Biochemistry, 4th Edition P57 pdf

b Plus inhibitor: In the presence of an inhibitor in this case, an inhibitor of enzyme 4 , intermediates upstream of the metabolic block B, C, and D accumulate, reveal-ing themselves as

NADPH Provides the Reducing Power for Anabolic Processes

Whereas catabolism is fundamentally an oxidative process, anabolism is, by its

con-trasting nature, reductive The biosynthesis of the complex constituents of the cell

begins at the level of intermediates derived from the degradative pathways of

catab-olism; or, less commonly, biosynthesis begins with oxidized substances available in

the inanimate environment, such as carbon dioxide When the hydrocarbon chains

of fatty acids are assembled from acetyl-CoA units, activated hydrogens are needed

photosyn-thesis in plants, reducing power is required These reducing equivalents are

pro-vided by NADPH, the usual source of high-energy hydrogens for reductive

hydride ions In heterotrophic organisms, these electrons are removed from fuel

viewed as the carrier of electrons from catabolic reactions to anabolic reactions

(Figure 17.12) In photosynthetic organisms, the energy of light is used to pull

elec-trons from water and transfer them to NADP; O2is a by-product of this process

Coenzymes and Vitamins Provide Unique Chemistry

and Essential Nutrients to Pathways

to metabolism Some of these are essential nutrients called vitamins (The name was

coined by Kazimierz Funk, who discovered thiamine as a cure for beriberi in 1912

and termed it a “vital amine.” He later proposed that other diseases might be cured

by “vitamins.”)

Vitamins are required in the diet, usually in trace amounts, because they cannot

be synthesized by the organism itself The requirement for any given vitamin

de-pends on the organism Not all vitamins are required by all organisms Vitamins

re-quired in the human diet are listed in Table 17.3 These important substances are

traditionally distinguished as being either water soluble or fat soluble

Except for vitamin C (ascorbic acid), the water-soluble vitamins are all

compo-nents or precursors of important biological substances known as coenzymes These

are low-molecular-weight molecules that bring unique chemical functionality to

certain enzyme reactions Coenzymes may also act as carriers of specific functional

groups, such as methyl groups and acyl groups The side chains of the common

amino acids provide only a limited range of chemical reactivities and carrier

prop-erties Coenzymes, acting in concert with appropriate enzymes, provide a broader

range of catalytic properties for the reactions of metabolism Coenzymes are

typi-cally modified by these reactions and are then converted back to their original

forms by other enzymes, so small amounts of these substances can be used

repeat-edly The coenzymes derived from the water-soluble vitamins are listed in Table

17.3 Each will be discussed in the context of the chemistry they provide to specific

pathways in Chapters 18 through 27 The fat-soluble vitamins are not directly

re-lated to coenzymes, but they play essential roles in a variety of critical biological

processes, including vision, maintenance of bone structure, and blood

coagula-tion The mechanisms of action of fat-soluble vitamins are not as well understood

as their water-soluble counterparts, but modern research efforts are gradually

clos-ing this gap

17.4 What Experiments Can Be Used to Elucidate

Metabolic Pathways?

Armed with the knowledge that metabolism is organized into pathways of successive

reactions, we can appreciate by hindsight the techniques employed by early

bio-chemists to reveal their sequence A major intellectual advance took place at the

Catabolism

Reductive biosynthetic reactions Reductive

biosynthetic product

Oxidized precursor

Reduced fuel

Oxidized product

FIGURE 17.12 Transfer of reducing equivalents from catabolism to anabolism via the NADPH cycle.

end of the 19th century when Eduard Buchner showed that the fermentation of glu-cose to yield ethanol and carbon dioxide can occur in extracts of broken yeast cells Until this discovery, many thought that metabolism was a vital property, unique to intact cells; even the eminent microbiologist Louis Pasteur, who contributed so

much to our understanding of fermentation, was a vitalist, one of those who

be-lieved that the processes of living substance transcend the laws of chemistry and physics After Buchner’s revelation, biochemists searched for intermediates in the transformation of glucose and soon learned that inorganic phosphate was essential

to glucose breakdown This observation gradually led to the discovery of a variety of phosphorylated organic compounds that serve as intermediates along the fermen-tative pathway

An important tool for elucidating the steps in the pathway was the use of

meta-bolic inhibitors Adding an enzyme inhibitor to a cell-free extract caused an

accumu-lation of intermediates in the pathway prior to the point of inhibition (Figure 17.13) Each inhibitor was specific for a particular site in the sequence of metabolic events As the arsenal of inhibitors was expanded, the individual steps in metabo-lism were revealed

Discussed

Water-Soluble

Thiamine (vitamin B1)

Niacin (nicotinic acid)

Riboflavin (vitamin B2)

Pantothenic acid

Pyridoxal, pyridoxine,

pyridoxamine (vitamin B6)

Cobalamin (vitamin B12)

Biotin

Lipoic acid

Folic acid

Fat-Soluble

Retinol (vitamin A)

Retinal (vitamin A)

Retinoic acid (vitamin A)

Ergocalciferol (vitamin D2)

Cholecalciferol (vitamin D3)

-Tocopherol (vitamin E)

Menaquinone (vitamin K)

Thiamine pyrophosphate

Nicotinamide adenine dinucleotide (NAD) Nicotinamide adenine dinucleo-tide phosphate (NADP) Flavin adenine dinucleotide (FAD)

Flavin mononucleotide (FMN) Coenzyme A

Pyridoxal phosphate

5-Deoxyadenosylcobalamin

Methylcobalamin Biotin–lysine complexes (biocytin)

Lipoyl–lysine complexes (lipoamide)

Tetrahydrofolate

Decarboxylation of -keto acids and formation and

cleavage of -hydroxyketones

Hydride transfer

Hydride transfer

One- and two-electron transfer

One- and two-electron transfer Activation of acyl groups for transfer by nucleophilic attack, and activation of the -hydrogen of the acyl

group for abstraction as a proton Formation of stable Schiff base (aldimine) adducts with-amino groups of amino acids; serving as an

electron sink to stabilize reaction intermediates Intramolecular rearrangement, reduction of ribonucleotides to deoxyribonucleotides, and methyl group transfer

Carrier of carboxyl groups in carboxylation reactions

Coupling acyl group transfer and electron transfer during oxidation and decarboxylation of -keto acids

Acceptor and donor of 1-C units for all oxidation levels of carbon except that of CO2

19, 22

18–27

21, 22, 24–26

19, 20, 23, 26

20

19, 23, 24, 27

25

23

22, 24

19

25, 26

E 1 E 2 E 3 E 4 E 5 E 6 E 1 E 2 E 3 E 4 E 5 E 6

Control:

Intermediate

Inhibitor

Intermediate

(b) (a)

FIGURE 17.13 The use of inhibitors to reveal the

se-quence of reactions in a metabolic pathway (a) Control:

Under normal conditions, the steady-state concentra-tions of a series of intermediates will be determined by the relative activities of the enzymes in the pathway.

(b) Plus inhibitor: In the presence of an inhibitor (in this

case, an inhibitor of enzyme 4 ), intermediates upstream

of the metabolic block (B, C, and D) accumulate, reveal-ing themselves as intermediates in the pathway The concentration of intermediates lying downstream (E and F) will fall.

24Na Radioactive , 15 hours

36Cl Radioactive  310,000 years

59Fe Radioactive , 45 days

131I Radioactive , 8 days

*The relative natural abundance of a stable isotope is important because, in tracer studies, the amount of stable isotope

in Metabolic Studies

Mutations Create Specific Metabolic Blocks

Genetics provides an approach to the identification of intermediate steps in

me-tabolism that is somewhat analogous to inhibition Mutation in a gene encoding an

enzyme often results in an inability to synthesize the enzyme in an active form Such

a defect leads to a block in the metabolic pathway at the point where the enzyme

acts, and the enzyme’s substrate accumulates Such genetic disorders are lethal if

the end product of the pathway is essential or if the accumulated intermediates have

toxic effects In microorganisms, however, it is often possible to manipulate the

growth medium so that essential end products are provided Then the biochemical

consequences of the mutation can be investigated Studies on mutations in genes of

the filamentous fungus Neurospora crassa led G W Beadle and E L Tatum to

hy-pothesize in 1941 that genes are units of heredity that encode enzymes (a principle

referred to as the “one gene–one enzyme” hypothesis)

Isotopic Tracers Can Be Used as Metabolic Probes

Another widely used approach to the elucidation of metabolic sequences is to “feed”

cells a substrate or metabolic intermediate labeled with a particular isotopic form of

an element that can be traced Two sorts of isotopes are useful in this regard:

radioac-tive isotopes, such as 14C, and stable “heavy” isotopes, such as 18O or 15N (Table 17.4)

Because the chemical behavior of isotopically labeled compounds is rarely distin-guishable from that of their unlabeled counterparts, isotopes provide reliable “tags” for observing metabolic changes The metabolic fate of a radioactively labeled sub-stance can be traced by determining the presence and position of the radioactive atoms in intermediates derived from the labeled compound (Figure 17.14)

Heavy Isotopes Heavy isotopes endow the compounds in which they appear with slightly greater masses than their unlabeled counterparts These compounds can be separated and quantitated by mass spectrometry (or density gradient centrifuga-tion, if they are macromolecules) For example, 18O was used in separate experi-ments as a tracer of the fate of the oxygen atoms in water and carbon dioxide to de-termine whether the atmospheric oxygen produced in photosynthesis arose from

H2O, CO2, or both:

CO2 H2O⎯⎯→ (CH2O) O2

none of the 18O was found in O2 Curiously, it was recovered as H218O In contrast, when plants fixing CO2were equilibrated with H218O,18O2was evolved These lat-ter labeling experiments established that photosynthesis is best described by the equation

C16O2 2 H218O⎯⎯→ (CH216O)18O2 H216O

accounted for in (CH2O), and two reduce the O lost from CO2to H2O

NMR Spectroscopy Is a Noninvasive Metabolic Probe

A technology analogous to isotopic tracers is provided by nuclear magnetic

resonance (NMR) spectroscopy.The atomic nuclei of certain isotopes, such as the naturally occurring isotope of phosphorus, 31P, have magnetic moments The

reso-nance frequency of a magnetic moment is influenced by the local chemical envi-ronment That is, the NMR signal of the nucleus is influenced in an identifiable way by the chemical nature of its neighboring atoms in the compound In many ways, these nuclei are ideal tracers because their signals contain a great deal of structural information about the environment around the atom and thus the

FIGURE 17.14 One of the earliest experiments using a

ra-dioactive isotope as a metabolic tracer Cells of Chlorella

(a green alga) synthesizing carbohydrate from carbon

iso-lated from the cells, separated by two-dimensional paper

chromatography, and observed via autoradiographic

ex-posure of the chromatogram Such experiments

identi-fied radioactive 3-phosphoglycerate (PGA) as the

was labeled in the 1-position (in its carboxyl group)

Ra-dioactive compounds arising from the conversion of

3-phosphoglycerate to other metabolic intermediates

included phosphoenolpyruvate (PEP), malic acid,

triose phosphate, alanine, and sugar phosphates and

nature of the compound containing the atom Transformations of substrates and

metabolic intermediates labeled with magnetic nuclei can be traced by following

changes in NMR spectra Furthermore, NMR spectroscopy is a noninvasive

proce-dure Whole-body NMR spectrometers are being used today in hospitals to directly

observe the metabolism (and clinical condition) of living subjects (Figure 17.15)

NMR promises to be a revolutionary tool for clinical diagnosis and for the

investi-gation of metabolism in situ (literally “in site,” meaning, in this case, “where and

as it happens”)

Metabolic Pathways Are Compartmentalized Within Cells

Although the interior of a prokaryotic cell is not subdivided into compartments by

internal membranes, the cell still shows some segregation of metabolism For

ex-ample, certain metabolic pathways, such as phospholipid synthesis and oxidative

phosphorylation, are localized in the plasma membrane Protein biosynthesis is

car-ried out on ribosomes

In contrast, eukaryotic cells are extensively compartmentalized by an

endo-membrane system Each of these cells has a true nucleus bounded by a double

membrane called the nuclear envelope The nuclear envelope is continuous with

the endomembrane system, which is composed of differentiated regions: the

en-doplasmic reticulum; the Golgi complex; various membrane-bounded vesicles

such as lysosomes, vacuoles, and microbodies; and, ultimately, the plasma

mem-brane itself Eukaryotic cells also possess mitochondria and, if they are

photo-synthetic, chloroplasts Disruption of the cell membrane and fractionation of the

cell contents into the component organelles have allowed an analysis of their

re-spective functions (Figure 17.16) Each compartment is dedicated to specialized

metabolic functions, and the enzymes appropriate to these specialized functions

are confined together within the organelle In many instances, the enzymes of a

metabolic sequence occur together within the organellar membrane Thus, the

flow of metabolic intermediates in the cell is spatially as well as chemically segregated For

example, the 10 enzymes of glycolysis are found in the cytosol, but pyruvate, the

product of glycolysis, is fed into the mitochondria These organelles contain the

energy released in the process is captured by the oxidative phosphorylation

system of mitochondrial membranes and used to drive the formation of ATP

(Figure 17.17)

(a)

31 P

Chemical shift

Before exercise

Phosphocreatine

ATP

Chemical shift

(b)

During exercise

Phosphocreatine

FIGURE 17.15 With NMR spectroscopy, one can observe the metabolism of a living subject in real time These NMR spectra show the changes in ATP, creatine-P

human subjected to 19 minutes of exercise Note that

chemical shifts, reflecting their different chemical environments.

600 rpm

Teflon pestle

Tissue–sucrose homogenate

(minced tissue + 0.25 M sucrose buffer)

Strain homogenate

to remove connective tissue and blood vessels.

Tube is

moved

slowly up

and down

as pestle

rotates.

Centrifuge homogenate

at 600 g × 10 min.

Centrifuge supernatant 1

Supernatant 1

Supernatant 2

Centrifuge

100,000 g × 60 min.

Nuclei and any unbroken cells

Mitochondria, lysosomes, and microbodies

Supernatant 3:

Soluble fraction

of cytoplasm (cytosol)

Ribosomes and microsomes,

consisting of endoplasmic reticulum, Golgi, and plasma membrane fragments

FIGURE 17.16 Fractionation of a cell extract by differential centrifugation It is possible to separate organelles and subcellular particles in a centrifuge because their inherent size and density differences give them different rates of sedimentation in an applied centrifugal field Nuclei are pelleted in relatively weak centrifugal fields and mitochondria in somewhat stronger fields, whereas very strong centrifugal fields are necessary to pellet ribo-somes and fragments of the endomembrane system.

17.5 What Can the Metabolome Tell Us

about a Biological System?

Rapid advances in chemical analysis have made it possible to carry out

comprehen-sive studies of the many metabolites in a living organism The metabolome is the

complete set of low-molecular-weight molecules present in an organism or excreted

by it under a given set of physiological conditions Metabolomics is the systematic

identification and quantitation of all these metabolites in a given organism or

sam-ple It is quite remarkable that biochemists can foresee the rise of a true systems

biology, where comprehensive information sets from the genome, the

transcrip-tome, the proteome, and the metabolome will combine to provide incisive

descrip-tions of biological systems and detailed understanding of many human diseases

Even simple organisms present daunting challenges for metabolomic analyses

There are more than 500 metabolites represented in Figure 17.2, but far more

ex-ist in a typical cell For example, the 40 or so fatty acids occurring in a cell can alone

account for thousands of different metabolites (Triglycerides, with three fatty acids

esterified to a glycerol backbone, could account for 40  40  40  64,000 species

by themselves!) The Human Metabolomics Database (www.hmdb.ca) provides data

on more than 2500 metabolites known in cells of the human body and human body

fluids (blood, urine, and so on) Metabolomic measurements must be able to

re-solve and discriminate this array of small molecules Moreover, concentrations of

metabolites vary widely, from 1012M (for many hormones) to 0.1 M (for Naions)

Comprehensive metabolomic analyses involve processing of many samples, so the

time and cost required per sample must be as low as possible

Mass spectrometry (MS) and nuclear magnetic resonance (NMR) are both

pow-erful techniques for metabolomic analysis Mass spectrometry offers unmatched

sen-sitivity for detection of metabolites at low concentrations (Figure 17.18), and NMR

spectroscopy can provide remarkable resolution and discrimination of metabolites

in complex mixtures (Figure 17.19) Combination of these techniques with a variety

of chromatographic separation protocols (Figure 17.20) makes it possible to analyze

thousands of metabolites in biological samples rapidly and at low cost

100

50

0

m/z

320

Control

100

50

0

m/z

320

PKU

100

50

0

m/z

320

HCY

100

50

0

m/z

320

MSUD

FIGURE 17.18Mass spectrometry offers remarkable sensitivity for metabolomic analyses Shown here are desorption electrospray ionization mass spectra for urine samples from individuals with inborn errors of

from Pan, Z., Gu, H., et al., 2007 Principal component analysis of urine metabolites by NMR and DESI-MS in patients with inborn

errors of metabolism Analytical and Bioanalytical Chemistry

387:539–549.)

ATP

2

+

Glucose

Glucose

2 Pyruvate

Glycolysis

in the cytosol

Acetyl-CoA

Citric acid cycle

Citric acid cycle and oxidative phosphoryla-tion in the mitochondria ATP

ATP

ATP

NADH

CO 2

O 2

2 NADH

NADH ADP

H 2 O P i

ATP

2

FIGURE 17.17 Compartmentalization of glycolysis, the citric acid cycle, and oxidative phosphorylation.

13 C

80 60 40

Arabidopsis extract

Mixture of standards

FIGURE 17.19 (a) One-dimensional1 H NMR

spectrum of an equimolar mixture of

26 small-molecule standards (b)

Two-dimensional NMR spectrum of the same

mixture (red) overlaid onto a spectrum of

aqueous whole-plant extract from

Arabidopsis thaliana, a model organism

for the study of plant molecular biology

Schommer, S., et al., 2007 Method for determining

molar concentrations of metabolites in complex

solutions from two-dimensional 1 H- 13 C NMR

spectra Analytical Chemistry 79:9385–9390.)

100

0 10.0 12.0 14.0 16.0 18.0 20.0 22.0 24.0 26.0 28.0 30.0 32.0 34.0 36.0 38.0 40.0 42.0 44.0 46.0 48.0 50.0

Time

FIGURE 17.20The combination of mass spectrometry

and gas chromatography makes it possible to separate

and identify hundreds of metabolites Shown is an ion

chromatogram of a human urine sample, with 1582

Aronov, P., 2007 Mass spectrometry-based metabolomics Mass

17.6 What Food Substances Form the Basis

of Human Nutrition?

The use of foods by organisms is termed nutrition The ability of an organism to use a

particular food material depends upon its chemical composition and upon the

meta-bolic pathways available to the organism In addition to essential fiber, food includes

the macronutrients—protein, carbohydrate, and lipid—and the micronutrients—

including vitamins and minerals

Humans Require Protein

Humans must consume protein in order to make new proteins Dietary protein is a

rich source of nitrogen, and certain amino acids—the so-called essential amino

acids—cannot be synthesized by humans (and various animals) and can be

ob-tained only in the diet The average adult in the United States consumes far more

protein than required for synthesis of essential proteins Excess dietary protein is

then merely a source of metabolic energy Some of the amino acids (termed

gluco-genic ) can be converted into glucose, whereas others, the ketogenic amino acids,

can be converted to fatty acids and/or keto acids If fat and carbohydrate are

al-ready adequate for the energy needs of the individual, then both kinds of amino

acids will be converted to triacylglycerol and stored in adipose tissue

An individual’s protein undergoes a constant process of degradation and

resyn-thesis Together with dietary protein, this recycled protein material participates in

a nitrogen equilibrium, or nitrogen balance A positive nitrogen balance occurs

whenever there is a net increase in the organism’s protein content, such as during

periods of growth A negative nitrogen balance exists when dietary intake of

nitrogen is insufficient to meet the demands for new protein synthesis

Carbohydrates Provide Metabolic Energy

The principal purpose of carbohydrate in the diet is production of metabolic energy

Simple sugars are metabolized in the glycolytic pathway (see Chapter 18) Complex

carbohydrates are degraded into simple sugars, which then can enter the glycolytic

pathway Carbohydrates are also essential components of nucleotides, nucleic

acids, glycoproteins, and glycolipids Human metabolism can adapt to a wide range

of dietary carbohydrate levels, but the brain requires glucose for fuel When

di-etary carbohydrate consumption exceeds the energy needs of the individual,

ex-cess carbohydrate is converted to triacylglycerols and glycogen for long-term

en-ergy storage On the other hand, when dietary carbohydrate intake is low, ketone

bodiesare formed from acetate units to provide metabolic fuel for the brain and

other organs

Lipids Are Essential, But in Moderation

Fatty acids and triacylglycerols can be used as fuel by many tissues in the human

body, and phospholipids are essential components of all biological membranes

Even though the human body can tolerate a wide range of fat intake levels, there

are disadvantages in either extreme Excess dietary fat is stored as triacylglycerols in

adipose tissue, but high levels of dietary fat can also increase the risk of

athero-sclerosis and heart disease Moreover, high dietary fat levels are also correlated with

increased risk for colon, breast, and prostate cancers When dietary fat

consump-tion is low, there is a risk of essential fatty acid deficiencies As will be seen in

Chap-ter 24, the human body cannot synthesize linoleic and linolenic acids, so these must

be acquired in the diet In addition, arachidonic acid can by synthesized in humans

only from linoleic acid, so it too is classified as essential The essential fatty acids are

key components of biological membranes, and arachidonic acid is the precursor to

prostaglandins, which mediate a variety of processes in the body

Fiber May Be Soluble or Insoluble

The components of food materials that cannot be broken down by human digestive

enzymes are referred to as dietary fiber There are several kinds of dietary fiber, each with its own chemical and biological properties Cellulose and hemicellulose are

in-soluble fiber materials that stimulate regular function of the colon They may play a

role in reducing the risk of colon cancer Lignins, another class of insoluble fibers,

ab-sorb organic molecules in the digestive system Lignins bind cholesterol and clear it from the digestive system, reducing the risk of heart disease Pectins and gums are wa-ter-soluble fiber materials that form viscous gel-like suspensions in the digestive system, slowing the rate of absorption of many nutrients, including carbohydrates, and lower-ing serum cholesterol in many cases The insoluble fibers are prevalent in vegetable grains Water-soluble fiber is a component of fruits, legumes, and oats

A DEEPER LOOK

A Popular Fad Diet—Low Carbohydrates, High Protein, High Fat

Possibly the most serious nutrition problem in the United States is

excessive food consumption, and many people have experimented

with fad diets in the hope of losing excess weight One of the most

popular of the fad diets has been the high-protein, high-fat

(low-carbohydrate) diet The presumed rationale is tantalizing:

Be-cause the tricarboxylic acid (TCA) cycle (see Chapter 19) plays a

key role in fat catabolism and because glucose is usually needed to

replenish intermediates in the TCA cycle, if carbohydrates are

re-stricted in the diet, dietary fat should merely be converted to

ke-tone bodies and excreted This so-called diet appears to work at

first because a low-carbohydrate diet results in an initial water

(and weight) loss This occurs because glycogen reserves are

de-pleted by the diet and because about 3 grams of water of hydration are lost for every gram of glycogen

However, the premise for such diets is erroneous for several reasons First, ketone body excretion by the human body usually does not exceed 20 grams (400 kJ) per day Second, amino acids can function effectively to replenish TCA cycle intermediates, making the reduced carbohydrate regimen irrelevant Third, the typical fare in a high-protein, high-fat, low-carbohydrate diet is expensive but not very tasty, and it is thus difficult to maintain

Finally, a diet high in saturated and trans fatty acids is a high risk

factor for atherosclerosis and coronary artery disease

SUMMARY

unifying principles of modern biology is that organisms show marked

similarity in their major pathways of metabolism Given the almost

un-limited possibilities within organic chemistry, this generality would

ap-pear most unlikely Yet it’s true, and it provides strong evidence that all

life has descended from a common ancestral form All forms of

nutri-tion and almost all metabolic pathways evolved in early prokaryotes

prior to the appearance of eukaryotes 1 billion years ago All organisms,

even those that can synthesize their own glucose, are capable of glucose

degradation and ATP synthesis via glycolysis Other prominent pathways

are also virtually ubiquitous among organisms.

repre-sents the sum of the chemical changes that convert nutrients, the “raw

materials” necessary to sustain living organisms, into energy and the

chemically complex finished products of cells Metabolism consists of

literally hundreds of enzymatic reactions organized into discrete

path-ways Metabolic maps portray the principal reactions of the

intermedi-ary metabolism of carbohydrates, lipids, amino acids, and their

deriva-tives In such maps, arrows connect metabolites and represent the

enzyme reactions that interconvert the metabolites Alternative

map-pings of biochemical pathways have been proposed in a response to the

emergence of genomic, transcriptomic, and proteomic perspectives on

the complexity of biological systems.

com-plex nutrient molecules (carbohydrates, lipids, and proteins) obtained

either from the environment or from cellular reserves The breakdown

of these molecules by catabolism leads to the formation of simpler mol-ecules such as lactic acid, ethanol, carbon dioxide, urea, or ammonia Catabolic reactions are usually exergonic, and often the chemical

en-ergy released is captured in the form of ATP Anabolism is a synthetic

process in which the varied and complex biomolecules (proteins, nu-cleic acids, polysaccharides, and lipids) are assembled from simpler pre-cursors Such biosynthesis involves the formation of new covalent bonds, and an input of chemical energy is necessary to drive such dergonic processes The ATP generated by catabolism provides this en-ergy Furthermore, NADPH is an excellent donor of high-energy elec-trons for the reductive reactions of anabolism.

An important tool for elucidating the steps in the pathway is the use of

metabolic inhibitors Adding an enzyme inhibitor to a cell-free extract

causes an accumulation of intermediates in the pathway prior to the point of inhibition Each inhibitor is specific for a particular site in the sequence of metabolic events Genetics provides an approach to the identification of intermediate steps in metabolism that is somewhat analogous to inhibition Mutation in a gene encoding an enzyme often results in an inability to synthesize the enzyme in an active form Such a defect leads to a block in the metabolic pathway at the point where the enzyme acts, and the enzyme’s substrate accumulates Such genetic dis-orders are lethal if the end product of the pathway is essential or if the accumulated intermediates have toxic effects In microorganisms, how-ever, it is often possible to manipulate the growth medium so that