 16-8). Flavins (substances that contain the isoallox-azine ring) can undergo two sequential one-electron trans-

fers or a simultaneous two-electron transfer that bypasses

the semiquinone state.

D. Eliminations, Isomerizations, and

Rearrangements

a. Elimination Reactions Form Carbon–Carbon

Double Bonds

Elimination reactions result in the formation of a dou-

ble bond between two previously single-bonded saturated

centers. The substances eliminated may be H2O, NH3, an

alcohol (ROH), or a primary amine (RNH2). The dehydra-

tion of an alcohol, for example, is an elimination reaction:

Bond breaking and bond making in this reaction may pro-

ceed via one of three mechanisms (Fig. 16-9a): (1) con-

certed; (2) stepwise with the bond breaking first to

form a carbocation; or (3) stepwise with the bond

breaking first to form a carbanion.

Enzymes catalyze dehydration reactions by either of

two simple mechanisms: (1) protonation of the OH group

by an acidic group (acid catalysis) or (2) abstraction of the

proton by a basic group (base catalysis). Moreover, in a step-

wise reaction, the charged intermediate may be stabilized

C¬H

C¬O

H

H HOH

H

R

R H

R�

R�

C C C C H2O�

566 Chapter 16. Introduction to Metabolism

Figure 16-8 The molecular formula and reactions of the coenzyme flavin adenine dinucleotide (FAD). The term “flavin”

is synonymous with the isoalloxazine system. The D-ribitol

residue is derived from the alcohol of the sugar D-ribose. The

FAD may be half-reduced to the stable radical FADH� or fully

reduced to FADH2 (boxes). Consequently, different

FAD-containing enzymes cycle between different oxidation

states of FAD. FAD is usually tightly bound to its enzymes, so

that this coenzyme is normally a prosthetic group rather than a

cosubstrate as is, for example, NAD�. Consequently, although

humans and other higher animals are unable to synthesize the

isoalloxazine component of flavins and hence must obtain it in

their diets [for example, in the form of riboflavin (vitamin B2)],

riboflavin deficiency is quite rare in humans. The symptoms of

riboflavin deficiency, which are associated with general

malnutrition or bizarre diets, include an inflamed tongue, lesions

in the corners of the mouth, and dermatitis.

CH2CH2

NH2

N

N

N

N

OOO

O

C O� O�

C

C

CH2

PP O

HHH

HO OH

O

D-Ribitol

Riboflavin

Flavin adenine dinucleotide (FAD)(oxidized or quinone form)

HO H

H

H

HO

HO

H

N

H

7, 8-Dimethylisoalloxazine

Adenosine

FADH2 (reduced or hydroquinone form)

R

N N O

O

H3C

H3C

NHN

O10 10a

4a

9a8a

7a 5a

8

76

9

5 4

1

3

2N N

O

H3C

H3C

NN H

H

R

N N O

O

H3C

H3C

NHN

H

H

JWCL281_c16_557-592.qxd 2/26/10 11:10 AM Page 566

by an oppositely charged active site group (electrostatic

catalysis). The glycolytic enzyme enolase (Section 17-2I) and

the citric acid cycle enzyme fumarase (Section 21-3G)

catalyze such dehydration reactions.

Elimination reactions may take one of two possible

stereochemical courses (Fig. 16-9b): (1) trans (anti) elimi-

nations, the most prevalent biochemical mechanism, and

(2) cis (syn) eliminations, which are biochemically less

common.

b. Biochemical Isomerizations Involve Intramolecular

Hydrogen Atom Shifts

Biochemical isomerization reactions involve the in-

tramolecular shift of a hydrogen atom so as to change the

location of a double bond. In such a process, a proton is re-

moved from one carbon atom and added to another. The

metabolically most prevalent isomerization reaction is the

aldose–ketose interconversion, a base-catalyzed reaction

that occurs via enediolate anion intermediates (Fig. 16-10).

The glycolytic enzyme phosphoglucose isomerase cat-

alyzes such a reaction (Section 17-2B).

Racemization is an isomerization reaction in which a hy-

drogen atom shifts its stereochemical position at a molecule’s

only chiral center so as to invert that chiral center (e.g., the

racemization of proline by proline racemase;Section 15-1Fa).

Such an isomerization is called an epimerization in a mole-

cule with more than one chiral center.

c. Rearrangements Produce Altered

Carbon Skeletons

Rearrangement reactions break and reform bonds

so as to rearrange a molecule’s carbon skeleton. There are

few such metabolic reactions. One is the conversion of

L-methylmalonyl-CoA to succinyl-CoA by methylmalonyl-CoA mutase, an enzyme whose prosthetic group is a

vitamin B12 derivative:

This reaction is involved in the oxidation of fatty acids with

an odd number of carbon atoms (Section 25-2Ec) and sev-

eral amino acids (Section 26-3Ec).

E. Reactions That Make and Break

Carbon–Carbon Bonds

Reactions that make and break carbon–carbon bonds formthe basis of both degradative and biosynthetic metabolism.

The breakdown of glucose to CO2 involves five such cleav-

ages, whereas its synthesis involves the reverse process.

Such reactions, considered from the synthetic direction, in-

volve addition of a nucleophilic carbanion to an elec-

trophilic carbon atom. The most common electrophilic

H

H

H HC C

COO�

C S CoA

O

H

H

H HC C

COO�

CC C

C

CC C

C

SCoA

methylmalonyl-CoA mutase

C

OL-Methylmalonyl-CoA Succinyl-CoA

Carbon skeleton rearrangement

C¬C

Section 16-2. Organic Reaction Mechanisms 567

Figure 16-9 Possible elimination reaction mechanisms usingdehydration as an example. Reactions may be (a) either

concerted, stepwise via a carbocation intermediate, or stepwise

via a carbanion intermediate; and may occur with (b) either trans

(anti) or cis (syn) stereochemistry.

Figure 16-10 Mechanism of aldose–ketose isomerization. The

reaction occurs with acid–base catalysis and proceeds via

cis-enediolate intermediates.

H

R H

R�

C C

H

R H

R�

C C

H

R H

R�

C C

R

H H

R�

C C

H

R H

R�

C C

H

H

C C R�

H

OH

R H�� OH�

OH�

�

H� OH��

H

H

C C R�

H

OH

R

Stepwise via a carbocation

Concerted

H

H

C C R� R�

H

OH

R

H

H

C C

H

R

Stepwise via a carbanion

H�

H�

H

H

C C R�

H

OH

R

OH�

C�

H

C R�

H

OH

R

�

(a)

(b)

trans (anti)

H� OH��

H

H

C C R�

H

OH

R cis (syn)

BH�

Ketose

Aldose

cis-Enediolate intermediates

B H

H C

CB �

�

R

H

O

O

H

H

H

C

R

C

O

O

H

H

C

R

C

O

O�

HOH

C

R

C

O�BH�

JWCL281_c16_557-592.qxd 2/26/10 11:10 AM Page 567

carbon atoms in such reactions are the sp2-hybridized car-

bonyl carbon atoms of aldehydes, ketones, esters, and CO2:

Stabilized carbanions must be generated to add to these

electrophilic centers. Three examples are the aldol con-densation (catalyzed, e.g., by aldolase; Section 17-2D),

Claisen ester condensation (citrate synthase; Section 21-3A),

and the decarboxylation of �-keto acids (isocitrate dehy-

C� C C OHO� C

drogenase, Section 21-3C; and fatty acid synthase, Section

25-4C). In nonenzymatic systems, both the aldol condensa-

tion and Claisen ester condensation involve the base-

catalyzed generation of a carbanion to a carbonyl group

(Fig. 16-11a,b). The carbonyl group is electron withdraw-

ing and thereby provides resonance stabilization by form-

ing an enolate (Fig. 16-12a). The enolate may be further

stabilized by neutralizing its negative charge. Enzymes do

so through hydrogen bonding or protonation (Fig. 16-12b),

conversion of the carbonyl group to a protonated Schiff

base (covalent catalysis; Fig. 16-12c), or by its coordination

568 Chapter 16. Introduction to Metabolism

Figure 16-11 Examples of C—C bond formation and cleavagereactions. (a) Aldol condensation, (b) Claisen ester

condensation, and (c) decarboxylation of a �-keto acid. All three

(a) Aldol condensation

(b) Claisen ester condensation

(c) Decarboxylation of a �-keto acid

B B �� R C

C

R�

H

O

H H�

R

R R�C C

C

C

R�

O

H

H

H O

R

C

C

R�

O�

H

B � R R�

R�

C C

H HR C

C

H HO

O

B B� �

�

H C

H

H

O

C SCoA H�

C

H

H

O

C SCoA

Addition toelectrophiliccenter [asin (a)]

Addition toelectrophiliccenter [asin (a)]

C

H

H

O�

C SCoA

Ketone

Acetyl-CoA

�-Keto acid

Resonance-stabilizedenolate

Resonance-stabilizedcarbanion(enolate)

Resonance-stabilized

enolate

Second ketone(electrophilic center)�

�

�

R C C O�

OO

CH2

R C

O

CH2

R C

O�

CH2

CO2

types of reactions involve generation of a resonance-stabilized

carbanion followed by addition of this carbanion to an

electrophilic center.

JWCL281_c16_557-592.qxd 2/26/10 11:10 AM Page 568

complex network of regulatory processes renders meta-

bolic pathways remarkably sensitive to the needs of the or-

ganism; the output of a pathway is generally only as great

as required.

As you might well imagine, the elucidation of a meta-

bolic pathway on all of these levels is a complex process, in-

volving contributions from a variety of disciplines. Most of

the techniques used to do so involve somehow perturbing

the system and observing the perturbation’s effect on

growth or on the production of metabolic intermediates.

One such technique is the use of metabolic inhibitors that

block metabolic pathways at specific enzymatic steps.

Another is the study of genetic abnormalities that interrupt

specific metabolic pathways. Techniques have also been de-

veloped for the dissection of organisms into their compo-

nent organs, tissues, cells, and subcellular organelles, and for

the purification and identification of metabolites as well as

the enzymes that catalyze their interconversions.The use of

isotopic tracers to follow the paths of specific atoms and

molecules through the metabolic maze has become routine.

Techniques utilizing NMR technology are able to trace

metabolites noninvasively as they react in vivo. This section

outlines the use of these various techniques.

A. Metabolic Inhibitors, Growth Studies, and

Biochemical Genetics

a. Pathway Intermediates Accumulate in the

Presence of Metabolic Inhibitors

The first metabolic pathway to be completely traced was

the conversion of glucose to ethanol in yeast by a process

known as glycolysis (Section 17-1A). In the course of these

studies, certain substances, called metabolic inhibitors, were

found to block the pathway at specific points, thereby caus-

ing preceding intermediates to build up. For instance,

iodoacetate causes yeast extracts to accumulate fructose-

1,6-bisphosphate, whereas fluoride causes the buildup of

two phosphate esters, 3-phosphoglycerate and 2-phospho-

glycerate. The isolation and characterization of these inter-

mediates was vital to the elucidation of the glycolytic path-

way: Chemical intuition combined with this information led

to the prediction of the pathway’s intervening steps. Each

of the proposed reactions was eventually shown to occur

in vitro as catalyzed by a purified enzyme.

b. Genetic Defects Also Cause Metabolic

Intermediates to Accumulate

Archibald Garrod’s realization, in the early 1900s, that

human genetic diseases are the consequence of deficien-

cies in specific enzymes (Section 1-4Cd) also contributed to

the elucidation of metabolic pathways. For example, on the

ingestion of either phenylalanine or tyrosine, individuals with

the largely harmless inherited condition known as alcap-tonuria, but not normal subjects, excrete homogentisic acidin their urine (Section 26-3Hd). This is because the liver of

alcaptonurics lacks an enzyme that catalyzes the breakdown

of homogentisic acid. Another genetic disease, phenylke-tonuria (Section 26-3Hd), results in the accumulation of

Section 16-3. Experimental Approaches to the Study of Metabolism 569

Figure 16-12 Stabilization of carbanions. (a) Carbanions

adjacent to carbonyl groups are stabilized by the formation of

enolates. (b) Carbanions adjacent to carbonyl groups hydrogen

bonded to general acids are stabilized electrostatically or by

charge neutralization. (c) Carbanions adjacent to protonated

imines (Schiff bases) are stabilized by the formation of enamines.

(d) Metal ions stabilize carbanions adjacent to carbonyl groups

by the electrostatic stabilization of the enolate.

to a metal ion (metal ion catalysis; Fig. 16-12d). The decar-

boxylation of a �-keto acid does not require base catalysis

for the generation of the resonance-stabilized carbanion;

the highly exergonic formation of CO2 provides its driving

force (Fig. 16-11c).

3 EXPERIMENTAL APPROACHES

TO THE STUDY OF METABOLISM

A metabolic pathway can be understood at several levels:

1. In terms of the sequence of reactions by which a spe-

cific nutrient is converted to end products, and the energet-

ics of these conversions.

2. In terms of the mechanisms by which each intermedi-

ate is converted to its successor. Such an analysis requires

the isolation and characterization of the specific enzymes

that catalyze each reaction.

3. In terms of the control mechanisms that regulate the

flow of metabolites through the pathway. An exquisitely

Carbanion

Carbanion Zn2�–stabilizedenolate

Enolate

Hydrogen-bondedcarbonyl

Hydrogen-bondedenolate or enol

Schiff basecarbanion (imine)

Schiff base(enamine)

(a)

(b)

(c)

(d)

C CH

O

C CH

O�

�

C

O

H �B

�

C CH or

O�

H �B

C CH

O

H B

C CH

NH� NH�

C CH

C

O

Zn2� Zn2�

�

C CH

O�

CH

CH

CH

JWCL281_c16_557-592.qxd 6/10/10 11:51 AM Page 569

phenylpyruvate in the urine (and which, if untreated, causes

severe mental retardation in infants). Ingested phenylala-

nine and phenylpyruvate appear as phenylpyruvate in the

urine of affected subjects, whereas tyrosine is metabolized

normally. The effects of these two abnormalities suggested

the pathway for phenylalanine metabolism diagrammed in

Fig. 16-13. However, the supposition that phenylpyruvate

but not tyrosine occurs on the normal pathway of pheny-

lalanine metabolism because phenylpyruvate accumulates

in the urine of phenylketonurics has proved incorrect.This

indicates the pitfalls of relying solely on metabolic blocks

and the consequent buildup of intermediates as indicators

of a metabolic pathway. In this case, phenylpyruvate for-

mation was later shown to arise from a normally minor

pathway that becomes significant only when the pheny-

lalanine concentration is abnormally high, as it is in

phenylketonurics.

c. Metabolic Blocks Can Be Generated by

Genetic Manipulation

Early metabolic studies led to the astounding discovery

that the basic metabolic pathways in most organisms are es-sentially identical. This metabolic uniformity has greatly fa-

cilitated the study of metabolic reactions. A mutation that

inactivates or deletes an enzyme in a pathway of interest can

be readily generated in rapidly reproducing microorganisms

through the use of mutagens (chemical agents that induce

genetic changes; Section 32-1A), X-rays, or genetic engineer-

ing techniques (Section 5-5). Desired mutants are identified

by their requirement of the pathway’s end product for

growth. For example, George Beadle and Edward Tatum

proposed a pathway of arginine biosynthesis in the mold

Neurospora crassa based on their analysis of three arginine-

requiring auxotrophic mutants (mutants requiring a specific

nutrient for growth), which were isolated after X-irradiation

(Fig. 16-14). This landmark study also conclusively demon-

strated that enzymes are specified by genes (Section 1-4Cd).

d. Genetic Manipulations of Higher Organisms

Provide Metabolic Insights

Transgenic organisms (Section 5-5H) constitute valu-

able resources for the study of metabolism. They can beused to both create metabolic blocks and to express genes intissues where they are not normally present. For example,

creatine kinase catalyzes the formation of phosphocreatine(Section 16-4Cd), a substance that functions to generate

ATP rapidly when it is in short supply. This enzyme is nor-

mally present in many tissues, including brain and muscle,

but not in liver. The introduction of the gene encoding cre-

atine kinase into the liver of a mouse causes the liver to

synthesize phosphocreatine when the mouse is fed crea-

tine, as demonstrated by localized in vivo NMR techniques

(Fig. 16-15; NMR is discussed below). The presence of

570 Chapter 16. Introduction to Metabolism

Figure 16-13 Pathway for phenylalanine degradation.It was originally hypothesized that phenylpyruvate was a

pathway intermediate based on the observation that

phenylketonurics excrete ingested phenylalanine

and phenylpyruvate as phenylpyruvate. Further studies, however,

demonstrated that phenylpyruvate is not a homogentisate

precursor; rather, phenylpyruvate production is significant only

when the phenylalanine concentration is abnormally high.

Instead, tyrosine is the normal product of phenylalanine

degradation.

H

C

NH+3

CH2

Phenylalanine

COO–

H

C

NH+3

CH2

Tyrosine

COO–HO

O

CCH2 COO–HO

p-Hydroxyphenylpyruvate Phenylpyruvate

CH2 COO–

HO

OH

Homogentisate

H2O + CO2

Defective inalcaptonuria

Originally unknown;defective inphenylketonuria

nonexistent:originallythought toexist and bedefective inphenylketonurics

secondarypathway

O

CCH2 COO–

JWCL281_c16_557-592.qxd 2/26/10 11:10 AM Page 570

phosphocreatine in a transgenic mouse liver protects the

animal against the sharp drop in [ATP] ordinarily caused

by fructose overload (Section 17-5Aa).This genetic manip-

ulation technique is being used to study mechanisms of

metabolic control in vivo.Metabolic pathways are regulated both by controlling the

activities of regulatory enzymes (Sections 17-4 and 18-3) and

by controlling their concentrations at the level of gene ex-

pression (Sections 31-3, 32-4, and 34-3).The important ques-

tion of how hormones and diet control metabolic processes

at the level of gene expression is being addressed through

the use of transgenic animals. Reporter genes (genes whose

products are easily detected; Section 5-5Gd) are placed un-

der the influence of promoters (genetic elements that regu-

late transcriptional initiation; Section 5-4Aa) that control

the expression of specific regulatory enzymes, and the re-

sulting composite gene is expressed in animals. The trans-

genic animals can then be treated with specific hormones

and/or diets and the production of the reporter gene prod-

uct measured. For instance, in an investigation by Richard

Hanson, the promoter for the enzyme phosphoenolpyru-vate carboxykinase (PEPCK) was attached to the structural

gene encoding growth hormone (GH). PEPCK, an impor-

tant regulatory enzyme in gluconeogenesis (the synthesis of

glucose from noncarbohydrate precursors; Section 23-1), is

normally present in liver and kidneys but not in blood. GH,

however, is secreted into the blood and its presence there

can be readily quantitated by an ELISA (Section 6-1Da).

Mice transgenic for PEPCK/GH were fed either a high-

carbohydrate/low-protein diet or a high-protein/low-carbo-

hydrate diet, which are known to decrease and increase

PEPCK activity, respectively. GH in high concentrations was

detected only in the serum of PEPCK/GH mice on a high-

protein diet, thereby indicating that the GH was synthesized

under the same dietary control as that of the PEPCK ex-

pressed by the normal gene. Thus, the activity of PEPCK in

PEPCK/GH mice can be continuously monitored, albeit in-

directly, through serum GH assays (the direct measurement

of PEPCK in mouse liver or kidney requires the sacrifice of

the animal and hence can be done only once). Such use of re-

porter genes has proved to be of great value in the study of

the genetic control of metabolism.

Section 16-3. Experimental Approaches to the Study of Metabolism 571

Figure 16-14 Pathway of arginine biosynthesis indicating thepositions of genetic blocks. All of these mutants grow in the

presence of arginine, but mutant 1 also grows in the presence of

the (nonstandard) -amino acids citrulline or ornithine and

mutant 2 grows in the presence of citrulline. This is because in

(a) Control liver

PME

PCr

15 10 5 0PPM

–5 –10 –15 –20

ATP

Pi

γα

β

(b) Creatine kinasepositive liver

Figure 16-15 The expression of creatine kinase in transgenicmouse liver as demonstrated by localized in vivo 31P NMR.(a) The spectrum of a normal mouse liver after the mouse had

been fed a diet supplemented with 2% creatine. The peaks

corresponding to inorganic phosphate (Pi), the , �, and �phosphoryl groups of ATP, and phosphomonoesters (PME) are

labeled. (b) The spectrum of the liver of a mouse transgenic for

creatine kinase that had been fed a diet supplemented with 2%

creatine. The phosphocreatine peak is labeled PCr. [After

Koretsky, A.P., Brosnan, M.J., Chen, L., Chen, J., and Van Dyke,

T.A., Proc. Natl. Acad. Sci. 87, 3114 (1990)].

Ornithine Citrulline Arginine

mutant 1 mutant 2 mutant 3

NH3�

NH3�

CH2

CH2

CH2

C

COO�

H

NH

NH3�

CH2

NH2

CH2

CH2

C

C O

COO�

H

NH

NH

NH2

CH2

NH2

CH2

CH2

C

C

COO�

H

mutant 1, an enzyme leading to the production of ornithine is

absent but enzymes farther along the pathway are normal. In

mutant 2, the enzyme catalyzing citrulline production is

defective, whereas in mutant 3 an enzyme involved in the

conversion of citrulline to arginine is lacking.

JWCL281_c16_557-592.qxd 2/26/10 11:10 AM Page 571

Modern techniques also make it possible to insert a muta-

tion that inactivates or deletes an enzyme or control protein in

a pathway of interest in higher organisms such as mice (knock-out mice; Section 5-5H). Knockout mice have proved useful

for studying metabolic control mechanisms. For example,

PEPCK activity is thought to be controlled exclusively by in-

creasing or decreasing its availability. Diet affects its produc-

tion, as we have seen. However, this demand-based control is

superimposed on the developmental regulation of PEPCK

production.The enzyme is not produced at all in early embryos

and only appears near birth,when gluconeogenesis is required

to supply the glucose that had been previously available inutero. One of the proteins thought to be responsible for the de-

velopmental regulation of PEPCK production is CCAAT/en-hancer-binding protein � (C/EBP�), a transcription factor(Section 5-4Aa; transcriptional regulation in eukaryotes is dis-

cussed in Section 34-3B). Newborn mice homozygous for

the targeted deletion of the c/ebp gene (c/ebp knockout

mice) do not produce C/EBP and therefore do not produce

PEPCK. Consequently, their livers cannot synthesize the

glucose necessary to maintain adequate blood glucose levels

once they are disconnected from the maternal circulation.

Indeed, these mice become so hypoglycemic that they die

within 8 hours of birth. Clearly C/EBP has an important

role in the developmental regulation of PEPCK.

B. Isotopes in Biochemistry

The specific labeling of metabolites such that their inter-

conversions can be traced is an indispensable technique for

elucidating metabolic pathways. Franz Knoop formulated

this technique in 1904 to study fatty acid oxidation. He fed

dogs fatty acids chemically labeled with phenyl groups

and isolated the phenyl-substituted end products from

their urine. From the differences in these products when

the phenyl-substituted starting material contained odd and

even numbers of carbon atoms he deduced that fatty acids

are degraded in C2 units (Section 25-2).

a. Isotopes Specifically Label Molecules without

Altering Their Chemical Properties

Chemical labeling has the disadvantage that the chemi-

cal properties of labeled metabolites differ from those of

normal metabolites. This problem is eliminated by labeling

molecules of interest with isotopes (atoms with the same

number of protons but a different number of neutrons in

their nuclei). Recall that the chemical properties of an

element are a consequence of its electron configuration

which, in turn, is determined by its atomic number, not its

atomic mass. The metabolic fate of a specific atom in a

metabolite can therefore be elucidated by isotopically

labeling that position and following its progress through the

metabolic pathway of interest.The advent of isotopic label-

ing and tracing techniques in the 1940s therefore revolu-

tionized the study of metabolism. (Isotope effects, which

are changes in reaction rates arising from the mass differ-

ences between isotopes, are in most instances negligible.

Where they are significant, most noticeably between hydro-

gen and its isotopes deuterium and tritium, they have been

used to gain insight into enzymatic reaction mechanisms.)

b. NMR Can Be Used to Study Metabolism

in Whole Animals

Nuclear magnetic resonance (NMR) detects specific

isotopes due to their characteristic nuclear spins. Among

the isotopes that NMR can detect are 1H, 13C, and 31P. Since

the NMR spectrum of a particular nucleus varies with its

immediate environment, it is possible to identify the peaks

corresponding to specific atoms even in relatively complex

mixtures.

The development of magnets large enough to accom-

modate animals and humans, and to localize spectra to

specific organs, has made it possible to study metabolic

pathways noninvasively by NMR techniques. Thus, 31P

NMR can be used to study energy metabolism in muscle by

monitoring the levels of ATP, ADP, inorganic phosphate,

and phosphocreatine (Figure 16-15). Indeed, a 31P NMR

system has been patented to measure the muscular meta-

bolic efficiency and maximum power of race horses while

they are walking or running on a motor-driven treadmill in

order to identify promising animals and to evaluate the

efficacy of their training and nutritional programs.

Isotopically labeling specific atoms of metabolites with13C (which is only 1.10% naturally abundant) permits the

metabolic progress of the labeled atoms to be followed by 13C

NMR. Figure 16-16 shows in vivo 13C NMR spectra of a rat

liver before and after an injection of D-[1-13C]glucose.The 13C

can be seen entering the liver and then being converted to

glycogen (the storage form of glucose; Chapter 18). 1H NMR

techniques are being used to determine the in vivo levels of a

variety of metabolites in tissues such as brain and muscle.

c. The Detection of Radioactive Isotopes

All elements have isotopes. For example, the atomic

mass of naturally occurring Cl is 35.45 D because, at least

on Earth, it is a mixture of 55% 35Cl and 45% 36Cl (other

isotopes of Cl are present in only trace amounts). Stable

isotopes are generally identified and quantitated by mass

spectrometry or NMR techniques. Many isotopes, how-

ever, are unstable; they undergo radioactive decay, a

process that involves the emission from the radioactive

nuclei of subatomic particles such as helium nuclei (� parti-cles), electrons (� particles), and/or photons (� radiation).Radioactive nuclei emit radiation with characteristic ener-

gies. For example, 3H, 14C, and 32P all emit � particles but

with respective energies of 0.018, 0.155, and 1.71 MeV. The

radiation from 32P is therefore highly penetrating, whereas

that from 3H and 14C is not. (3H and 14C, as all radioactive

isotopes, must, nevertheless, be handled with great caution

because they can cause genetic damage on ingestion.)

Radiation can be detected by a variety of techniques.

Those most commonly used in biochemical investigations

are proportional counting (known in its simplest form as

Geiger counting), liquid scintillation counting, and autora-diography. Proportional counters electronically detect the

ionizations in a gas caused by the passage of radiation.

Moreover, they can also discriminate between particles of

different energies and thus simultaneously determine the

amounts of two or more different isotopes present.

Although proportional counters are quite simple to use,

the radiation from two of the most widely used isotopes in

572 Chapter 16. Introduction to Metabolism

JWCL281_c16_557-592.qxd 2/26/10 11:10 AM Page 572

biochemical analysis, 3H and 14C, have insufficient pene-

trating power to enter a proportional counter’s detection

chamber with reasonable efficiency. This limitation is cir-

cumvented through liquid scintillation counting. In this

Section 16-3. Experimental Approaches to the Study of Metabolism 573

Figure 16-16 The conversion of [1-13C]glucose to glycogen as observed bylocalized in vivo 13C NMR. (a) The natural

abundance 13C NMR spectrum of the liver

of a live rat. Note the resonance

corresponding to C1 of glycogen. (b) The13C NMR spectrum of the liver of the same

rat �5 min after it was intravenously

injected with 100 mg of [1-13C]glucose (90%

enriched). The resonances of the C1 atom

of both the and � anomers of glucose are

clearly distinguishable from each other and

from the resonance of the C1 atom of

glycogen. (c) The 13C NMR spectrum of the

liver of the same rat �30 min after the

[1-13C]glucose injection. The C1 resonances

of both the and � glucose anomers are

much reduced while the C1 resonance of

glycogen has increased. [After Reo, N.V.,

Siegfried, B.A., and Acherman, J.J.H., J.Biol. Chem. 259, 13665 (1984)].

Table 16-2 Some Trace Isotopes of BiochemicalImportance

Stable Isotopes

Nucleus Natural Abundance (%)

2H 0.01213C 1.0715N 0.3618O 0.20

Radioactive Isotopes

Nucleus Radiation Type Half-Life

3H � 12.31 years14C � 5715 years22Na � �, � 2.60 years32P � 14.28 days35S � 87.2 days45Ca � 162.7 days60Co �, � 5.271 years125I � 59.4 days131I �, � 8.02 days

Source: Holden, N.E., in Lide, D.R. (Ed.), Handbook of Chemistry andPhysics (90th ed.), pp. 11–57 to 266, CRC Press (2009–2010).

technique, a radioactive sample is dissolved or suspended

in a solution containing fluorescent substances that emit a

pulse of light when struck by radiation. The light is de-

tected electronically so that the number of light pulses can

be counted. The emitting nucleus can also be identified

because the intensity of a light pulse is proportional to the

radiation energy (the number of fluorescent molecules

excited by a radioactive particle is proportional to the

particle’s energy).

In autoradiography, radiation is detected by its blacken-

ing of photographic film. The radioactive sample is laid on,

or in some cases mixed with, the photographic emulsion and,

after sufficient exposure time (from minutes to months),

the film is developed. Autoradiography is widely used to

locate radioactive substances in polyacrylamide gels (e.g.,

Fig. 6-27). Position-sensitive radiation counters (electronic

film) are similarly employed.

d. Radioactive Isotopes Have Characteristic

Half-Lives

Radioactive decay is a random process whose rate for a

given isotope depends only on the number of radioactive

atoms present. It is therefore a simple first-order process

whose half-life, t1/2, is a function only of the rate constant, k,for the decay process (Section 14-1Ba):

[14.5]

Because k is different for each radioactive isotope, each

has a characteristic half-life. The properties of some iso-

topes in common biochemical use are listed in Table 16-2.

t1>2 ln 2

k

0.693

k

(a)

RCOOR′

180 120 60 0ppm

C1 Glycogen

C1–βGlucose

C1–α

Glucose andglycogen

Choline N(CH3)3

CH2

C2–C5

C6

C C

(b)

(c)

JWCL281_c16_557-592.qxd 6/10/10 11:52 AM Page 573

e. Isotopes Are Indispensable for Establishing the

Metabolic Origins of Complex Metabolites and

Precursor–Product Relationships

The metabolic origins of complex molecules such as

heme, cholesterol, and phospholipids may be determined

by administering isotopically labeled starting materials to

animals and isolating the resulting products. One of the

early advances in metabolic understanding resulting from

the use of isotopic tracers was the demonstration, by David

Shemin and David Rittenberg in 1945, that the nitrogen

atoms of heme are derived from glycine rather than from

ammonia,glutamic acid,proline,or leucine (Section 26-4Aa).

They showed this by feeding rats these 15N-labeled nutri-

ents, isolating the heme in their blood, and analyzing it for15N content. Only when the rats were fed [15N]glycine did

the heme contain 15N (Fig. 16-17). This technique was also

used to demonstrate that all of cholesterol’s carbon atoms

are derived from acetyl-CoA (Section 25-6A).

Isotopic tracers are also useful in establishing the order

of appearance of metabolic intermediates, their so-called

precursor–product relationships. An example of such an

analysis concerns the biosynthesis of the complex phos-

pholipids called plasmalogens and alkylacylglycerophos-pholipids (Section 25-8Ab). Alkylacylglycerophospholipids

are ethers, whereas the closely related plasmalogens are

vinyl ethers.Their similar structures brings up the interesting

question of their biosynthetic relationship: Which is the

precursor and which is the product? Two possible modes of

synthesis can be envisioned (Fig. 16-18):

I. The starting material is converted to the vinyl ether

(plasmalogen), which is then reduced to yield the ether

(alkylacylglycerophospholipid). Accordingly, the vinyl

ether would be the precursor and the ether the product.

574 Chapter 16. Introduction to Metabolism

Figure 16-17 The metabolic origin of the nitrogen atoms in heme. Only [15N]glycine, of many15N-labeled metabolites, is an 15N-labeled heme precursor.

Figure 16-18 Two possible pathways for the biosynthesis ofether– and vinyl ether–containing phospholipids. (I) The vinyl

ether is the precursor and the ether is the product. (II) The ether

is the precursor and the vinyl ether is the product.

Glutamate

Proline

Leucine

Glycine

H2C

NH3�

COO�

15

NH4�15

�OOCCH2CH2CH

CH2 CH2 CH3

CH2

CH2

CH3

CH3CH2

CH2

COO�

�OOC

CHCH2CH

NH3�

COO�

15

NH3�15

NH15

COO�

CH2

CH2C

H3C

H3C

H3C

H2C

COO�

HHC

CH

CH

NN Fe

N

N

CH

Heme

CH2

Starting materials

Scheme I

reduction

Scheme II

Vinyl ether

Vinyl ether

Ether

Ether

CHCHO

R

CH2 R�

CHCHO

R

CH2 R�

CH2CH2O

R

CH2 R�

CH2CH2O

R

CH2 R�

oxidation

JWCL281_c16_557-592.qxd 2/26/10 11:10 AM Page 574

II. The ether is formed first and then oxidized to yield

the vinyl ether. The ether would then be the precursor and

the vinyl ether the product.

Precursor–product relationships can be most easilysorted out through the use of radioactive tracers. A pulse of

the labeled starting material is administered to an organ-

ism and the specific radioactivities of the resulting meta-

bolic products are followed with time (Fig. 16-19):

(here the * represents the radioactive label). Metabolic

pathways, as we shall see in Section 16-6Ba, normally oper-

ate in a steady state; that is, the throughput of metabolites

in each of its reaction steps is equal. Moreover, the rates of

most metabolic reactions are first order for a given sub-

strate. Making these assumptions, we note that the rate of

change of B’s radioactivity, [B*], is equal to the rate of pas-

sage of label from A* to B* less the rate of passage of label

from B* to the pathway’s next product:

[16.1]

where k is the pseudo-first-order rate constant for both the

conversion of A to B and the conversion of B to its prod-

uct, and t is time. Inspection of this equation indicates the

criteria that must be met to establish that A is the precur-

sor of B (Fig. 16-19):

1. Before the radioactivity of the product [B*] is maxi-

mal, d[B*]>dt � 0, so [A*] � [B*]; that is, while the radioac-tivity of a product is rising, it should be less than that of itsprecursor.

2. When [B*] is maximal, d[B*]>dt 0, so [A*] [B*];

that is, when the radioactivity of a product is at its peak, itshould be equal to that of its precursor. This result also im-

plies that the radioactivity of a product peaks after that of itsprecursor.

d [B*]

dt k [A*] � k [B*] k( [A*] � [B*] )

Starting material* ¡ A* ¡ B* ¡ later products*

3. After [B*] begins to decrease, d[B*]>dt � 0, so [A*] �[B*]; that is, after the radioactivity of a product has peaked,it should remain greater than that of its precursor.

Such a determination of the precursor–product rela-

tionship between alkylacylglycerophospholipid and plas-

malogen, using 14C-labeled starting materials, indicated

that the ether is the precursor and the vinyl ether is the

product (Fig. 16-18, Scheme II).

C. Isolated Organs, Cells, and Subcellular Organelles

In addition to understanding the chemistry and catalytic

events that occur at each step of a metabolic pathway, it is

important to learn where a given pathway occurs within an

organism. Early workers studied metabolism in whole ani-

mals. For example, the role of the pancreas in diabetes was

established by Frederick Banting and Charles Best in 1921

by surgically removing that organ from dogs and observing

that these animals then developed the disease.

The metabolic products produced by a particular organ

can be studied by organ perfusion or in tissue slices. In or-

gan perfusion, a specific organ is surgically removed from

an animal and the organ’s arteries and veins are connected

to an artificial circulatory system. The composition of the

material entering the organ can thereby be controlled and

its metabolic products monitored. Metabolic processes can

be similarly studied in slices of tissue thin enough to be

nourished by free diffusion in an appropriate nutrient solu-

tion. Otto Warburg pioneered the tissue slice technique in

the early twentieth century through his studies of respiration,

in which he used a manometer to measure the changes in

gas volume above tissue slices as a consequence of their O2

consumption.

A given organ or tissue generally contains several cell

types. Cell sorters are devices that can separate cells ac-

cording to type once they have been treated with the en-

zymes trypsin and collagenase to destroy the intercellular

matrix that binds them into a tissue. This technique allows

further localization of metabolic function. A single cell

type may also be grown in tissue culture for study. Al-

though culturing cells often results in their loss of differen-

tiated function, techniques have been developed for main-

taining several cell types that still express their original

characteristics.

As discussed in Section 16-1, metabolic pathways in eu-

karyotes are compartmentalized in various subcellular or-

ganelles (Table 16-1). For example, oxidative phosphoryla-

tion occurs in the mitochondrion, whereas glycolysis and

fatty acid biosynthesis occur in the cytosol. Such observa-

tions are made by breaking cells open and fractionating

their components by differential centrifugation (Section

6-1B),possibly followed by zonal ultracentrifugation through

a sucrose density gradient or by equilibrium density gradi-

ent ultracentrifugation in a CsCl density gradient, which, re-

spectively, separate particles according to their size and

density (Section 6-5B). The cell fractions are then analyzed

for biochemical function.

Section 16-3. Experimental Approaches to the Study of Metabolism 575

Figure 16-19 The flow of a pulse of radioactivity from precursorto product. At point 1, product radioactivity (B*, purple) is

increasing and is less than that of its precursor (A*, orange); at

point 2, product radioactivity is maximal and is equal to that of

its precursor; and at point 3, product radioactivity is decreasing

and is greater than that of its precursor.

Time after addition of labeled starting material

[A*]

1

[B*]

Spe

cific

rad

ioac

tivity

2 3

JWCL281_c16_557-592.qxd 2/26/10 11:10 AM Page 575

D. Systems Biology

Metabolism has traditionally been studied by hypothesis-

based research: isolating individual enzymes and metabo-

lites and assembling them into metabolic pathways as

guided by experimentally testable hypotheses. This is a re-

ductionist approach: the explanation of the workings of a

system in terms of its component parts. A different, so-

called integrative approach, systems biology, has recently

emerged with the advent of complete genome sequences,

the development of rapid and sensitive techniques for ana-

lyzing large numbers of gene transcripts, proteins, and

metabolites all at once, and the development of new com-

putational and mathematical tools. Systems biology is

discovery-based: collecting and integrating enormous

amounts of data in searchable databases so that the prop-

erties and dynamics of entire biological networks can be

analyzed. As a result, our understanding of the path from

genotype to phenotype has expanded. In addition to the

central dogma of molecular biology (Section 5-4), that a

single gene composed of DNA is transcribed to mRNA,

which is translated to a single protein that influences

metabolism, we are increasingly taking into account the

genome, transcriptome, proteome, and metabolome (the

complete set of a cell’s metabolites) and their interrelation-

ships (Fig. 16-20). The term bibliome (Greek: biblion,book) has even been coined to denote the systematic

incorporation of pre-existing information about reaction

mechanisms and metabolic pathways. In the following

paragraphs we discuss some of these emerging technolo-

gies and new fields of study.

a. Transcriptomics

The overall metabolic capabilities of an organism are

encoded by its genome (its entire complement of genes). In

principle, it should be possible to reconstruct a cell’s meta-

bolic activities from its genomic sequence. However, at

present, this can be done only in a general sense. For exam-

ple, the 4.0-Mb genome of Vibrio cholerae, the bacterium

that causes cholera, contains a large repertoire of genes en-

coding transport proteins and enzymes for catabolizing a

576 Chapter 16. Introduction to Metabolism

Figure 16-20 The relationship between genotype and phenotype. The path from genetic information (genotype) to

metabolic function (phenotype) has several steps. Portions of the

genome are transcribed to produce the transcriptome, which

wide range of nutrients. This is consistent with the compli-

cated lifestyle of V. cholerae, which can live on its own, in

association with zooplankton, or in the human gastroin-

testinal tract (where it causes cholera; Section 19-2Cd).

However, a simple catalog of an organism’s genes does not

reveal how these genes function. Thus, some genes are

expressed continuously at high levels, whereas others are

expressed rarely, for example, only when the organism

encounters a particular metabolite.

Creating an accurate picture of gene expression is the

goal of transcriptomics, the study of a cell’s transcriptome

(its entire complement of mRNAs). Identifying and quan-

tifying all the transcripts from a single cell type reveals

which genes are active. Cells transcribe thousands of genes

at once so this study requires the use of DNA microarray

technology (Section 7-6B). For example, Fig. 7-39 shows a

DNA microarray that indicates the differences in gene ex-

pression between yeast grown in the presence and absence

of glucose.

Differences in the expression of particular genes have

been correlated with many developmental processes or

growth patterns. For example, DNA microarrays have been

used to profile the patterns of gene expression in tumor

cells because different types of tumors express different

types and amounts of proteins (Section 34-3B). This infor-

mation is useful in choosing how best to treat a cancer.

b. Proteomics

The correlation between the amount of a particular

mRNA and the amount of its protein product is imperfect.

This is because the various mRNAs and their correspon-

ding proteins are synthesized and degraded at different

rates. Furthermore, many proteins are post-translationally

modified, sometimes in several different ways (e.g., by

phosphorylation or glycosylation). Consequently, the num-

ber of unique proteins in a cell exceeds the number of

unique mRNAs.

A more reliable way than transcriptomics to assess gene

expression is to examine a cell’s proteome, the complete

set of proteins that the cell synthesizes. This proteomics

Metabolome

Genome

DNAGenotype

Transcriptome

Proteome

Phenotype Metabolites

Substrates

mRNA

Enzyme

directs the synthesis of the proteome, whose various activities are

responsible for synthesizing and degrading the components of

the metabolome.

JWCL281_c16_557-592.qxd 2/26/10 11:10 AM Page 576

approach requires that the proteins first be separated,usually

by two-dimensional (2D) gel electrophoresis (Section 6-4D).

Individual proteins are then identified by using tandem

mass spectrometry to obtain amino acid sequence infor-

mation (Section 7-1Ia) and correlating it with protein

sequence databases. All the proteins that are contained in

a cell or tissue under a given set of conditions can thereby

be catalogued.

One can compare all the proteins synthesized by a cell

under two different sets of conditions by using different

isotopically labeled reagents that are either contained in

the growth medium (e.g., deuterated amino acids) or that

are reacted with the cell extract. One technique for label-

ing cellular proteins uses isotope-coded affinity tags(ICATs), which are analogous to the differently fluoresc-

ing dyes that are used to label cDNAs.

An ICAT contains three functional elements: an

iodoacetyl group to react with Cys residues, a linker that

contains either 8 hydrogen (light) or 8 deuterium (heavy)

atoms, and biotin, a coenzyme (Section 23-1Ab) that is

used as a biotechnology tool because of its extremely tight

binding to the protein avidin (K 10�15 M; Fig. 16-21a).

Avidin is immobilized on a chromatographic resin so that

the ICAT-labeled peptides can be isolated by biotin/avidin

affinity chromatography (Section 6-3C).

The ICAT procedure is illustrated in Fig. 16-21b. Two

protein mixtures representing two different growth condi-

tions are treated with light (d0) or heavy (d8) versions of

the ICAT reagent. The labeled protein mixtures are com-

bined and digested with trypsin to form Cys-containing la-

beled peptides, which are then isolated by biotin/avidin

affinity chromatography. Individual peptides are separated

by liquid chromatography and detected by mass spectrom-

etry (LC/MS). The ratio of the intensities of the light and

heavy peptide signals indicates the relative peptide abun-

dance in the two samples. Tandem mass spectrometry

Section 16-3. Experimental Approaches to the Study of Metabolism 577

Figure 16-21 The isotope-coded affinity tag (ICAT) methodfor quantitative proteome analysis. (a) An example of an ICAT

reagent that contains an iodoacetyl reactive group, a linker, and a

biotin residue. X denotes the position of hydrogen (d0) or

deuterium (d8). (b) The ICAT strategy for differential labeling of

proteins expressed by cells under two different sets of conditions.

(1) Proteins from states A and B are respectively treated with

the light (d0) and heavy (d8) forms of the ICAT reagent. (2) The

labeled protein mixtures are combined. (3) The labeled proteins

are digested with trypsin to form Cys-containing labeled

peptides. These peptides are then purified by biotin/avidin

Two cell states:Reduce and label cysteineswith ICAT reagent

Digest and affinitypurify labeled peptides

Analyze byLC/MS and

MS/MS

200

1405.0 1426.0

1417

1409

Perc

enta

ge in

tens

ityPe

rcen

tage

inte

nsity

0

100

100

400 600Mass (m/z)

Mass (m/z)

800

Biotin Reactive groupLinker

O

XX

XX

XX

XX

R(d0)-biotin(b)

(a)

R(d8)-biotin1

Quantify by MS

Identify by MS/MS

CysA

B Cys

NHHN

SONH

NHOI

O

O

32

1

4

5

affinity chromatography. The purified peptides are analyzed by

mass spectrometry in two ways: (4) Liquid chromatography

followed by mass spectrometry (LC/MS) is used to quantitate

the peptides. The ratio of the signal intensities from the

corresponding light and heavy peptides indicates the relative

peptide abundance in the two mixtures. (5) Tandem mass

spectrometry (MS/MS) is used to determine the amino acid

sequence of each peptide and to thereby identify the protein

from which it is derived by comparing the peptide’s sequence to

those in a database of all known proteins.

JWCL281_c16_557-592.qxd 2/26/10 11:10 AM Page 577

(MS/MS) is then used to sequence each peptide and deter-

mine its identity. This method was used to identify many of

the yeast proteins whose mRNA concentrations increased

or decreased when glucose was absent from the growth

medium (Fig. 7-39). A hope for the future is that samples

from diseased and normal subjects can be compared in this

manner to find previously undetected disease markers that

would allow early diagnosis of various diseases.

c. Metabolomics

In order to describe a cell’s functional state (its pheno-

type) we need, in addition to the cell’s genome, transcrip-

tome, and proteome, a quantitative description of all of the

metabolites it contains under a given set of conditions, its

metabolome. However, a cell or tissue contains thousands

of metabolites with greatly varying properties, so that iden-

tifying and quantifying all these substances is a daunting

task, requiring many different analytical tools. Conse-

quently, this huge undertaking is often subdivided. For ex-

ample, lipidomics is the subdiscipline of metabolomicsaimed at identifying and characterizing all lipids in a cell un-

der a particular set of conditions, including how these lipids

influence membrane structure, cell signaling, gene expres-

sion, cell–cell interactions, etc., whereas glycomics similarly

identifies and characterizes all the carbohydrates in a cell.

A recently constructed model of the human

metabolome—based on 1496 protein-encoding genes, 2004

proteins, 2766 metabolites, and 3311 metabolic and trans-

port reactions—has been used to simulate 288 known meta-

bolic functions in a variety of cell and tissue types. This insilico (computerized) model is expected to provide a frame-

work for future advances in human systems biology.

4 THERMODYNAMICS OF

PHOSPHATE COMPOUNDS

The endergonic processes that maintain the living state aredriven by the exergonic reactions of nutrient oxidation. This

coupling is most often mediated through the syntheses of a

few types of “high-energy” intermediates whose exergonic

consumption drives endergonic processes. These intermedi-

ates therefore form a sort of universal free energy “currency”

through which free energy–producing reactions “pay for” the

free energy–consuming processes in biological systems.

Adenosine triphosphate (ATP; Fig. 16-22), which occurs

in all known life-forms, is the “high-energy” intermediate

that constitutes the most common cellular energy currency.

Its central role in energy metabolism was first recognized in

1941 by Fritz Lipmann and Herman Kalckar. ATP consists

of an adenosine moiety to which three phosphoryl groups( ) are sequentially linked via a phosphoester bondfollowed by two phosphoanhydride bonds. Adenosinediphosphate (ADP) and 5�-adenosine monophosphate(AMP) are similarly constituted but with only two and

one phosphoryl units, respectively.

In this section we consider the nature of phosphoryl-trans-

fer reactions, discuss why some of them are so exergonic, and

outline how the cell consumes and regenerates ATP.

¬PO2�3

A. Phosphoryl-Transfer Reactions

Phosphoryl-transfer reactions,

are of enormous metabolic significance. Some of the most

important reactions of this type involve the synthesis and

hydrolysis of ATP:

where Pi and PPi, respectively, represent orthophosphateand pyrophosphate in any of their ioniza-

tion states. These highly exergonic reactions are coupled tonumerous endergonic biochemical processes so as to drivethem to completion. Conversely, ATP is regenerated by cou-pling its formation to a more highly exergonic metabolicprocess (the thermodynamics of coupled reactions is dis-

cussed in Section 3-4C).

To illustrate these concepts, let us consider two exam-

ples of phosphoryl-transfer reactions.The initial step in the

metabolism of glucose is its conversion to glucose-6-phos-

phate (Section 17-2A). Yet the direct reaction of glucose

and Pi is thermodynamically unfavorable (Fig. 16-23a). In

biological systems, however, this reaction is coupled to the

exergonic hydrolysis of ATP, so the overall reaction is ther-

modynamically favorable. ATP can be similarly rege-

nerated by coupling its synthesis from ADP and Pi to the

even more exergonic hydrolysis of phosphoenolpyruvate(Fig. 16-23b; Section 17-2J).

The bioenergetic utility of phosphoryl-transfer reactionsstems from their kinetic stability to hydrolysis combinedwith their capacity to transmit relatively large amounts offree energy. The G°¿ values of hydrolysis of several phos-

phorylated compounds of biochemical importance are tab-

ulated in Table 16-3. The negatives of these values are often

referred to as phosphate group-transfer potentials; they

(P2O4�7 )(PO4

3�)

ATP � H2O Δ AMP � PPi

ATP � H2O Δ ADP � Pi

R1¬O¬PO32� � R2¬OH Δ R1¬OH � R2¬O¬PO3

2�

578 Chapter 16. Introduction to Metabolism

Figure 16-22 The structure of ATP indicating its relationshipto ADP, AMP, and adenosine. The phosphoryl groups, starting with

that on AMP, are referred to as the , �, and � phosphates. Note the

difference between phosphoester and phosphoanhydride bonds.

N

CH2

N

NH2

N

N

OO

O

O–

H HHH

HO

Adenosine

OH

POPO

Phosphoanhydridebonds

Phosphoesterbond

P

OO

O–O–

–Oγ β α

AMP

ADP

ATP

JWCL281_c16_557-592.qxd 2/26/10 11:10 AM Page 578

are a measure of the tendency of phosphorylated com-

pounds to transfer their phosphoryl groups to water. Note

that ATP has an intermediate phosphate group-transfer

potential. Under standard conditions, the compounds

above ATP in Table 16-3 can spontaneously transfer a

phosphoryl group to ADP to form ATP, which can, in turn,

spontaneously transfer a phosphoryl group to the hydroly-

sis products (ROH form) of the compounds below it.

a. �G of ATP Hydrolysis Varies with pH, Divalent

Metal Ion Concentration, and Ionic Strength

The G of a reaction varies with the total concentra-

tions of its reactants and products and thus with their ionic

states (Eq. [3.15]). The G’s of hydrolysis of phosphory-

lated compounds are therefore highly dependent on pH,

divalent metal ion concentration (divalent metal ions such

as Mg2� have high phosphate-binding affinities), and ionic

strength. Reasonable estimates of the intracellular values

of these quantities as well as of [ATP], [ADP], and [Pi]

(which are generally on the order of millimolar) indicate

that ATP hydrolysis under physiological conditions has

G � �50 kJ � mol�1 rather than the �30.5 kJ � mol�1 of its

G°¿. Nevertheless, for the sake of consistency in compar-

ing reactions, we shall usually refer to the latter value.

The above situation for ATP is not unique. It is impor-

tant to keep in mind that within a given cell, the concentra-tions of most substances vary both with location and time.Indeed, the concentrations of many ions, coenzymes, andmetabolites commonly vary by several orders of magnitudeacross membranous organelle boundaries. Unfortunately, it

is usually quite difficult to obtain an accurate measurement

of the concentration of any particular chemical species in a

specific cellular compartment. The G’s for most in vivoreactions are therefore little more than estimates.

Section 16-4. Thermodynamics of Phosphate Compounds 579

Figure 16-23 Some overall coupled reactions involving ATP.(a) The phosphorylation of glucose to form glucose-6-

phosphate and ADP. (b) The phosphorylation of ADP by

phosphoenolpyruvate to form ATP and pyruvate. Each reaction

Phosphoenolpyruvate Pyruvate

Endergonichalf-reaction 1

(a)

(b)

Exergonichalf-reaction 2

Exergonichalf-reaction 1

Overallcoupled reaction

Pi

Pi

glucose

glucose

glucose-6-P

glucose-6-P

ATP

H2O

CH2 CH3 C

O

C

COO–

COO–

OPO2–

+13.8

–30.5

+30.5

–31.4

–16.7

– 61.9

H2O

H2O

++

+

ATP

ATP

H2O+

ATP +

+

+

ADP

+

CH3 C

O

COO– +

Pi+

Pi+

ADP +

ADP

ADPOverallcoupled reaction

3

CH2 C

COO–

OPO2–3

Endergonichalf-reaction 2

ΔG°� (kJ • mol–1)

ΔG°� (kJ • mol–1)

Table 16-3 Standard Free Energies of Phosphate Hydrolysisof Some Compounds of Biological Interest

Compound G°¿ (kJ � mol�1)

Phosphoenolpyruvate �61.9

1,3-Bisphosphoglycerate �49.4

ATP (S AMP � PPi) 45.6

Acetyl phosphate �43.1

Phosphocreatine �43.1

ATP (S ADP � Pi) 30.5

Glucose-1-phosphate �20.9

PPi �19.2

Fructose-6-phosphate �13.8

Glucose-6-phosphate �13.8

Glycerol-3-phosphate �9.2

Source: Mostly from Jencks, W.P., in Fasman, G.D. (Ed.), Handbook ofBiochemistry and Molecular Biology (3rd ed.), Physical and ChemicalData, Vol. I, pp. 296–304, CRC Press (1976).

has been conceptually decomposed into a direct phosphorylation

step (half-reaction 1) and a step in which ATP is hydrolyzed

(half-reaction 2). Both half-reactions proceed in the direction in

which the overall reaction is exergonic ( G � 0).

JWCL281_c16_557-592.qxd 6/10/10 11:52 AM Page 579

B. Rationalizing the “Energy”

in “High-Energy” Compounds

Bonds whose hydrolysis proceeds with large negative values

of G°¿ (customarily more negative than �25 kJ � mol�1)

are often referred to as “high-energy” bonds or “energy-rich” bonds and are frequently symbolized by the squiggle

(�).Thus ATP may be represented as AR¬P�P�P, where

A, R, and P symbolize adenyl, ribosyl, and phosphoryl

groups, respectively.Yet, the phosphoester bond joining the

adenosyl group of ATP to its -phosphoryl group appears

to be not greatly different in electronic character from the

so-called “high-energy” bonds bridging its � and � phos-

phoryl groups. In fact, none of these bonds have any un-

usual properties, so the term “high-energy” bond is some-

what of a misnomer. (In any case, it should not be confused

with the term “bond energy,” which is defined as the energy

required to break, not hydrolyze, a covalent bond.) Why

then, should the phosphoryl-transfer reactions of ATP be so

exergonic? The answer comes from the comparison of the

stabilities of the reactants and products of these reactions.

Several different factors appear to be responsible for

the “high-energy” character of phosphoanhydride bonds

such as those in ATP (Fig. 16-24):

1. The resonance stabilization of a phosphoanhydride

bond is less than that of its hydrolysis products. This is

because a phosphoanhydride’s two strongly electron-

withdrawing phosphoryl groups must compete for the lone

pair of electrons of its bridging oxygen atom, whereas this

competition is absent in the hydrolysis products. In other

words, the electronic requirements of the phosphoryl

groups are less satisfied in a phosphoanhydride than in its

hydrolysis products.

2. Of perhaps greater importance is the destabilizing

effect of the electrostatic repulsions between the charged

groups of a phosphoanhydride in comparison to that of its

hydrolysis products. In the physiological pH range, ATP

has three to four negative charges whose mutual electro-

static repulsions are partially relieved by ATP hydrolysis.

3. Another destabilizing influence, which is difficult to as-

sess, is the smaller solvation energy of a phosphoanhydride in

comparison to that of its hydrolysis products. Some estimates

suggest that this factor provides the dominant thermody-

namic driving force for the hydrolysis of phosphoanhydrides.

A further property of ATP that suits it to its role as an

energy intermediate stems from the relative kinetic stabil-

ity of phosphoanhydride bonds to hydrolysis. Most types of

anhydrides are rapidly hydrolyzed in aqueous solution.

Phosphoanhydride bonds, however, have unusually large

free energies of activation. Consequently, ATP is reason-

ably stable under physiological conditions but is readily

hydrolyzed in enzymatically mediated reactions.

a. Other “High-Energy” Compounds

The compounds in Table 16-3 with phosphate group-

transfer potentials significantly greater than that of ATP

have additional destabilizing influences:

1. Acyl phosphates. The hydrolysis of acyl phosphates(mixed phosphoric–carboxylic anhydrides), such as acetylphosphate and 1,3-bisphosphoglycerate,

is driven by the same competing resonance and differential

solvation influences that function in the hydrolysis of phos-

phoanhydrides. Apparently these effects are more pro-

nounced for acyl phosphates than for phosphoanhydrides.

2. Enol phosphates. The high phosphate group-transfer

potential of an enol phosphate, such as phosphoenolpyru-

vate (Fig. 16-23b), derives from its enol hydrolysis product

being less stable than its keto tautomer. Consider the hy-

drolysis reaction of an enol phosphate as occurring in two

steps (Fig. 16-25). The hydrolysis step is subject to the

driving forces discussed above. It is therefore the highlyexergonic enol–keto conversion that provides phospho-enolpyruvate with the added thermodynamic impetus tophosphorylate ADP to form ATP.

3. Phosphoguanidines. The high phosphate group-trans-

fer potentials of phosphoguanidines, such as phosphocrea-tine and phosphoarginine, largely result from the compet-

ing resonances in their guanidino group, which are even

more pronounced than they are in the phosphate group of

phosphoanhydrides (Fig. 16-26). Consequently, phospho-

creatine can phosphorylate ADP (see Section 16-4Cd).

Compounds such as glucose-6-phosphate or glycerol-3-phosphate,

H

H

OH

OH

α-D-Glucose-6-phosphate L-Glycerol-3-phosphate

OH

CH2OPO2 3–

CH2OHO

H

H

HCHO HO

H

CH2OPO2 3–

1,3-Bisphosphoglycerate

Acetyl phosphateCH3 OPO3

2�C

O

CH OPO32��2O3POCH2 C

OOH

580 Chapter 16. Introduction to Metabolism

O O

O O

�O O�

OP P

H2O

or

or

O

O O

�O O�

�P PH H OO O

Figure 16-24 Resonance and electrostatic stabilization in aphosphoanhydride and its hydrolysis products. The competing

resonances (curved arrows from the central O) and

charge–charge repulsions (zigzag line) between the phosphoryl

groups of a phosphoanhydride decrease its stability relative to its

hydrolysis products.

JWCL281_c16_557-592.qxd 6/10/10 11:52 AM Page 580

which are below ATP in Table 16-3, have no significantly

different resonance stabilization or charge separation in

comparison with their hydrolysis products. Their free ener-

gies of hydrolysis are therefore much less than those of the

preceding “high-energy” compounds.

C. The Role of ATP

As Table 16-3 indicates, in the thermodynamic hierarchy ofphosphoryl-transfer agents, ATP occupies the middle rank.This enables ATP to serve as an energy conduit between

“high-energy” phosphate donors and “low-energy” phos-

phate acceptors (Fig. 16-27). Let us examine the general

biochemical scheme of how this occurs.

In general, the highly exergonic phosphoryl-transfer

reactions of nutrient degradation are coupled to the for-

mation of ATP from ADP and Pi through the auspices of

various enzymes known as kinases, enzymes that catalyze

the transfer of phosphoryl groups between ATP and other

molecules. Consider the two reactions in Fig. 16-23b. If

carried out independently, these reactions would not influ-

ence each other. In the cell, however, the enzyme pyruvate

Section 16-4. Thermodynamics of Phosphate Compounds 581

Figure 16-25 Hydrolysis of phosphoenolpyruvate. The reaction is broken down into two steps,

hydrolysis and tautomerization.

Phosphoenol-pyruvate

COO–

C O + H2OPO2 3–

CH H

Hydrolysis

COO–

C O H

H

+ HPO2 4–

CH H

Pyruvate(enol form)

Pyruvate(keto form)

COO–

C O

CH H

Tautomerization

COO–

C O

HCH

H

COO–

C O + H2OPO2 3–

CH H

Overall reaction + HPO2 4–

COO–

C O

HCH

H

�

� ΔG°� = –61.9 kJ • mol–1

ΔG°� = –46 kJ • mol–1

ΔG°� = –16 kJ • mol–1

Phosphocreatine

N

R

CO–

H2N

NH

NH+

P

Oor or

O–C

X O–

O

+

R = CH2 X = CH32

3

2

;

PhosphoarginineCO–CHCH2CH2R = CH2 X = H;

Figure 16-26 Competing resonances in phosphoguanidines.

Figure 16-27 The flow of phosphoryl groups from “high-energy” phosphate donors, via the ATP–ADP system, to “low-energy” phosphate acceptors.

–60

–50

–40

–30

–20

–10

0

Phosphoenolpyruvate

1,3-Bisphosphoglycerate

Phosphocreatine

Glucose-6-phosphate

Glycerol-3-phosphate

“High-energy”phosphate compounds

“Low-energy”phosphate compounds

ΔG°′

of h

ydro

lysi

s (k

J • m

ol–1

)

~P~P

~P

P

P

ATP

JWCL281_c16_557-592.qxd 2/26/10 11:10 AM Page 581

kinase couples the two reactions by catalyzing the trans-

fer of the phosphoryl group of phosphoenolpyruvate di-

rectly to ADP to result in an overall exergonic reaction

(Section 17-2J).

a. Consumption of ATP

In its role as the universal energy currency of living sys-

tems, ATP is consumed in a variety of ways:

1. Early stages of nutrient breakdown. The exergonic

hydrolysis of ATP to ADP may be enzymatically coupled

to an endergonic phosphorylation reaction to form “low-

energy” phosphate compounds.We have seen one example

of this in the hexokinase-catalyzed formation of glucose-

6-phosphate (Fig. 16-23a). Another example is the phos-phofructokinase-catalyzed phosphorylation of fructose-6-phosphate to form fructose-1,6-bisphosphate (Fig. 16-28).

Both of these reactions occur in the first stage of glycolysis

(Section 17-2).

2. Interconversion of nucleoside triphosphates. Many

biosynthetic processes, such as the synthesis of proteins

and nucleic acids, require nucleoside triphosphates other

than ATP. These include the ribonucleoside triphosphates

CTP, GTP, and UTP, which, together with ATP, are utilized,

for example, in the biosynthesis of RNA (Section 31-2) and

the deoxyribonucleoside triphosphate DNA precursors

dATP, dCTP, dGTP, and dTTP (Section 5-4C). All these

nucleoside triphosphates (NTPs) are synthesized from

ATP and the corresponding nucleoside diphosphate(NDP) in reactions catalyzed by the nonspecific enzyme

nucleoside diphosphate kinase:

The G°¿ values for these reactions are nearly zero, as

might be expected from the structural similarities among

the NTPs. These reactions are driven by the depletion

of the NTPs through their exergonic hydrolysis in the biosyn-

thetic reactions in which they participate (Section 3-4C).

3. Physiological processes. The hydrolysis of ATP to

ADP and Pi energizes many essential endergonic physio-

logical processes such as chaperone-assisted protein fold-

ing (Section 9-2C), muscle contraction (Section 35-3B),

and the transport of molecules and ions against concentra-

tion gradients (Section 20-3). In general, these processes

result from conformational changes in proteins (enzymes)

that occur in response to their binding of ATP. This is fol-

lowed by the exergonic hydrolysis of ATP and release of

ATP � NDP Δ ADP � NTP

ADP and Pi, thereby causing these processes to be unidi-

rectional (irreversible).

4. Additional phosphoanhydride cleavage in highlyendergonic reactions. Although many reactions involving

ATP yield ADP and Pi (orthophosphate cleavage), others

yield AMP and PPi (pyrophosphate cleavage). In these lat-

ter cases, the PPi is rapidly hydrolyzed to 2Pi by inorganicpyrophosphatase ( G°¿ �19.2 kJ � mol�1) so that thepyrophosphate cleavage of ATP ultimately results in thehydrolysis of two “high-energy” phosphoanhydride bonds.The attachment of amino acids to tRNA molecules for pro-

tein synthesis is an example of this phenomenon (Fig. 16-29

and Section 32-2C).The two steps of the reaction involving

the amino acid are readily reversible because the free ener-

gies of hydrolysis of the bonds formed are comparable to

that of ATP hydrolysis. The overall reaction is driven to

completion by the hydrolysis of PPi, which is essentially ir-

reversible. Nucleic acid biosynthesis from the appropriate

NTPs also releases PPi (Sections 30-1A and 31-2). The free

energy changes of these vital reactions are around zero, so

the subsequent hydrolysis of PPi is essential to drive the

synthesis of nucleic acids.

b. Formation of ATP

To complete its intermediary metabolic function, ATP

must be replenished. This is accomplished through three

types of processes:

1. Substrate-level phosphorylation. ATP may be

formed, as is indicated in Fig. 16-23b, from phospho-

enolpyruvate by direct transfer of a phosphoryl group from

a “high-energy” compound to ADP. Such reactions, which

are referred to as substrate-level phosphorylations, most

commonly occur in the early stages of carbohydrate me-

tabolism (Section 17-2).

2. Oxidative phosphorylation and photophosphoryla-tion. Both oxidative metabolism and photosynthesis act to

generate a proton (H�) concentration gradient across a

membrane (Sections 22-3 and 24-2D). Discharge of this

gradient is enzymatically coupled to the formation of ATP

from ADP and Pi (the reverse of ATP hydrolysis). In oxida-

tive metabolism, this process is called oxidative phosphory-lation, whereas in photosynthesis it is termed photophos-phorylation. Most of the ATP produced by respiring and

photosynthesizing organisms is generated in this manner.

3. Adenylate kinase reaction. The AMP resulting from

pyrophosphate cleavage reactions of ATP is converted to

582 Chapter 16. Introduction to Metabolism

Figure 16-28 The phosphorylation of fructose-6-phosphate by ATP to form fructose-1,6-bisphosphate and ADP.

Fructose-6-phosphate Fructose-1,6-bisphosphate

ATP� ADP�

�2O3P CH2 CH2O O OH

H OH

HO H

H HO

PO32�

phosphofructokinase

G�� = –14.2 kJ •mol–1

�2O3P CH2 CH2O O O

H OH

HO H

H HO

JWCL281_c16_557-592.qxd 2/26/10 11:10 AM Page 582

ADP in a reaction catalyzed by the enzyme adenylatekinase (Section 17-4Fe):

The ADP is subsequently converted to ATP through

substrate-level phosphorylation, oxidative phosphorylation,

or photophosphorylation.

c. Rate of ATP Turnover

The cellular role of ATP is that of a free energy transmit-ter rather than a free energy reservoir. The amount of ATP

in a cell is typically only enough to supply its free energy

needs for a minute or two. Hence,ATP is continually being

hydrolyzed and regenerated. Indeed, 32P-labeling experi-

ments indicate that the metabolic half-life of an ATP mole-

cule varies from seconds to minutes depending on the cell

type and its metabolic activity. For instance, brain cells

have only a few seconds’ supply of ATP (which, in part, ac-

counts for the rapid deterioration of brain tissue by oxygen

deprivation). An average person at rest consumes and re-generates ATP at a rate of �3 mol (1.5 kg) � h�1 and as muchas an order of magnitude faster during strenuous activity.

d. Phosphocreatine Provides a “High-Energy”

Reservoir for ATP Formation

Muscle and nerve cells, which have a high ATP turnover

(a maximally exerting muscle has only a fraction of a

second’s ATP supply), have a free energy reservoir that

functions to regenerate ATP rapidly. In vertebrates, phos-

phocreatine (Fig. 16-26) functions in this capacity. It is

synthesized by the reversible phosphorylation of creatine

by ATP as catalyzed by creatine kinase:

Note that this reaction is endergonic under standard condi-

tions. However, the intracellular concentrations of its reac-

tants and products (typically 4 mM ATP and 0.013 mM

¢G°¿ �12.6 kJ � mol�1

ATP � creatine Δ phosphocreatine � ADP

AMP � ATP Δ 2ADP

ADP) are such that it operates close to equilibrium ( G � 0).

Accordingly, when the cell is in a resting state, so that

[ATP] is relatively high, the reaction proceeds with net

synthesis of phosphocreatine, whereas at times of high

metabolic activity, when [ATP] is low, the equilibrium shifts

so as to yield net synthesis of ATP. Phosphocreatine therebyacts as an ATP “buffer” in cells that contain creatine kinase.A resting vertebrate skeletal muscle normally has suffi-

cient phosphocreatine to supply its free energy needs for

several minutes (but for only a few seconds at maximum

exertion). In the muscles of some invertebrates, such as

lobsters, phosphoarginine performs the same function.

These phosphoguanidines are collectively named phos-phagens.

5 OXIDATION–REDUCTION REACTIONS

Oxidation–reduction reactions, processes involving thetransfer of electrons, are of immense biochemical signifi-cance; living things derive most of their free energy fromthem. In photosynthesis (Chapter 24), CO2 is reduced(gains electrons) and H2O is oxidized (loses electrons) to

yield carbohydrates and O2 in an otherwise endergonic

process that is powered by light energy. In aerobic metabo-

lism, which is carried out by all eukaryotes and many

prokaryotes, the overall photosynthetic reaction is essen-

tially reversed so as to harvest the free energy of oxidation

of carbohydrates and other organic compounds in the form

of ATP (Chapter 22). Anaerobic metabolism generates

ATP, although in lower yields, through intramolecular

oxidation–reductions of various organic molecules, for

example, glycolysis (Chapter 17). In certain anaerobic

bacteria,ATP is generated through the use of non-O2 oxidiz-

ing agents such as sulfate or nitrate. In this section we out-

line the thermodynamics of oxidation–reduction reactions

in order to understand the quantitative aspects of these

crucial biological processes.

Section 16-5. Oxidation–Reduction Reactions 583

Figure 16-29 Pyrophosphate cleavage in the synthesis of anaminoacyl–tRNA. Here the squiggle (�) represents a “high-

energy” bond. In the first reaction step, the amino acid is

adenylylated by ATP. In the second step, a tRNA molecule

AMP P

H

C + � �P

ATP

P �P 2Pi

inorganicpyrophosphatase

H2O

�O

C

Aminoacyl–adenylate

tRNA AMP

C

NH3+ NH3

+ NH3+

Amino acid

O–

O

PPi

H

CR AMP

O

C

H

CR tRNA

Aminoacyl–tRNA

R

displaces the AMP moiety to form an aminoacyl–tRNA. The

exergonic hydrolysis of pyrophosphate ( G°¿ �19.2

kJ � mol�1) drives the reaction forward.

JWCL281_c16_557-592.qxd 2/26/10 11:10 AM Page 583

A. The Nernst Equation

Oxidation–reduction reactions (also known as redox or ox-idoreduction reactions) resemble other types of chemical

reactions in that they involve group transfer. For instance,

hydrolysis transfers a functional group to water. In oxidation–

reduction reactions, the “groups” transferred are electrons,

which are passed from an electron donor (reductant or re-ducing agent) to an electron acceptor (oxidant or oxidizingagent). For example, in the reaction

Cu�, the reductant, is oxidized to Cu2� while Fe3�, the oxi-

dant, is reduced to Fe2�.

Redox reactions may be divided into two half-reactionsor redox couples, such as

whose sum is the above whole reaction.These half-reactions

occur during oxidative metabolism in the vital mitochon-

drial electron transfer mediated by cytochrome c oxidase(Section 22-2C5). Note that for electrons to be transferred,

both half-reactions must occur simultaneously. In fact,

the electrons are the two half-reactions’ common interme-

diate.

a. Electrochemical Cells

A half-reaction consists of an electron donor and its

conjugate electron acceptor; in the oxidation half-reaction

shown above, Cu� is the electron donor and Cu2� is its con-

jugate electron acceptor.Together these constitute a conju-gate redox pair analogous to the conjugate acid–base pair

(HA and A�) of a Brønsted acid (Section 2-2A).An impor-

tant difference between redox pairs and acid–base pairs,

however, is that the two half-reactions of a redox reaction,each consisting of a conjugate redox pair, may be physicallyseparated so as to form an electrochemical cell (Fig. 16-30).

In such a device, each half-reaction takes place in its sepa-

rate half-cell, and electrons are passed between half-cells

as an electric current in the wire connecting their two elec-

trodes. A salt bridge is necessary to complete the electrical

circuit by providing a conduit for ions to migrate in the

maintenance of electrical neutrality.

The free energy of an oxidation–reduction reaction is

particularly easy to determine through a simple measure-

ment of the voltage difference between its two half-cells.

Consider the general redox reaction:

in which n electrons per mole of reactants are transferred

from reductant (Bred) to oxidant ( ). The free energy of

this reaction is expressed, according to Eq. [3.15], as

[16.2]

Equation [3.12] indicates that, under reversible conditions,

[16.3]¢G �w¿ �wel

¢G ¢G° � RT ln a [Ared] [Boxn� ]

[Aoxn� ] [Bred]

b

An�ox

An�ox � Bred Δ Ared � Bn�

ox

Cu� Δ Cu2� � e� (oxidation)

Fe3� � e� Δ Fe2� (reduction)

Fe3� � Cu� Δ Fe2� � Cu2�

where w¿, the non-pressure–volume work, is, in this case,

wel, the electrical work required to transfer the n moles of

electrons through the electric potential difference .This,

according to the laws of electrostatics, is

[16.4]

where f, the faraday, is the electrical charge of 1 mol of

electrons (1 f 96,485 C � mol�1 96,485 J � V�1 � mol�1,

where C and V are the symbols for coulomb and volt).

Thus, substituting Eq. [16.4] into Eq. [16.3],

[16.5]

Combining Eqs. [16.2] and [16.5], and making the analo-

gous substitution for G°, yields the Nernst equation:

[16.6]

which was originally formulated in 1881 by Walther Nernst.

Here e, the electromotive force (emf) or redox potential,may be described as the “electron pressure” that the elec-

trochemical cell exerts. The quantity e°, the redox poten-

tial when all components are in their standard states, is

called the standard redox potential. If these standard states

refer to biochemical standard states (Section 3-4Ba), then

e° is replaced by e°¿. Note that a positive e in

Eq. [16.5] results in a negative G; in other words, a posi-tive e is indicative of a spontaneous reaction, one that cando work.

B. Measurements of Redox Potentials

The free energy change of a redox reaction may be deter-

mined, as Eq. [16.5] indicates, by simply measuring its redox

potential with a voltmeter (Fig. 16-30). Consequently,

voltage measurements are commonly employed to charac-

terize the sequence of reactions comprising a metabolic

¢

¢¢¢

¢

¢

¢e ¢e° �RTnf

ln a [Ared] [Boxn� ]

[Aoxn� ] [Bred]

b

¢G �nf ¢e

wel nf ¢e

¢e

584 Chapter 16. Introduction to Metabolism

Figure 16-30 Example of an electrochemical cell. The half-cell

undergoing oxidation (here Cu� S Cu2� � e�) passes the

liberated electrons through the wire to the half-cell undergoing

reduction (here e� � Fe3� S Fe2�). Electroneutrality in the two

half-cells is maintained by the transfer of ions through the

electrolyte-containing salt bridge.

Saltbridge

e– + Fe3+ Cu+Fe2+

Pt Pt

Cu2+ + e–

Voltmeter

JWCL281_c16_557-592.qxd 6/10/10 11:52 AM Page 584

electron-transport pathway (such as mediates, e.g., oxida-

tive metabolism; Chapter 22).

Any redox reaction can be divided into its component

half-reactions:

where, by convention, both half-reactions are written as re-

ductions. These half-reactions can be assigned reductionpotentials,eA and eB, in accordance with the Nernst equation:

[16.7a]

[16.7b]

For the redox reaction of any two half-reactions:

[16.8]

Thus, when the reaction proceeds with A as the electron

acceptor and B as the electron donor, and

similarly for .¢e¢e° � e°A � e°B

¢e° � e°(e�acceptor) � e°(e�donor)

eB � eB° �RTnf

ln a [Bred]

[Boxn� ]b

eA � eA° �RTnf

ln a [Ared]

[Aoxn� ]b

Bn�ox � ne� Δ Bred

An�ox � ne� Δ Ared

Reduction potentials, like free energies, must be defined

with respect to some arbitrary standard. By convention,

standard reduction potentials are defined with respect to

the standard hydrogen half-reaction

in which H� at pH 0, 25°C, and 1 atm is in equilibrium with

H2(g) that is in contact with a Pt electrode. This half-cell is

arbitrarily assigned a standard reduction potential of � 0 V

(1 V � 1 J � C�1). For the biochemical convention, we like-

wise define the standard (pH � 0) hydrogen half-reaction

as having so that the hydrogen half-cell at the bio-

chemical standard state (pH � 7) has

(Table 16-4). When is positive, �G is negative (Eq.

[16.5]), indicating a spontaneous process. In combining

two half-reactions under standard conditions, the direc-

tion of spontaneity therefore involves the reduction of the

redox couple with the more positive standard reduction

potential. In other words, the more positive the standard re-duction potential, the greater the tendency for the redoxcouple’s oxidized form to accept electrons and thus becomereduced.

¢ee°¿ � �0.421 V

e¿ � 0

e°

2H� � 2e� Δ H2(g)

Section 16-5. Oxidation–Reduction Reactions 585

Half-Reaction (V)

0.815

0.42

0.385

0.295

0.29

0.235

0.22

0.077

0.045

0.031

�0.040

�0.166

�0.185

�0.197

�0.219

�0.23

�0.29

�0.315

�0.320

�0.340

�0.346

�0.421

�0.454

�0.581Acetate� � 3H� � 2e� Δ acetaldehyde � H2O

SO2�4 � 2H� � 2e� Δ SO2�

3 � H2O

H� � e� Δ 12H2

Acetoacetate� � 2H� � 2e� Δ �-hydroxybutyrate�

Cystine � 2H� � 2e� Δ 2 cysteine

NADP� � H� � 2e� Δ NADPH

NAD� � H� � 2e� Δ NADH

Lipoic acid � 2H� � 2e� Δ dihydrolipoic acid

S � 2H� � 2e� Δ H2S

FAD � 2H� � 2e� Δ FADH2 (free coenzyme)

Acetaldehyde � 2H� � 2e� Δ ethanol

Pyruvate� � 2H� � 2e� Δ lactate�

Oxaloacetate� � 2H� � 2e� Δ malate�

FAD � 2H� � 2e� Δ FADH2 (in flavoproteins)

Fumarate� � 2H� � 2e� Δ succinate�

Ubiquinone � 2H� � 2e� Δ ubiquinol

Cytochrome b(Fe3� ) � e� Δ cytochrome b(Fe2� ) (mitochondrial)

Cytochrome c1(Fe3� ) � e� Δ cytochrome c1(Fe2� )

Cytochrome c(Fe3� ) � e� Δ cytochrome c(Fe2� )

Cytochrome a(Fe3� ) � e� Δ cytochrome a(Fe2� )

O2(g) � 2H� � 2e� Δ H2O2

Cytochrome a3(Fe3� ) � e� Δ cytochrome a3(Fe2� )

NO�3 � 2H� � 2e� Δ NO�

2 � H2O

12O2 � 2H� � 2e� Δ H2O

e°¿

Table 16-4 Standard Reduction Potentials of Some Biochemically Important Half-Reactions

Source: Mostly from Loach, P.A., in Fasman, G.D. (Ed.), Handbook of Biochemistry and Molecular Biology(3rd ed.), Physical and Chemical Data, Vol. I, pp. 123–130, CRC Press (1976).

JWCL281_c16_557-592.qxd 6/30/10 10:34 AM Page 585

a. Biochemical Half-Reactions Are

Physiologically Significant

The biochemical standard reduction potentials of

some biochemically important half-reactions are listed in

Table 16-4. The oxidized form of a redox couple with a

large positive standard reduction potential has a high affin-

ity for electrons and is a strong electron acceptor (oxidizing

agent), whereas its conjugate reductant is a weak electron

donor (reducing agent). For example, O2 is the strongest

oxidizing agent in Table 16-4, whereas H2O, which tightly

holds its electrons, is the table’s weakest reducing agent.

The converse is true of half-reactions with large negative

standard reduction potentials. Since electrons sponta-

neously flow from low to high reduction potentials, they

are transferred, under standard conditions, from the re-

duced products in any half-reaction in Table 16-4 to the ox-

idized reactants of any half-reaction above it (although this

may not occur at a measurable rate in the absence of a suit-

able enzyme). Thus, in biological systems, the approximate

lower limit for a standard reduction potential is �0.421 V

because reductants with a lesser value of would reduce

protons to H2. However, reducing centers in proteins that

are protected from water may have lower potentials. Note

that the Fe3� ions of the various cytochromes tabulated in

Table 16-4 have significantly different redox potentials.

This indicates that the protein components of redoxenzymes play active roles in electron-transfer reactions bymodulating the redox potentials of their bound redox-activecenters.

Electron-transfer reactions are of great biological

importance. For example, in the mitochondrial electron-

transport chain (Section 22-2), the primary source of ATP

in eukaryotes, electrons are passed from NADH (Fig. 13-2)

along a series of electron acceptors of increasing reduction

potential (many of which are listed in Table 16-4) to O2.

ATP is generated from ADP and Pi by coupling its synthe-

sis to this free energy cascade. NADH thereby functions asan energy-rich electron-transfer coenzyme. In fact, the oxi-

dation of one NADH to NAD� supplies sufficient free en-

ergy to generate 2.5 ATPs (Section 22-2Bb). The NAD�/

NADH redox couple functions as the electron acceptor in

many exergonic metabolite oxidations. In serving as the

electron donor in ATP synthesis, it fulfills its cyclic role as a

free energy conduit in a manner analogous to ATP. The

metabolic roles of redox coenzymes are further discussed

in succeeding chapters.

C. Concentration Cells

A concentration gradient has a lower entropy (greater or-der) than the corresponding uniformly mixed solution andtherefore requires the input of free energy for its formation.Consequently, discharge of a concentration gradient is anexergonic process that may be harnessed to drive an ender-gonic reaction. For example, discharge of a proton concen-

tration gradient (generated by the reactions of the electron-

transport chain) across the inner mitochondrial

membrane drives the enzymatic synthesis of ATP from

ADP and Pi (Section 22-3). Likewise, nerve impulses,

e°¿

(e°¿)

which require electrical energy, are transmitted through

the discharge of [Na�] and [K�] gradients that nerve cells

generate across their cell membranes (Section 20-5B).

Quantitation of the free energy contained in a concentra-

tion gradient is accomplished by use of the concepts of

electrochemical cells.

The reduction potential and free energy of a half-cell

vary with the concentrations of its reactants. An electro-

chemical cell may therefore be constructed from two

half-cells that contain the same chemical species but at

different concentrations. The overall reaction for such an

electrochemical cell may be represented

[16.9]

and, according to the Nernst equation, since when

the same reaction occurs in both cells,

Such concentration cells are capable of generating electri-

cal work until they reach equilibrium.This occurs when the

concentration ratios in the half-cells become equal (Keq 1).

The reaction constitutes a sort of mixing of the two half-

cells; the free energy generated is a reflection of the en-

tropy of this mixing.The thermodynamics of concentration

gradients as they apply to membrane transport is discussed

in Section 20-1.

6 THERMODYNAMICS OF LIFE

One of the last refuges of vitalism, the doctrine that biolog-

ical processes are not bound by the physical laws that gov-

ern inanimate objects, was the belief that living things can

somehow evade the laws of thermodynamics.This view was

partially refuted by elaborate calorimetric measurements

on living animals that are entirely consistent with the

energy conservation predictions of the first law of thermo-

dynamics. However, the experimental verification of the

second law of thermodynamics in living systems is more

difficult. It has not been possible to measure the entropy of

living matter because the heat, qp, of a reaction at a con-

stant T and P is only equal to T S if the reaction is carried

out reversibly (Eq. [3.8]). Obviously, the dismantling of a

living organism to its component molecules for such a

measurement would invariably result in its irreversible

death. Consequently, the present experimentally verified

state of knowledge is that the entropy of living matter is

less than that of the products to which it decays.

In this section we consider the special aspects of the

thermodynamics of living systems. Knowledge of these

matters, which is by no means complete, has enhanced our

understanding of how metabolic pathways are regulated,

how cells respond to stimuli, and how organisms grow and

change with time.

¢e RTnf

ln a [An�ox (half-cell 2) ][Ared(half-cell 1) ]

[An�ox (half-cell 1) ] [Ared(half-cell 2) ]

b

¢e° 0

An�ox (half-cell 2) � Ared(half-cell 1)

An�ox (half-cell 1) � Ared(half-cell 2) Δ

586 Chapter 16. Introduction to Metabolism

JWCL281_c16_557-592.qxd 2/26/10 11:10 AM Page 586

A. Living Systems Cannot Be at Equilibrium

Classical or equilibrium thermodynamics (Chapter 3)

applies largely to reversible processes in closed systems.

The fate of any isolated system, as we discussed in Section

3-4A, is that it must inevitably reach equilibrium. For ex-

ample, if its reactants are in excess, the forward reaction

will proceed faster than the reverse reaction until equilib-

rium is attained ( G 0). In contrast, open systems may

remain in a nonequilibrium state as long as they are able to

acquire free energy from their surroundings in the form of

reactants, heat, or work. While classical thermodynamics

provides invaluable information concerning open systems

by indicating whether a given process can occur sponta-

neously, further thermodynamic analysis of open systems

requires the application of the more recently elucidated

principles of nonequilibrium or irreversible thermodynamics.In contrast to classical thermodynamics, this theory explic-

itly takes time into account.

Living organisms are open systems and therefore cannever be at equilibrium. As indicated above, they continu-

ously ingest high-enthalpy, low-entropy nutrients, which

they convert to low-enthalpy, high-entropy waste products.

The free energy resulting from this process is used to do

work and to produce the high degree of organization char-

acteristic of life. If this process is interrupted, the organism

ultimately reaches equilibrium, which for living things is

synonymous with death. For example, one theory of aging

holds that senescence results from the random but in-

evitable accumulation in cells of genetic defects that inter-

fere with and ultimately disrupt the proper functioning of

living processes. [The theory does not, however, explain

how single-celled organisms or the germ cells of multicellu-

lar organisms (sperm and ova), which are in effect immor-

tal, are able to escape this so-called error catastrophe.]Living systems must maintain a nonequilibrium state

for several reasons:

1. Only a nonequilibrium process can perform useful

work.

2. The intricate regulatory functions characteristic of

life require a nonequilibrium state because a process at

equilibrium cannot be controlled (similarly, a ship that is

dead in the water will not respond to its rudder).

3. The complex cellular and molecular systems that

conduct biological processes can be maintained only in the

nonequilibrium state. Living systems are inherently unsta-

ble because they are degraded by the very biochemical re-

actions to which they give rise. Their regeneration, which

must occur almost simultaneously with their degradation,

requires the continuous influx of free energy. For example,

the ATP-generating consumption of glucose (Section 17-2),

as has been previously mentioned, occurs with the initial

consumption of ATP through its reactions with glucose to

form glucose-6-phosphate and with fructose-6-phosphate

to form fructose-1,6-bisphosphate. Consequently, if metab-

olism is suspended long enough to exhaust the available

ATP supply, glucose metabolism cannot be resumed. Life

therefore differs in a fundamental way from a complex

machine such as a computer. Both require a throughput of

free energy to be active. However, the function of the ma-

chine is based on a static structure, so that the machine can

be repeatedly switched on and off. Life, in contrast, is based

on a self-destructing but self-renewing process, which once

interrupted, cannot be reinitiated.

B. Nonequilibrium Thermodynamics and

the Steady State

In a nonequilibrium process, something (such as matter,

electrical charge, or heat) must flow, that is, change its spa-

tial distribution. In classical mechanics, the acceleration of

mass occurs in response to force. Similarly, flow in a ther-modynamic system occurs in response to a thermodynamicforce (driving force), which results from the system’snonequilibrium state. For example, the flow of matter in

diffusion is motivated by the thermodynamic force of a con-

centration gradient; the migration of electrical charge (elec-

tric current) occurs in response to a gradient in an electric

field (a voltage difference); the transport of heat results

from a temperature gradient; and a chemical reaction

results from a difference in chemical potential. Such flows

are said to be conjugate to their thermodynamic force.

A thermodynamic force may also promote a nonconju-gate flow under the proper conditions. For example, a gra-

dient in the concentration of matter can give rise to an elec-

tric current (a concentration cell), heat (such as occurs on

mixing H2O and HCl), or a chemical reaction (the mito-

chondrial production of ATP through the dissipation of a

proton gradient). Similarly, a gradient in electrical poten-

tial can motivate a flow of matter (electrophoresis), heat

(resistive heating), or a chemical reaction (the charging of

a battery). When a thermodynamic force stimulates a non-

conjugate flow, the process is called energy transduction.

a. Living Things Maintain the Steady State

Living systems are, for the most part, characterized bybeing in a steady state. By this it is meant that all flows in the

system are constant, so that the system does not change with

time. Some environmental steady-state processes are

schematically illustrated in Fig. 16-31. Ilya Prigogine, a

pioneer in the development of irreversible thermodynamics,

has shown that a steady-state system produces the maximum

amount of useful work for a given energy expenditure under

the prevailing conditions. The steady state of an open systemis therefore its state of maximum thermodynamic efficiency.Furthermore, in analogy with Le Châtelier’s principle (Sec-

tion 3-4A), slight perturbations from the steady state give

rise to changes in flows that counteract these perturbations

so as to return the system to the steady state. The steady stateof an open system is therefore analogous to the equilibriumstate of an isolated system; both are stable states.

In the following chapters we shall see that many biological

regulatory mechanisms function to maintain a steady state.

For example, the flow of reaction intermediates through a

metabolic pathway is often inhibited by an excess of final prod-

uct and stimulated by an excess of starting material through

the allosteric regulation of its key enzymes (Section 13-4).

Section 16-6. Thermodynamics of Life 587

JWCL281_c16_557-592.qxd 2/26/10 11:10 AM Page 587

Living things have apparently evolved so as to take maximum

thermodynamic advantage of their environments.

C. Thermodynamics of Metabolic Control

a. Enzymes Selectively Catalyze Required Reactions

Biological reactions are highly specific; only reactions thatlie on metabolic pathways take place at significant rates de-spite the many other thermodynamically favorable reactions

that are also possible. As an example, let us consider the re-

actions of ATP, glucose, and water. Two thermodynamically

favorable reactions that ATP can undergo are phosphoryl

transfer to form ADP and glucose-6-phosphate, and hydroly-

sis to form ADP and Pi (Fig. 16-23a).The free energy profiles

of these reactions are diagrammed in Fig. 16-32.ATP hydrol-

ysis is thermodynamically favored over the phosphoryl

transfer to glucose. However, their relative rates are deter-

mined by their free energies of activation to their transition

588 Chapter 16. Introduction to Metabolism

Figure 16-31 Two examples of open systems in a steady state.(a) A constant flow of water in the river occurs under the

influence of the force of gravity. The water level in the reservoir

is maintained by rain, the major source of which is the

evaporation of seawater. Hence the entire cycle is ultimately

powered by the sun. (b) The steady state of the biosphere is

Figure 16-32 Reaction coordinate diagrams. These are (1) the

reaction of ATP and water (purple curve), and the reaction of

ATP and glucose (2) in the presence (orange curve) and (3) in

the absence (yellow curve) of an appropriate enzyme. Although

(a)Radiant energyfrom the sun

Rain

Heat loss

Watervapor

Sea

River flowingunder steadystate conditions(gravity)

Heat loss

Breakdown ofcarbohydrates

PhotosynthesisCO2

+H2O

(b)Radiant energyfrom the sun

ATP + H2O+ glucose

Reaction coordinate

G

ADP + H2O + glucose-6-P

ADP + Pi + glucose

(ATP•Glucose)enzymatic + H2O

(ATP•Glucose)enzymatic

(ADP•Glucose-6-P)enzymatic

=

(ATP•Glucose)uncatalyzed + H2O=

ΔG3=

ΔG1

ΔG1

=

ΔG2

ΔG2, ΔG3

=

similarly maintained by the sun. Plants harness the sun’s radiant

energy to synthesize carbohydrates from CO2 and H2O. The

eventual metabolism of the carbohydrates by the plants or by the

animals that eat them results in the release of their stored free

energy and the return of the CO2 and H2O to the environment to

complete the cycle.

the hydrolysis of ATP is a more exergonic reaction than the

phosphorylation of glucose ( G1 is more negative than G2), the

latter reaction is predominant in the presence of a suitable

enzyme because it is kinetically favored .(¢G‡2 � ¢G‡

1 )

JWCL281_c16_557-592.qxd 6/10/10 11:52 AM Page 588

states ( G‡ values; Section 14-1Cb) and the relative concen-

trations of glucose and water.The larger G‡, the slower the

reaction. In the absence of enzymes, G‡ for the phosphoryl-

transfer reaction is greater than that for hydrolysis, so the

hydrolysis reaction predominates (although neither reaction

occurs at a biologically significant rate).

The free energy barriers of both of the nonenzymatic re-

actions are far higher than that of the enzyme-catalyzed

phosphoryl transfer to glucose. Hence enzymatic forma-

tion of glucose-6-phosphate is kinetically favored over the

nonenzymatic hydrolysis of ATP. It is the role of an enzyme,in this case hexokinase, to selectively reduce the free energyof activation of a chemically coupled reaction so that it ap-proaches equilibrium faster than the more thermodynami-cally favored uncoupled reaction.

b. Many Enzymatic Reactions Are Near Equilibrium

Although metabolism as a whole is a nonequilibrium

process, many of its component reactions function close to

equilibrium. The reaction of ATP and creatine to form

phosphocreatine (Section 16-4Cd) is an example of such a

reaction. The ratio [creatine]/[phosphocreatine] depends

on [ATP] because creatine kinase, the enzyme catalyzing

this reaction, has sufficient activity to equilibrate the reac-

tion rapidly. The net rate of such an equilibrium reaction is

effectively controlled by varying the concentrations of its

reactants and/or products.

c. Pathway Throughput Is Regulated by Controlling

Enzymes Operating Far from Equilibrium

Other biological reactions function far from equilibrium.

For example, the phosphofructokinase reaction (Fig. 16-28)

has an equilibrium constant of but under physio-

logical conditions in rat heart muscle has the mass action ra-

tio [fructose-1,6-bisphosphate][ADP]/[fructose-6-phos-

phate][ATP] 0.03, which corresponds to G �25.7

kJ � mol�1 (Eq. [3.15]). This situation arises from a buildup

of reactants because there is insufficient phosphofructoki-

nase activity to equilibrate the reaction. Changes in sub-

strate concentrations therefore have relatively little effect

on the rate of the phosphofructokinase reaction; the en-

zyme is close to saturation. Only changes in the activity of

the enzyme, through allosteric interactions, for example, can

significantly alter this rate.An enzyme such as phosphofruc-

tokinase is therefore analogous to a dam on a river. Sub-

strate flux (rate of flow) is controlled by varying its activity

(allosterically or by other means), much as a dam controls

the flow of a river below the dam by varying the opening of

its floodgates (when the water levels on the two sides of the

dam are different, that is, when they are not at equilibrium).

Understanding of how reactant flux in a metabolic path-

way is controlled requires knowledge of which reactions

are functioning near equilibrium and which are far from it.

Most enzymes in a metabolic pathway operate near equi-

librium and therefore have net rates that are sensitive only

to their substrate concentrations. However, as we shall see

in the following chapters (particularly in Section 17-4), cer-tain enzymes, which are strategically located in a metabolicpathway, operate far from equilibrium. These enzymes,which are targets for metabolic regulation by allosteric inter-actions and other mechanisms, are responsible for the main-tenance of a stable steady-state flux of metabolites throughthe pathway. This situation, as we have seen, maximizes the

pathway’s thermodynamic efficiency.

K¿eq 300

Chapter Summary 589

1 Metabolic Pathways Metabolic pathways are series of

consecutive enzymatically catalyzed reactions that produce

specific products for use by an organism. The free energy re-

leased by degradation (catabolism) is, through the intermedi-

acy of ATP and NADPH, used to drive the endergonic

processes of biosynthesis (anabolism). Carbohydrates, lipids,

and proteins are all converted to the common intermediate

acetyl-CoA, whose acetyl group is then converted to CO2 and

H2O through the action of the citric acid cycle and oxidative

phosphorylation.A relatively few metabolites serve as starting

materials for a host of biosynthetic products. Metabolic path-

ways have five principal characteristics: (1) Metabolic path-

ways are irreversible; (2) if two metabolites are interconvertible,

the synthetic route from the first to the second must differ

from the route from the second to the first; (3) every meta-

bolic pathway has an exergonic first committed step; (4) all

metabolic pathways are regulated, usually at the first commit-

ted step; and (5) metabolic pathways in eukaryotes occur in

specific subcellular compartments.

2 Organic Reaction Mechanisms Almost all metabolic

reactions fall into four categories: (1) group-transfer reactions;

(2) oxidation–reduction reactions; (3) eliminations, isomeriza-

tions, and rearrangements; and (4) reactions that make or break

carbon–carbon bonds. Most of these reactions involve het-

erolytic bond cleavage or formation occurring through the addi-

tion of nucleophiles to electrophilic carbon atoms. Group-trans-

fer reactions therefore involve transfer of an electrophilic group

from one nucleophile to another.The main electrophilic groups

transferred are acyl groups, phosphoryl groups, and glycosyl

groups.The most common nucleophiles are amino, hydroxyl, im-

idazole, and sulfhydryl groups. Electrophiles participating in

metabolic reactions are protons, metal ions, carbonyl carbon

atoms, and cationic imines. Oxidation–reduction reactions

involve loss or gain of electrons. Oxidation at carbon usually

involves bond cleavage, with the ultimate loss by C of

the two bonding electrons through their transfer to an elec-

tron acceptor such as NAD�. The terminal electron acceptor

in aerobes is O2. Elimination reactions are those in which a

double bond is created from two saturated carbon cen-

ters with the loss of H2O, NH3, ROH, or RNH2. Dehydration

reactions are the most common eliminations. Isomerizations

involve shifts of double bonds within molecules. Rearrange-

ments are biochemically uncommon reactions in which in-

tramolecular bonds are broken and reformed to produce

new carbon skeletons. Reactions that make and break

bonds form the basis of both degradative and biosynthetic

C¬C

C¬C

C“C

C¬H

CHAPTER SUMMARY

JWCL281_c16_557-592.qxd 6/10/10 11:52 AM Page 589

590 Chapter 16. Introduction to Metabolism

metabolism. In the synthetic direction, these reactions involve

addition of a nucleophilic carbanion to an electrophilic carbon

atom. The most common electrophilic carbon atom is the car-

bonyl carbon, whereas carbanions are usually generated by re-

moval of a proton from a carbon atom adjacent to a carbonyl

group or by decarboxylation of a �-keto acid.

3 Experimental Approaches to the Study of Metabolism

Experimental approaches employed in elucidating metabolic

pathways include the use of metabolic inhibitors, growth stud-

ies, and biochemical genetics. Metabolic inhibitors block path-

ways at specific enzymatic steps. Identification of the resulting

intermediates indicates the course of the pathway. Mutations,

which occur naturally in genetic diseases or can be induced by

mutagens, X-rays, or genetic engineering, may also result in the

absence or inactivity of an enzyme. Modern genetic techniques

make it possible to express foreign genes in higher organisms

(transgenic animals) or inactivate (knock out) a gene and study

the effects of these changes on metabolism. When isotopic la-

bels are incorporated into metabolites and allowed to enter a

metabolic system, their paths may be traced from the distribu-

tion of label in the intermediates. NMR is a noninvasive tech-

nique that may be used to detect and study metabolites in vivo.Studies on isolated organs, tissue slices, cells, and subcellular or-

ganelles have contributed enormously to our knowledge of the

localization of metabolic pathways. Systems biology endeavors

to quantitatively describe the properties and dynamics of bio-

logical networks as a whole through the integration of genomic,

transcriptomic, proteomic, and metabolomic information.

4 Thermodynamics of Phosphate Compounds Free en-

ergy is supplied to endergonic metabolic processes by the ATP

produced via exergonic metabolic processes. ATP’s �30.5 kJ �mol�1 G°¿ of hydrolysis is intermediate between those of

“high-energy” metabolites such as phosphoenolpyruvate and

“low-energy” metabolites such as glucose-6-phosphate. The

“high-energy” phosphoryl groups are enzymatically trans-

ferred to ADP, and the resulting ATP, in a separate reaction,

phosphorylates “low-energy” compounds. ATP may also un-

dergo pyrophosphate cleavage to yield PPi, whose subsequent

hydrolysis adds further thermodynamic impetus to the reaction.

ATP is present in too short a supply to act as an energy reser-

voir. This function, in vertebrate nerve and muscle cells, is car-

ried out by phosphocreatine, which under low-ATP conditions

readily transfers its phosphoryl group to ADP to form ATP.

5 Oxidation–Reduction Reactions The half-reactions of

redox reactions may be physically separated to form two elec-

trochemical half-cells.The redox potential for the reduction of

A by B,

in which n electrons are transferred, is given by the Nernst

equation

The redox potential of such a reaction is related to the reduc-

tion potentials of its component half-reactions, and , by

If , then has a greater electron affinity than does

.The reduction potential scale is defined by arbitrarily set-

ting the reduction potential of the standard hydrogen half-cell

to zero. Redox reactions are of great metabolic importance.

For example, the oxidation of NADH yields 2.5 ATPs through

the mediation of the electron-transport chain.

6 Thermodynamics of Life Living organisms are open sys-

tems and therefore cannot be at equilibrium.They must contin-

uously dissipate free energy in order to carry out their various

functions and preserve their highly ordered structures. The

study of nonequilibrium thermodynamics has indicated that the

steady state, which living processes maintain, is the state of max-

imum efficiency under the constraints governing open systems.

Control mechanisms that regulate biological processes preserve

the steady state by regulating the activities of enzymes that are

strategically located in metabolic pathways.

Bn�ox

An�oxeA � eB

¢e eA � eB

eBeA

¢e ¢e° �RTnf

lna [Ared] [Bn�ox ]

[An�ox ] [Bred]

b

An�ox � Bred Δ Ared � Bn�

ox

Metabolic Studies

Aebersold, R., Quantitative proteome analysis: Methods and ap-

plications, J. Infect. Dis. 182 (supplement 2), S315–S320 (2003).

Beadle, G.W., Biochemical genetics, Chem. Rev. 37, 15–96 (1945).

[A classic review summarizing the “one gene–one enzyme”

hypothesis.]

Campbell A.M. and Heyer L.J., Discovering Genomics, Pro-teomics and Bioinformatics (2nd ed.), Pearson Benjamin Cum-

mings, New York (2007). [An interactive introduction to these

subjects.]

Cerdan, S. and Seelig, J., NMR studies of metabolism, Annu. Rev.Biophys. Biophys. Chem. 19, 43–67 (1990).

Choi, S. (Ed.), Introduction to Systems Biology, Humana Press

(2007).

Cooper, T.G., The Tools of Biochemistry, Chapter 3, Wiley-

Interscience (1977). [A presentation of radiochemical tech-

niques.]

Duarte, N.C., Becker, S.A., Jamshidi, N., Thiele, I., Mo, M.L., Vo,

T.D., Srivas, R., and Palsson, B. Ø., Global reconstruction of the

human metabolic network based on genomic and bibliomic

data, Proc. Natl. Acad. Sci. 104, 1777–1782 (2007).

Freifelder, D., Biophysical Chemistry (2nd ed.), Chapters 5 and 6,

Freeman (1982). [A discussion of the principles of radioactive

counting and autoradiography.]

Go,V.L.W.,Nguyen,C.T.H.,Harris,D.M.,and Lee, W.-N.P.,Nutrient-

gene interaction: Metabolic genotype-phenotype relationship,

J. Nutr. 135, 2016s–3020s (2005).

Hevesy, G., Historical sketch of the biological application of tracer

elements, Cold Spring Harbor Symp. Quant. Biol. 13, 129–150

(1948).

Jeffrey, F.M.H., Rajagopal, A., Malloy, C.R., and Sherry, A.D.,13C-NMR:A simple yet comprehensive method for analysis of in-

termediary metabolism, Trends Biochem. Sci. 16, 5–10 (1991).

Michal, G. (Ed.), Biochemical Pathways. An Atlas of Biochemistryand Molecular Biology, Wiley (1999). [An encyclopedic com-

pendium of metabolic pathways.]

Shemin, D. and Rittenberg, D.,The biological utilization of glycine

for the synthesis of the protoporphyrin of hemoglobin, J. BiolChem. 166, 621–625 (1946).

Shulman, R.G. and Rothman, D.L., 13C NMR of intermediary me-

tabolism: Implications for systematic physiology, Annu. Rev.Physiol. 63, 15–48 (2001).

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Smolin, L.A. and Grosvenor, M.B, Nutrition: Science and Applica-tions, Wiley (2008). [A good text for those interested in pursu-

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Walsh, C., Enzymatic Reaction Mechanisms, Chapter 1, Freeman

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Bioenergetics

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adenosine phosphates, J. Biol. Chem. 244, 3290–3302 (1969).

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(2000).

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Problems 591

1. Glycolysis (glucose breakdown) has the overall stoichiome-

try:

whereas that of gluconeogenesis (glucose synthesis) is

What is the overall stoichiometry of the glycolytic breakdown of

1 mol of glucose followed by its gluconeogenic synthesis? Explain

why it is necessary that the pathways of these two processes be in-

dependently controlled and why they must differ by at least one

reaction.

2. It has been postulated that a trigonal bipyramidal penta-

covalent phosphorus intermediate can undergo a vibrational de-

formation process known as pseudorotation in which its apical

ligands exchange with two of its equatorial ligands via a tetragonal

pyramidal transition state:

Trigonal bipyramid[X and Y apical]

Trigonal bipyramid[O2 and O3 apical]

Tetragonal pyramidaltransition state

O1

O2

O3

X

Y

P

O1 O1O2

O3

X

Y

P

O1X

Y

O3

O2

P

O2

O3X

YP

glucose � 6ADP � 6Pi � 2NAD�

2 pyruvate � 6ATP � 2NADH � 4H� � 6H2O ¡

2 pyruvate� � 2ATP � 2NADH � 4H� � 2H2O

Glucose � 2ADP � 2Pi � 2NAD� ¡

In a nucleophilic substitution reaction, would two cycles of

pseudorotation, so as to place the leaving group (X) in an apical

position and the attacking group (Y) in an equatorial position,

lead to retention or inversion of configuration on the departure of

the leaving group?

3. One Curie (Ci) of radioactivity is defined as 3.70 � 1010 dis-

integrations per second, the number that occurs in 1 g of pure226Ra. A sample of 14CO2 has a specific radioactivity of 5 �Ci ��mol�1. What percentage of its C atoms are 14C?

4. In the hydrolysis of ATP to ADP and Pi, the equilibrium

concentration of ATP is too small to be measured accurately. A

better way of determining and hence G°¿ of this reaction, is

to break it up into two steps whose values of G°¿ can be accu-

rately determined. This has been done using the following pair of

reactions (the first being catalyzed by glutamine synthetase):

What is the G°¿ of ATP hydrolysis according to these data?

*5. Consider the reaction catalyzed by hexokinase:

A mixture containing 40 mM ATP and 20 mM glucose was incu-

bated with hexokinase at pH 7 and 25°C. Calculate the equilibrium

concentrations of the reactants and products (see Table 16-3).

6. In aerobic metabolism, glucose is completely oxidized in

the reaction

with the coupled generation of 32 ATP molecules from 32 ADP

and 32 Pi.Assuming the G for the hydrolysis of ATP to ADP and

Glucose � 6 O2 Δ 6CO2 � 6H2O

ATP � glucose Δ ADP � glucose-6-phosphate

¢G°¿2 �14.2 kJ � mol�1

(2) Glutamate � NH�4 Δ glutamine � H2O � H�

¢G°¿1 �16.3 kJ � mol�1

(1) ATP � glutamate � NH4� Δ ADP � Pi � glutamine � H�

K¿eq,

PROBLEMS

JWCL281_c16_557-592.qxd 6/10/10 11:52 AM Page 591

592 Chapter 16. Introduction to Metabolism

Pi under intracellular conditions is �50 kJ � mol�1 and that for the

combustion of glucose is �2823.2 kJ � mol�1, what is the efficiency

of the glucose oxidation reaction in terms of the free energy se-

questered in the form of ATP?

7. Typical intracellular concentrations of ATP, ADP, and Pi in

muscles are 5.0, 0.5, and 1.0 mM, respectively. At 25°C and pH 7:

(a) What is the free energy of hydrolysis of ATP at these concen-

trations? (b) Calculate the equilibrium concentration ratio of

phosphocreatine to creatine in the creatine kinase reaction:

if ATP and ADP have the above concentrations. (c) What concen-

tration ratio of ATP to ADP would be required under the forego-

ing conditions to yield an equilibrium concentration ratio of phos-

phocreatine to creatine of 1? Assuming the concentration of Pi

remained 1.0 mM, what would the free energy of hydrolysis of

ATP be under these latter conditions?

*8. Assuming the intracellular concentrations of ATP, ADP,

and Pi, are those given in Problem 7: (a) Calculate the concen-

tration of AMP at pH 7 and 25°C under the condition that the

adenylate kinase reaction:

is at equilibrium. (b) Calculate the equilibrium concentration of

AMP when the free energy of hydrolysis of ATP to ADP and Pi is

�55 kJ � mol�1. Assume [Pi] and ([ATP] � [ADP]) remain

constant.

9. Using the data in Table 16-4, list the following substances in

order of their decreasing oxidizing power: (a) fumarate�, (b) cys-

tine, (c) O2, (d) NADP�, (e) cytochrome c (Fe3�), and (f) lipoic

acid.

10. Calculate the equilibrium concentrations of reactants and

products for the reaction:

�-hydroxybutyrate� � NAD�

Acetoacetate� � NADH � H� Δ

2ADP Δ ATP � AMP

Creatine � ATP Δ phosphocreatine � ADP

when the initial concentrations of acetoacetate� and NADH are

0.01 and 0.005M, respectively, and �-hydroxybutyrate� and NAD�

are initially absent. Assume the reaction takes place at 25°C and

pH 7.

11. In anaerobic bacteria, the final metabolic electron accep-

tor is some molecule other than O2. A major requirement for any

redox pair utilized as a metabolic free energy source is that it pro-

vides sufficient free energy to generate ATP from ADP and Pi. In-

dicate which of the following redox pairs are sufficiently exer-

gonic to enable a properly equipped bacterium to utilize them as

a major energy source. Assume that redox reactions forming ATP

require two electrons and that .

(a) Ethanol � NO�3 (c) H2 � S

(b) Fumarate� � SO2�3 (d) Acetaldehyde � acetaldehyde

12. Calculate �G°¿ for the following pairs of half-reactions at

pH 7 and 25°C.Write a balanced equation for the overall reaction

and indicate the direction in which it occurs spontaneously under

standard conditions.

(a) and

(b) (Pyruvate� � 2H�/lactate�) and (NAD� � H�/NADH)

*13. The chemiosmotic hypothesis (Section 22-3A) postulates

that ATP is generated in the two-electron reaction:

which is driven by a metabolically generated pH gradient in the

mitochondria. What is the magnitude of the pH gradient required

for net synthesis of ATP at 25°C and pH 7, if the steady-state

concentrations of ATP, ADP, and Pi are 0.01, 10, and 10 mM,

respectively?

14. Gastric juice is 0.15M HCl.The blood plasma, which is the

source of this H� and Cl�, is 0.10M in Cl� and has a pH of 7.4. Cal-

culate the free energy necessary to produce the HCl in 0.1 L of

gastric juice at 37°C.

ATP � H2O � 2H� (high pH)

ADP � Pi � 2H� (low pH) Δ

(12 O2 � 2H�>H2O)(H�>12H2)

¢e � ¢e°¿

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