Voet D., Voet Ju.G. Biochemistry

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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



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. Relatively

few metabolites, mainly pyruvate, acetyl-CoA, and the citric

acid cycle intermediates, serve as starting materials for a host

of 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 two

metabolites are metabolically interconvertible, the pathway

from the first to the second must differ from the pathway

from 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

Section 16-1. Metabolic Pathways 561

Figure 16-2 ATP and NADPH are the sources of free energy

for 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 CO

via the citric acid cycle with the concomitant

reduction of NAD



and FAD. Reoxidation of these latter

coenzymes by O

via the electron-transport chain and oxidative

phosphorylation yields H

O and ATP.

ATP

NADPH

NADP

ADP + HPO

2–

Degradation

Simple products

Complex metabolites

Biosynthesis

Citric

Acid

Cycle

Oxidative

phosphorylation

Pyruvate

Acetyl-CoA

Glycolysis

GlucoseAmino acids Fatty acids & Glycerol

CarbohydratesProteins Lipids

FAD

NAD

ADP

NADH

ATP

FADH

NADH

FAD

NAD

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 property

of metabolic pathways because it allows independent control

of 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



) 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

Nucleophilic

reaction of an

amine

Basic reaction

of an amine RRNH



R CCO OH

R

R

R

R



RNH



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 Hydride

ion

CCCH









homolytic

cleavage

(i)

(ii)

(a) Nucleophiles

ROH

RSH

RNH



RNH



Nucleophilic

form



Hydroxyl group



Sulfhydryl group



Amino group



Imidazole group

(b) Electrophiles



Protons

n

Metal ions

R

Carbonyl carbon atom

R

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:

R

R

COH

R+

Amine Aldehyde

ketone

Carbinolamine

intermediate

R

R

R +

Imine

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 all

times, 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

YYAAXX

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

Y X



XRY



Tetrahedral

intermediate

(a)



(c)

O O



double

displacement

Resonance-stabilized

carbocation (oxonium ion)

single

displacement (S



P



(b)



Trigonal

bipyramid

intermediate



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 (O

). 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 O

can only ac-

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

ferred to O

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 O

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;

General

base

Alcohol NAD

ⴙ

General

acid

Ketone NADH

B H H







C¬H

Section 16-2. Organic Reaction Mechanisms 565

Figure 16-7 The phosphoryl-transfer reaction catalyzed by

hexokinase. 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.



Glucose

O ADP

ADP



Glucose

ATP

Trigonal

bipyramid

intermediate

Glucose-6-phosphate



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 H

O, NH

,an

alcohol (ROH), or a primary amine (RNH

).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

R

CC CC H



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 FADH

(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 B

)],

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.

OOO



HO OH

D-Ribitol

Riboflavin

Flavin adenine dinucleotide (FAD)

(oxidized or quinone form)

HO H

7, 8-Dimethylisoalloxazine

Adenosine

FADH

(reduced or hydroquinone form)

10a

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 B

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 form

the basis of both degradative and biosynthetic metabolism.

The breakdown of glucose to CO

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

HHCC

COO



C S CoA

HHCC

COO



CCC

SCoA

methylmalonyl-

CoA mutase

-Methylmalonyl-CoA Succinyl-CoA

Carbon skeleton rearrangement

C¬C

Section 16-2. Organic Reaction Mechanisms 567

Figure 16-9 Possible elimination reaction mechanisms using

dehydration 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.

R



 OH









R

Stepwise via a carbocation

Concerted

R R

Stepwise via a carbanion



R



R



(a)

(b)

trans (anti)







R

cis (syn)



Ketose

Aldose

cis-Enediolate intermediates

B H

H C

CB 







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

carbon atoms in such reactions are the sp

-hybridized car-

bonyl carbon atoms of aldehydes, ketones, esters, and CO

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-



CCOHO

 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 cleavage

reactions. (a) Aldol condensation, (b) Claisen ester

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

(a) Aldol condensation

(b) Claisen ester condensation

BB RC

R

H H



RRCC

R



B  RR

R

H HRC

H H

BB



H C

C SCoA H



C SCoA

Addition to

electrophilic

center [as

in (a)]

Addition to

electrophilic

center [as

in (a)]



C SCoA

Ketone

Acetyl-CoA

␤-Keto acid

Resonance-stabilized

enolate

Resonance-

stabilized

carbanion

(enolate)

Resonance-

stabilized

enolate

Second ketone

(electrophilic center)



RC CO



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 acid

in 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 CO

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 Zn

2ⴙ

–stabilized

enolate

Enolate

Hydrogen-bonded

carbonyl

Hydrogen-bonded

enolate or enol

Schiff base

carbanion (imine)

Schiff base

(enamine)

(a)

(b)

(c)

(d)

CCH







CCH or





CCH





2



CCH



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 be

used to both create metabolic blocks and to express genes in

tissues 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.

Phenylalanine

COO

–

Tyrosine

COO

–

COO

–

p-Hydroxyphenylpyruvate Phenylpyruvate

COO

–

Homogentisate

O + CO

Defective in

alcaptonuria

Originally unknown;

defective in

phenylketonuria

nonexistent:

originally

thought to

exist and be

defective in

phenylketonurics

secondary

pathway

COO

–

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