Voet D., Voet Ju.G. Biochemistry

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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 the

positions 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 0

PPM

–5 –10 –15 –20

ATP

(b) Creatine kinase

positive liver

Figure 16-15 The expression of creatine kinase in transgenic

mouse liver as demonstrated by localized in vivo

P 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 (P

), 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



COO





COO



COO



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 in

utero. 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 C

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

C,and

P. 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,

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

P 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 with

C (which is only 1.10% naturally abundant) permits the

metabolic progress of the labeled atoms to be followed by

NMR. Figure 16-16 shows in vivo

C NMR spectra of a rat

liver before and after an injection of

D-[1-

C]glucose.The

can be seen entering the liver and then being converted to

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

H 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%

Cl and 45%

Cl (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,

C, and

P all emit  particles but

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

radiation from

P is therefore highly penetrating, whereas

that from

H and

C is not. (

H and

C, 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,

H and

C, 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-

C]glucose to glycogen as observed by

localized in vivo

C NMR. (a) The natural

abundance

C NMR spectrum of the liver

of a live rat. Note the resonance

corresponding to C1 of glycogen. (b) The

C NMR spectrum of the liver of the same

rat ⬃5 min after it was intravenously

injected with 100 mg of [1-

C]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

C NMR spectrum of the

liver of the same rat ⬃30 min after the

[1-

C]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 Biochemical

Importance

Stable Isotopes

Nucleus Natural Abundance (%)

H 0.012

C 1.07

N 0.36

O 0.20

Radioactive Isotopes

Nucleus Radiation Type Half-Life

H  12.31 years

C  5715 years

Na 



,  2.60 years

P  14.28 days

S  87.2 days

Ca  162.7 days

Co ,  5.271 years

125

I  59.4 days

131

I ,  8.02 days

Source: Holden, N.E., in Lide, D.R. (Ed.), Handbook of Chemistry and

Physics (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, t

1/2

, 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.

1>2



ln 2



0.693

(a)

RCOOR′

180 120 60 0

ppm

C1 Glycogen

C1–β

Glucose

C1–α

Glucose and

glycogen

Choline

N(CH

)

–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

N-labeled nutri-

ents, isolating the heme in their blood, and analyzing it for

N content. Only when the rats were fed [

N]glycine did

the heme contain

N (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 [

N]glycine, of many

N-labeled metabolites, is an

N-labeled heme precursor.

Figure 16-18 Two possible pathways for the biosynthesis of

ether– 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



COO







OOCCH

COO



OOC

CHCH



COO





COO



COO



NN Fe

Heme

Starting materials

Scheme I

reduction

Scheme II

Vinyl ether

Ether

CHCHO

R

CHCHO

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 easily

sorted 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 its

precursor.

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

that is, when the radioactivity of a product is at its peak, it

should be equal to that of its precursor. This result also im-

plies that the radioactivity of a product peaks after that of its

precursor.

d[B*]

 k[A*]  k[B*]  k([A*]  [B*])

Starting material*

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

C-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 O

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 precursor

to 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*]

[B*]

Specific radioactivity

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

DNA

Genotype

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) method

for 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 cysteines

with ICAT reagent

Digest and affinity

purify labeled peptides

Analyze by

LC/MS and

MS/MS

200

1405.0 1426.0

1417

1409

Percentage intensityPercentage intensity

100

400 600

Mass (m/z)

800

Biotin Reactive group

Linker

R(d0)-biotin

(b)

(a)

R(d8)-biotin

Quantify by MS

Identify by MS/MS

Cys

NHHN

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 metabolomics

aimed 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 in

silico (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 are

driven 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 bond

followed by two phosphoanhydride bonds. Adenosine

diphosphate (ADP) and 5ⴕ-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.

¬PO

2

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 P

and PP

, respectively, represent orthophosphate

and pyrophosphate in any of their ioniza-

tion states. These highly exergonic reactions are coupled to

numerous endergonic biochemical processes so as to drive

them to completion. Conversely, ATP is regenerated by cou-

pling its formation to a more highly exergonic metabolic

process (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 P

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 P

to the

even more exergonic hydrolysis of phosphoenolpyruvate

(Fig. 16-23b; Section 17-2J).

The bioenergetic utility of phosphoryl-transfer reactions

stems from their kinetic stability to hydrolysis combined

with their capacity to transmit relatively large amounts of

free 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

4

)(PO

3

)

ATP  H

O Δ AMP  PP

ATP  H

O Δ ADP  P

¬O¬PO

2

 R

¬OH Δ R

¬OH  R

¬O¬PO

2

578 Chapter 16. Introduction to Metabolism

Figure 16-22 The structure of ATP indicating its relationship

to 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.

–

Adenosine

POPO

Phosphoanhydride

bonds

Phosphoester

bond

–

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 Mg

2

have high phosphate-binding affinities), and ionic

strength. Reasonable estimates of the intracellular values

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

]

(which are generally on the order of millimolar) indicate

that ATP hydrolysis under physiological conditions has

G ⬇ 50 kJ ⴢ mol

1

rather than the 30.5 kJ ⴢ 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, and

metabolites commonly vary by several orders of magnitude

across 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 vivo

reactions 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

Endergonic

half-reaction 1

(a)

(b)

Exergonic

half-reaction 2

Exergonic

half-reaction 1

Overall

coupled reaction

glucose

glucose-6-P

ATP

COO

–

COO

–

+13.8

–30.5

+30.5

–31.4

–16.7

– 61.9

ATP

O+

ATP +

ADP

COO

–

ADP +

ADP

Overall

coupled reaction

COO

–

Endergonic

half-reaction 2

ΔG° (kJ

•

mol

–1

)

ΔG° (kJ

•

mol

–1

)

Table 16-3 Standard Free Energies of Phosphate Hydrolysis

of Some Compounds of Biological Interest

Compound G°¿ (kJ ⴢ mol

1

)

Phosphoenolpyruvate 61.9

1,3-Bisphosphoglycerate 49.4

ATP (S AMP ⴙ PP

) ⴚ45.6

Acetyl phosphate 43.1

Phosphocreatine 43.1

ATP (S ADP ⴙ P

) ⴚ30.5

Glucose-1-phosphate 20.9

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 of

Biochemistry and Molecular Biology (3rd ed.), Physical and Chemical

Data,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 ⴢ 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 acetyl

phosphate 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 highly

exergonic enol–keto conversion that provides phospho-

enolpyruvate with the added thermodynamic impetus to

phosphorylate 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,

α-

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

OPO

–

OPO

–

1,3-Bisphosphoglycerate

Acetyl phosphate

OPO

2

CH OPO

22

POCH

OOH

580 Chapter 16. Introduction to Metabolism



OPP



PPHH OO O

Figure 16-24 Resonance and electrostatic stabilization in a

phosphoanhydride 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.

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