introduction to metabolism 16 - kasetsart...
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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
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[A classic review summarizing the “one gene–one enzyme”
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Campbell A.M. and Heyer L.J., Discovering Genomics, Pro-teomics and Bioinformatics (2nd ed.), Pearson Benjamin Cum-
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Cerdan, S. and Seelig, J., NMR studies of metabolism, Annu. Rev.Biophys. Biophys. Chem. 19, 43–67 (1990).
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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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