Voet D., Voet Ju.G. Biochemistry

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pathways among these various intermediates (including ade-

nine), but all of these pathways lead to uric acid. Of course,

the intermediates in these processes may instead be reused to

form nucleotides via salvage reactions. In addition, ribose-1-

phosphate, a product of the reaction catalyzed by purine

nucleoside phosphorylase (PNP), is isomerized by phospho-

ribomutase to the PRPP precursor ribose-5-phosphate.

Adenosine and deoxyadenosine are not degraded by

mammalian PNP. Rather, adenine nucleosides and nu-

cleotides are deaminated by adenosine deaminase (ADA)

and AMP deaminase to their corresponding inosine deriv-

atives, which, in turn, may be further degraded. The X-ray

structure of murine ADA that was crystallized in the pres-

ence of its inhibitor purine ribonucleoside was determined

by Florante Quiocho (Fig. 28-24a). The enzyme forms an

eight-stranded ␣/␤ barrel with its active site in a pocket at the

C-terminal end of the ␤ barrel, as occurs in nearly all known

␣/␤ barrel enzymes (Section 8-3Bh). Purine ribonucleoside

Section 28-4. Nucleotide Degradation 1131

Figure 28-24 X-ray structure and mechanism of adenosine deaminase. (a) A

ribbon diagram of murine adenosine deaminase in complex with its transition

state analog 6-hydroxy-1,6-dihydropurine ribonucleoside (HDPR).The

polypeptide is drawn in ribbon form colored according to its secondary structure

(helices cyan, ␤ strands magenta, and loops salmon) and viewed approximately

down the axis of the enzyme’s ␣/␤ barrel from the N-terminal ends of its ␤

strands.The HDPR is shown in stick form with its C, N, and O atoms green, blue,

and red.The enzyme-bound Zn

2⫹

ion, which is coordinated by HDPR’s

6-hydroxyl group, is represented by a silver sphere. [Based on an X-ray structure

by Florante Quiocho, Baylor College of Medicine. PDBid 1ADA.] (b) The

proposed catalytic mechanism of adenosine deaminase. A Zn

2⫹

-polarized H

molecule (Section 15-1Cb) nucleophilically attacks C6 of the enzyme-bound

adenosine molecule in a process that is facilitated by His 238 acting as a general

base, Glu 217 acting as a general acid, and Asp 295 acting to orient the water

molecule via hydrogen bonding.The resulting tetrahedral intermediate

decomposes by the elimination of ammonia in a reaction that is aided by the now

imidazolium and carboxyl side chains of His 238 and Glu 217 acting as a general

acid and a general base, respectively. This yields inosine in its enol tautomeric

form, which, on its release from the enzyme, largely assumes its dominant keto

form.The Zn

2⫹

is coordinated by three His side chains that are not shown. [After

Wilson, D.K. and Quiocho, F.A., Biochemistry 32, 1692 (1993).]

See

Interactive Exercise 30

Inosine (enol tautomer)

Glu 217

Asp 295

–

H His 238

Ribose

Glu 217

–

Asp 295

–

sp 295

His 238

Glu 217

Ribose

Tetrahedral intermediate

H His 238

Ribose

Adenosine

(a) (b)

JWCL281_c28_1107-1142.qxd 10/19/10 9:59 AM Page 1131

binds to ADA in a normally rare hydrated form, 6-hydroxy-1,

6-dihydropurine ribonucleoside (HDPR),

a nearly ideal transition state analog of the ADA reaction.

Although it had been previously reported that ADA does

not require a cofactor, its X-ray structure clearly reveals

that a zinc ion is bound in the deepest part of the active site

pocket, where it is pentacoordinated by three His side

chains, a carboxyl oxygen of Asp 295, and the O6 atom of

HDPR. ADA’s active site complex suggests a catalytic

mechanism (Fig. 28-24b) reminiscent of that of carbonic

anhydrase (Section 15-1Cb): His 238, which is properly

positioned to act as a general base, abstracts a proton from

a bound Zn

2

-activated water molecule, which nucleophili-

cally attacks the adenine C6 atom to form a tetrahedral

intermediate. Products are then formed by the elimination

of ammonia.

a. Genetic Defects in ADA Result in Severe

Combined Immunodeficiency Disease

Abnormalities in purine nucleoside metabolism arising

from rare genetic defects in ADA selectively kill lympho-

cytes (a type of white blood cell). Since lymphocytes medi-

ate much of the immune response (Section 35-2A), ADA

deficiency results in severe combined immunodeficiency

disease (SCID) that, without special protective measures, is

invariably fatal in infancy due to overwhelming infection.

The mutations in all eight known ADA variants obtained

from SCID patients appear to structurally perturb the

active site of ADA.

Biochemical considerations provide a plausible expla-

nation of SCID’s etiology (causes). In the absence of active

ADA, deoxyadenosine is phosphorylated to yield levels of

dATP that are 50-fold greater than normal. This high con-

centration of dATP inhibits ribonucleotide reductase (Sec-

tion 28-3Ad), thereby preventing the synthesis of the other

dNTPs, choking off DNA synthesis and thus cell prolifera-

tion. The tissue-specific effect of ADA deficiency on the

immune system may be explained by the observation that

lymphoid tissue is particularly active in deoxyadenosine

phosphorylation.

SCID caused by ADA defects does not respond to treat-

ment by the intravenous injection of ADA because the

liver clears this enzyme from the bloodstream within min-

utes. If, however, several molecules of the biologically inert

polymer polyethylene glycol (PEG)

Polyethylene glycol

HO [¬CH

¬CH

¬O¬]

Ribose

6-Hydroxy-1,6-dihydropurine

ribonucleoside (HDPR)

Purine ribonucleoside

are covalently linked to surface groups on ADA,the resulting

PEG–ADA remains in the blood for 1 to 2 weeks, thereby

largely resuscitating the SCID victim’s immune system. The

protein-linked PEG only reduces the catalytic activity of

ADA by ⬃40% but, evidently, masks it from the receptors

that filter it out of the blood. SCID can therefore be treated

effectively by PEG–ADA.This treatment, however,is expen-

sive and not entirely satisfactory. Consequently, ADA defi-

ciency was selected as one of the first genetic diseases to be

treated by gene therapy (Section 5-5Hb): Lymphocytes were

extracted from the blood of an ADA-deficient child and

grown in the laboratory, had a normal ADA gene inserted

into them via genetic engineering techniques (Section 5-5),

and were then returned to the child.After 12 years, 20 to 25%

of the patient’s lymphocytes contained the introduced ADA

gene. However, ethical considerations have mandated that

the patient continue receiving injections of PEG–ADA so

that the efficacy of this gene therapy protocol is unclear.

b. The Purine Nucleotide Cycle

The deamination of AMP to IMP, when combined with

the synthesis of AMP from IMP (Fig. 28-4,left), has the effect

of deaminating aspartate to yield fumarate (Fig. 28-25). John

Lowenstein demonstrated that this purine nucleotide cycle

has an important metabolic role in skeletal muscle. An in-

crease in muscle activity requires an increase in the activity

of the citric acid cycle. This process usually occurs through

the generation of additional citric acid cycle intermediates

(Section 21-4). Muscles, however, lack most of the enzymes

that catalyze these anaplerotic (filling up) reactions in other

tissues. Rather, muscle replenishes its citric acid cycle inter-

mediates as fumarate generated in the purine nucleotide cy-

cle.The importance of the purine nucleotide cycle in muscle

metabolism is indicated by the observation that the activities

of the three enzymes involved are all severalfold higher in

muscle than in other tissues. In fact, individuals with an in-

herited deficiency in muscle AMP deaminase (myoadeny-

late deaminase deficiency) are easily fatigued and usually

suffer from cramps after exercise.

c. Xanthine Oxidase Is a Mini-Electron-

Transport Protein

Xanthine oxidase (XO) converts hypoxanthine to xan-

thine, and xanthine to uric acid (Fig. 28-23, bottom). In

mammals, this enzyme occurs mainly in the liver and the

small intestinal mucosa. XO is a homodimer of ⬃1330-

residue subunits, each of which binds a variety of electron-

transfer agents: an FAD, two spectroscopically distinct

[2Fe–2S] clusters, and a molybdopterin complex (Mo-pt)

OPO

–

Molybdopterin complex (Mo-pt)

....

1132 Chapter 28. Nucleotide Metabolism

JWCL281_c28_1107-1142.qxd 6/8/10 10:39 AM Page 1132

in which the Mo atom cycles between its Mo(VI) and

Mo(IV) oxidation states. The final electron acceptor is O

which is converted to H

, a potentially harmful oxidizing

agent that is subsequently disproportionated to H

O and

by catalase (Section 1-2Ad). In XO, the polypeptide has

been proteolytically cleaved into three segments (the un-

cleaved enzyme, which is known as xanthine dehydroge-

nase, preferably uses NAD



as its electron acceptor,

whereas XO does not react with NAD



The X-ray structure of XO from cow’s milk in complex

with the competitive inhibitor salicylic acid (Fig. 25-74),

determined by Emil Pai, reveals that the FAD and the molyb-

dopterin complex are interposed by the two [2Fe–2S] clusters

to form a mini-electron-transport chain (Fig. 28-26). Each of

its three peptide segments forms a separate domain with the

N-terminal domain binding the two [2Fe–2S] clusters, the cen-

tral domain binding the FAD, and the C-terminal domain

binding the Mo-pt complex. Although the salicylic acid does

not contact the Mo-pt complex, it binds to XO in a way that

blocks the approach of substrates to the metal center.

XO hydroxylates xanthine at its C8 position (and hy-

poxanthine at its C2 position), yielding uric acid in its enol

form that tautomerizes to the more stable keto form:

Uric acid (enol tautomer) Uric acid (keto tautomer)

Urate

pK = 5.4

HNHN

Section 28-4. Nucleotide Degradation 1133

Figure 28-25 The purine nucleotide cycle. This pathway

functions, in muscle, to prime the citric acid cycle by generating

fumarate.

Figure 28-26 X-ray structure of

xanthine oxidase from cow’s milk in

complex with salicylic acid.

(a) Ribbon diagram of its

1332-residue subunit in which the

N-terminal domain (residues 2–165)

is cyan, the central domain (residues

224–528) is gold, and the C-terminal

domain (residues 571–1315) is

lavender.The enzyme’s redox

cofactors and bound salicylic acid

are shown in space-filling form with

C green, N blue, O red, S yellow, P

magenta, Fe orange, and Mo light

blue. The ⬃50-residue peptide

segments spanning domains are

disordered and are apparently

highly flexible. (b) The enzyme’s

redox cofactors and salicylic acid

(Sal) drawn in stick form with their

S, Fe, and Mo atoms represented by

spheres.The atoms are colored as in

Part a and viewed from the same

direction but with greater

magnification. [Based on an X-ray

structure by Emil Pai, University of

Toronto,Toronto, Ontario, Canada.

PDBid 1FIQ.]

AMP

GTP

Aspartate

GDP P

Adenylosuccinate

adenylosuccinate

lyase

adenylosuccinate

synthetase

AMP

deaminase

IMP

Fumarate

Net:

O + Aspartate + GTP + GDP P

++fumarate

(a) (b)

JWCL281_c28_1107-1142.qxd 4/22/10 9:17 AM Page 1133

(its enol form ionizes with a pK of 5.4; hence, the name uric

acid).

O-labeling experiments have demonstrated that

the C8 keto oxygen of uric acid is derived from H

whereas the oxygen atoms of H

come from O

. Chemi-

cal and spectroscopic studies suggest that the enzyme has

the following mechanism (Fig. 28-27):

1. The reaction is initiated by the attack of an enzyme

nucleophile, X, on the C8 position of xanthine.

2. The C8¬H atom is eliminated as a hydride ion that

combines with the Mo(VI) complex, thereby reducing it to

the Mo(IV) state.

3. Water displaces the enzyme nucleophile producing

uric acid.

In the second stage of the reaction, the now reduced en-

zyme is reoxidized to its original Mo(VI) state by reaction

with O

. This complex process, not surprisingly, is but

poorly understood. EPR measurements indicate that elec-

trons are funneled from the Mo(IV) through the two

[2Fe–2S] clusters to the flavin and ultimately to O

, yield-

ing H

and regenerated enzyme.

B. Fate of Uric Acid

In humans and other primates, the final product of purine

degradation is uric acid, which is excreted in the urine. The

same is true of birds, terrestrial reptiles, and many insects,

but these organisms, which do not excrete urea, also catab-

olize their excess amino acid nitrogen to uric acid via

purine biosynthesis. This complicated system of nitrogen

excretion has a straightforward function: It conserves water.

Uric acid is only sparingly soluble in water, so that its ex-

cretion as a paste of uric acid crystals is accompanied by

very little water. In contrast, the excretion of an equivalent

amount of the much more water-soluble urea osmotically

sequesters a significant amount of water.

In all other organisms, uric acid is further processed be-

fore excretion (Fig. 28-28). Mammals other than primates

oxidize it to their excretory product, allantoin, in a reaction

catalyzed by the Cu-containing enzyme urate oxidase. A

further degradation product, allantoic acid, is excreted by

teleost (bony) fish. Cartilaginous fish and amphibia further

degrade allantoic acid to urea prior to excretion. Finally,

marine invertebrates decompose urea to their nitrogen

excretory product, NH



a. Gout Is Caused by an Excess of Uric Acid

Gout is a disease characterized by elevated levels of uric

acid in body fluids. Its most common manifestation is ex-

crutiatingly painful arthritic joint inflammation of sudden

onset, most often in the big toe (Fig. 28-29), caused by

deposition of nearly insoluble crystals of sodium urate.

Sodium urate and/or uric acid may also precipitate in the

kidneys and ureters as stones, resulting in renal damage

and urinary tract obstruction. Gout, which affects ⬃3 per

1000 persons, predominantly males, has been tradition-

ally, although inaccurately, associated with overindulgent

eating and drinking. The probable origin of this associa-

tion is that in previous centuries, when wine was often

contaminated with lead during its manufacture and stor-

age, heavy drinking resulted in chronic lead poisoning,

which, among other things, decreases the kidney’s ability

to excrete uric acid.

1134 Chapter 28. Nucleotide Metabolism

Figure 28-27 Mechanism of xanthine oxidase. The reduced enzyme is subsequently reoxidized

by O

, yielding H

Uric acid

(enol tautomer)

Enzyme

nucleophile

Xanthine

Reduced

enzyme

Reduced

enzyme

Enzyme–Mo

complex

(fully oxidized form)

Mo(VI)

Mo(IV)

••

JWCL281_c28_1107-1142.qxd 4/22/10 9:17 AM Page 1134

The most prevalent cause of gout is impaired uric acid

excretion (although usually for other reasons than lead

poisoning). Gout may also result from a number of meta-

bolic insufficiencies, most of which are not well character-

ized. One well-understood cause is HGPRT deficiency

(Lesch–Nyhan syndrome in severe cases), which leads to

excessive uric acid production through PRPP accumula-

tion (Section 28-1D). Uric acid overproduction is also

caused by glucose-6-phosphatase deficiency (von Gierke’s

glycogen storage disease; Section 18-4): The increased

availability of glucose-6-phosphate stimulates the pentose

phosphate pathway (Section 23-4), increasing the rate of

ribose-5-phosphate production and consequently that of

PRPP, which in turn stimulates purine biosynthesis.

Gout may be treated by administration of the xanthine

oxidase inhibitor allopurinol, a hypoxanthine analog with

interchanged N7 and C8 positions.

Xanthine oxidase hydroxylates allopurinol, as it does hy-

poxanthine, yielding alloxanthine,

which remains tightly bound to the reduced form of the en-

zyme, thereby inactivating it. Allopurinol consequently al-

leviates the symptoms of gout by decreasing the rate of uric

acid production while increasing the levels of the more sol-

uble hypoxanthine and xanthine. Although allopurinol

controls the gouty symptoms of Lesch–Nyhan syndrome, it

has no effect on its neurological symptoms.

Alloxanthine

Allopurinol Hypoxanthine

Section 28-4. Nucleotide Degradation 1135

Figure 28-28 Degradation of uric acid to ammonia. The

process is arrested at different stages in the indicated species and

the resulting nitrogen-containing product is excreted.

Figure 28-29 The Gout, a cartoon by James Gillray (1799).

[Yale University Medical Historical Library.]

Primates

Birds

Reptiles

Insects

Uric acid

2 H

O + O

+ H

urate oxidase

allantoinase

Urea

COOH

CHO

Glyoxylic acid

allantoicase

2CO

urease

Other mammals

Allantoin

Excreted by

4 NH

Teleost fish

Allantoic acid

Cartilaginous fish

Amphibia

Marine

invertebrates

COOH

JWCL281_c28_1107-1142.qxd 4/23/10 10:38 AM Page 1135

C. Catabolism of Pyrimidines

Animal cells degrade pyrimidine nucleotides to their compo-

nent bases (Fig. 28-30, top). These reactions, like those of

purine nucleotides, occur through dephosphorylation, deam-

ination, and glycosidic bond cleavages. The resulting uracil

and thymine are then broken down in the liver through

reduction (Fig. 28-30, middle) rather than by oxidation, as

occurs in purine catabolism. The end products of pyrimidine

catabolism, -alanine and -aminoisobutyrate, are amino

acids and are metabolized as such. They are converted,

through transamination and activation reactions, to malonyl-

CoA and methylmalonyl-CoA (Fig. 28-30, bottom left) for

further utilization (Sections 25-4A and 25-2Ea).

5 BIOSYNTHESIS OF NUCLEOTIDE

COENZYMES

In this section we outline the assembly, in animals, of the

nucleotide coenzymes NAD



and NADP



, FMN and

FAD, and coenzyme A, from their vitamin precursors.

These vitamins are synthesized de novo only by plants and

microorganisms.

A. Nicotinamide Coenzymes

The nicotinamide moiety of the nicotinamide coenzymes

(NAD



and NADP



) is derived, in humans, from dietary

nicotinamide, nicotinic acid,or the essential amino acid tryp-

tophan (Fig. 28-31). Nicotinate phosphoribosyltransferase,

1136 Chapter 28. Nucleotide Metabolism

Figure 28-30 Major pathways of pyrimidine catabolism in ani-

mals. The amino acid products of these reactions are taken up in

other metabolic processes. UMP and dTMP are degraded by the

same enzymes; the pathway for dTMP degradation is given in

parentheses.

nucleotidase

Cytidine Uridine (Deoxythymidine)

cytidine

deaminase

uridine

phosphorylase

(d)Ribose-1-P

Rib P

CMP

(d)Rib

UMP (dTMP)

Uracil (Thymine)

dihydrouracil

dehydrogenase

NADP + H

NADP

()

Dihydrouracil

(Dihydrothymine)

()

hydropyrimidine

hydratase

-Ureidopropionate

( -Ureidoisobutyrate)

()

–

ONH

-ureidopropionaseβ

()

OOC

-Alanine

( -Aminoisobutyrate)

–

Glutamate

-Ketoglutarate

Malonic semialdehyde

(Methylmalonic semialdehyde)

()

COO

–

NADH + H

NAD

+CoA

Malonyl-CoA

(Methylmalonyl-CoA)

()

COO

–

CoA

aminotransferase

JWCL281_c28_1107-1142.qxd 4/22/10 9:17 AM Page 1136

Section 28-5. Biosynthesis of Nucleotide Coenzymes 1137

Figure 28-31 Pathways for the biosynthesis of NAD



and NADP



. These nicotinamide coenzymes are

synthesized from their vitamin precursors,

nicotinate and nicotinamide, and from the

tryptophan degradation product, quinolinate.

Nicotinamide adenine dinucleotide phosphate (NADP

)

Nicotinamide adenine dinucleotide (NAD

)

Nicotinate adenine dinucleotide

Nicotinate mononucleotide

Nicotinamide mononucleotide (NMN)

Tryptophan

Quinolinate

Nicotinate

Nicotinamide

PRPP

nicotinate

phosphoribosyl

transferase

ATP

pyrophosphorylase

PRPP

nicotinamide

phosphoribosyl

transferase

Ribose Ribose

Adenine

PRPP

Glutamine

COO

OHOH

quinolinate

phosphoribosyl

transferase

NAD

+ ATP

pyrophosphorylase

NAD

ATP

NAD

synthetase

NAD

kinase

Glutamate

ATP

ADP

P P

Ribose Ribose

Adenine

OH O

OHOH

P P

JWCL281_c28_1107-1142.qxd 4/22/10 9:17 AM Page 1137

which occurs in most mammalian tissues, catalyzes the

formation of nicotinate mononucleotide from nicotinate

and PRPP. This intermediate may also be synthesized

from quinolinate, a degradation product of tryptophan

(Section 26-3G), in a reaction mediated by quinolinate

phosphoribosyltransferase, which occurs mainly in liver

and kidney. A poor diet, nevertheless, may result in pella-

gra (nicotinic acid deficiency; Section 13-3), since, under

such conditions, tryptophan will be almost entirely uti-

lized in protein biosynthesis. Nicotinate mononucleotide

is joined via a pyrophosphate linkage to an ATP-derived

AMP residue by NAD

⫹

pyrophosphorylase to yield

nicotinate adenine dinucleotide (desamido NAD

ⴙ

). Finally,

NAD

ⴙ

synthetase converts this intermediate to NAD

⫹

a transamidation reaction in which glutamine is the NH

donor.

NAD

⫹

may also be synthesized from nicotinamide. This

vitamin is converted to nicotinamide mononucleotide

(NMN) by nicotinamide phosphoribosyltransferase, a

widely occurring enzyme distinct from nicotinate phospho-

ribosyltransferase. However, NAD

⫹

is synthesized from

NMN and ATP by NAD pyrophosphorylase, the same

enzyme that synthesizes nicotinate adenine dinucleotide.

NADP

⫹

is formed via the ATP-dependent phosphoryla-

tion of the NAD

⫹

adenosine residue’s C2¿ OH group by

NAD

ⴙ

kinase.

B. Flavin Coenzymes

FAD is synthesized from riboflavin in a two-reaction path-

way (Fig. 28-32). First, the 5¿-OH group of riboflavin’s

ribityl side chain is phosphorylated by flavokinase, yielding

flavin mononucleotide (FMN; not a true nucleotide since

its ribityl residue is not a true sugar). FAD may then be

formed by the coupling of FMN and ATP-derived AMP in

a pyrophosphate linkage in a reaction catalyzed by FAD

pyrophosphorylase. Both of these enzymes are widely

distributed in nature.

C. Coenzyme A

Coenzyme A is synthesized in mammalian cells according

to the pathway diagrammed in Fig. 28-33. Pantothenate,

an essential vitamin, is phosphorylated by pantothenate

kinase and then coupled to cysteine, the future business

end of CoA, by phosphopantothenoylcysteine synthetase.

After decarboxylation by phosphopantothenoylcysteine

decarboxylase, the resulting 4ⴕ-phosphopantethiene is

coupled to AMP in a pyrophosphate linkage by dephospho-

CoA pyrophosphorylase and then phosphorylated at its

adenosine 3¿ OH group by dephospho-CoA kinase to

form CoA. The latter two enzymatic activities occur on a

single protein.

1138 Chapter 28. Nucleotide Metabolism

Figure 28-32 Biosynthesis of FMN and FAD from the vitamin

precursor riboflavin.

FAD

pyrophosphorylase

ATP

OCH

OHH

OHOH

OHH

Flavin adenine dinucleotide (FAD)

Flavin mononucleotide (FMN)

flavokinase

ATP

ADP

OHH

Riboflavin

JWCL281_c28_1107-1142.qxd 8/13/10 6:46 PM Page 1138

Section 28-5. Biosynthesis of Nucleotide Coenzymes 1139

Figure 28-33 Biosynthesis of coenzyme A from pantothenate, its vitamin precursor.

–

OHO

Adenine

pantothenate kinase

ATP

ADP

CH C

COO

–

Pantothenate

phosphopantothenoylcysteine

synthetase

ATP  Cysteine

 ADP

CH C

–2

COO

–

4′-Phosphopantothenate

phosphopantothenoylcysteine

decarboxylase

CH C

–2

COO

–

4′-Phosphopantothenoylcysteine

dephospho-CoA

pyrophosphorylase

ATP

CH C

–2

4′-Phosphopantetheine

dephospho-CoA

kinase

ADP

ATP

CH C

POPAdenosine O

Dephosphocoenzyme A

–

SHCH

Coenzyme A (CoA)

JWCL281_c28_1107-1142.qxd 4/22/10 9:17 AM Page 1139

1140 Chapter 28. Nucleotide Metabolism

1 Synthesis of Purine Ribonucleotides Almost all cells

synthesize purine nucleotides de novo via similar metabolic

pathways.The purine ring is constructed in an 11-step reaction

sequence that yields IMP. AMP and GMP are then synthe-

sized from IMP in separate pathways. Nucleoside diphos-

phates and triphosphates are sequentially formed from these

products via phosphorylation reactions. The rates of synthesis

of these various nucleotides are interrelated through feedback

inhibition mechanisms that monitor their concentrations.

Purine nucleotides may also be synthesized from free purines

salvaged from nucleic acid degradation processes. The impor-

tance of these salvage reactions is demonstrated, for example,

by the devastating and bizarre consequences of Lesch–Nyhan

syndrome.

2 Synthesis of Pyrimidine Ribonucleotides Cells also

synthesize pyrimidines de novo but, in this six-step process, a

free base is formed before it is converted to a nucleotide,

UMP. UTP is then formed by phosphorylation of UMP, and

CTP is synthesized by the amination of UTP. Pyrimidine

biosynthesis is regulated by feedback inhibition as well as by

the concentrations of purine nucleotides.

3 Formation of Deoxyribonucleotides Deoxyribonu-

cleotides are formed by reduction of the corresponding ri-

bonucleotides. Three classes of ribonucleotide reductase

(RNR) have been characterized: Class I RNR, which occurs in

nearly all eukaryotes and many prokaryotes, contains an

Fe(III)¬O

2–

¬Fe(III) group and a tyrosyl free radical; Class II

and III RNRs, which occur only in prokaryotes, contain,

respectively, a coenzyme B

cofactor, and a [4Fe–4S] cluster

together with a glycyl radical.All of them catalyze free radical–

based reductions. The substrates for Class I and II RNRs are

NDPs, whereas those for Class III RNRs are NTPs. Class I

RNR has three independent regulatory sites that control its

substrate specificity and its catalytic activity in part via its

oligomerization state, thereby generating deoxynucleotides in

the amounts required for DNA synthesis. The E. coli Class I

RNR is reduced to its original state by electron-transport

chains involving either thioredoxin, thioredoxin reductase,

and NADPH; or glutaredoxin, glutathione, glutathione reduc-

tase, and NADPH. Thymine is synthesized by the methylation

of dUMP by thymidylate synthase to form dTMP. The reac-

tion’s methyl source, N

-methylene-THF, is oxidized in the

reaction to yield dihydrofolate. N

-Methylene-THF is sub-

sequently regenerated through the sequential actions of dihy-

drofolate reductase and serine hydroxymethyltransferase.

Since this sequence of reactions is required for DNA biosyn-

thesis, it presents an excellent target for chemotherapy.

FdUMP, a mechanism-based inhibitor of thymidylate syn-

thase, and methotrexate, an antifolate that essentially irre-

versibly inhibits dihydrofolate reductase, are both highly ef-

fective anticancer agents.

4 Nucleotide Degradation Purine nucleotides are catab-

olized to yield uric acid. Depending on the species, the uric

acid is either directly excreted or first degraded to simpler

nitrogen-containing substances. Overproduction or underex-

cretion of uric acid in humans causes gout. Pyrimidines are

catabolized in animal cells to amino acids.

5 Biosynthesis of Nucleotide Coenzymes The nucleotide

coenzymes NAD



and NADP



, FMN and FAD, and coenzyme

A are synthesized in animals from vitamin precursors.

CHAPTER SUMMARY

General

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