Voet D., Voet Ju.G. Biochemistry

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PurK catalyzes the ATP-dependent carboxylation of AIR

to yield N

-CAIR, which Class I PurE rearranges to yield

CAIR. Class I PurE is homologous to AIR carboxylase,

which is therefore also called Class II PurE. Class I PurE

alone can catalyze the AIR carboxylase reaction but since

its K

for HCO



is 110 mM, it requires an unphysiologi-

cally high (⬃100 mM) HCO



concentration to do so at a

significant rate. However, the action of PurK decreases the

HCO



concentration required for the PurE-catalyzed re-

action by 1000-fold, presumably through the ATP-driven

formation of carbonyl phosphate, as is also postulated to

occur in the carbamoyl phosphate synthetase reaction

(Section 26-2A). The observation that N

-CAIR is chemi-

cally unstable (it decomposes to AIR with a half-life of 15 s

at pH 7.5 and 25°C) suggests that N

-CAIR is channeled

between PurK and Class I PurE. In fact, in yeast and plants,

the N-terminus of Class I PurE is fused to the C-terminus

of PurK. However, in E. coli, these two enzymatic activities

occur on separate proteins for which there is no evidence

of association.

8. Acquisition of N1. Purine atom N1 is contributed by

aspartate in an amide-forming condensation reaction

yielding 5-aminoimidazole-4-(N-succinylocarboxamide)

ribotide (SAICAR) that is catalyzed by SAICAR syn-

thetase (PurC).The reaction,which is driven by the hydrol-

ysis of ATP to ADPP

, chemically resembles Reaction 3.

9. Elimination of fumarate. SAICAR is cleaved with

the release of fumarate, yielding 5-aminoimidazole-4-

carboxamide ribotide (AICAR) in a reaction catalyzed by

adenylosuccinate lyase (PurB). Reactions 8 and 9 chemically

resemble the reactions in the urea cycle in which citrulline

is aminated to form arginine (Sections 26-2C and 26-2D).

In both pathways, aspartate’s amino group is transferred to

an acceptor through an ATP-driven coupling reaction fol-

lowed by the elimination of the aspartate carbon skeleton

as fumarate. In plants and microorganisms, AICAR is also

formed in the biosynthesis of histidine (Section 26-5Be)

but since in that process the AICAR is derived from ATP,

it provides for no net purine biosynthesis.

10. Acquisition of C2. The final purine ring atom is ac-

quired through formylation by N

-formyltetrahydrofolate,

yielding 5-formaminoimidazole-4-carboxamide ribotide

(FAICAR) in a reaction catalyzed by AICAR transform-

ylase (PurH). In bacteria, this reaction and that of Reaction

4 are indirectly inhibited by sulfonamides, which, it will be

recalled, prevent the synthesis of folate by competing with

its p-aminobenzoate component (Section 26-4D).Animals,

including humans, must acquire folate through the diet,

since they are incapable of synthesizing it. They are there-

fore unaffected by sulfonamides. The antibiotic properties

of sulfonamides are therefore largely a result of their inhi-

bition of nucleic acid biosynthesis in susceptible bacteria.

11. Cyclization to form IMP. The final reaction in the

pathway, ring closure to form IMP, occurs through the elim-

ination of water as catalyzed by IMP cyclohydrolase

(PurJ). In contrast to Reaction 6, the cyclization that forms

the imidazole ring, this reaction does not entail ATP

hydrolysis.

In animals, the activities catalyzing Reactions 3, 4, and 6,

Reactions 7 and 8, and Reactions 10 and 11 occur on single

polypeptides. The intermediate products of these multi-

functional enzymes are not readily released to the medium

but are channeled to the succeeding enzymatic activities of

the pathway, thereby increasing the overall rates of these

multistep processes and protecting the intermediates from

degradation by other cellular enzymes. We have previously

seen, for example, that the formation of acetyl-CoA from

pyruvate takes place on the pyruvate dehydrogenase mul-

tienzyme complex, which contains three enzymes catalyz-

ing five consecutive reactions (Section 21-2A); that all

seven enzymatic activities catalyzing fatty acid synthesis

in animals occur on a single protein molecule (Section

25-4Ca,b); and that the multifunctional enzymes carbamoyl

phosphate synthase I (Section 26-2Aa), glutamate synthase

(Section 26-5Aa), tryptophan synthase (Section 26-5Bd),

and amidophosphoribosyltransferase (see above) pass re-

active intermediate products between their active sites via

protein tunnels. It is becoming increasingly apparent that

the association of functionally related enzymes is a wide-

spread phenomenon.

B. Synthesis of Adenine and Guanine

Ribonucleotides

IMP does not accumulate in the cell but is rapidly con-

verted to AMP and GMP. AMP, which differs from IMP

only in the replacement of its 6-keto group by an amino

The aromatization of the imidazole ring is facilitated by the

tautomeric shift of the reactant from its imine to its ena-

mine form.

7. Acquisition of C6. In higher eukaryotes, purine C6 is

introduced as HCO



(CO

) in an ATP-dependent reaction

catalyzed by AIR carboxylase that yields carboxyaminoim-

idazole ribotide (CAIR) and ADP  P

. However, in yeast,

plants, and most prokaryotes (including E. coli), this over-

all reaction occurs in two steps that are mediated by sepa-

rate enzymatic activities: PurK and Class I PurE.

AIR carboxylase

(Class II PurE)

N R5P H

R5P

AIR CAIR

-CAIR

TP + HCO

ADP + P

PurK

Class I PurE

Section 28-1. Synthesis of Purine Ribonucleotides 1111

JWCL281_c28_1107-1142.qxd 6/8/10 10:39 AM Page 1111

Ribose-5-phosphate

IMP

Ribose-5-phosphate

Adenylosuccinate

Ribose-5-phosphate

Xanthosine monophosphate (XMP)

CH COO

––

OOC

Aspartate + GTP

GDP + P

adenylosuccinate

synthetase

NAD

+ H

NADH + H

IMP dehydrogenase

Glutamine + ATP + H

Glutamate + AMP + PP

Ribose-5-phosphate

AMP

Ribose-5-phosphate

GMP

Fumarate

adenylosuccinate

lyase

GMP

synthetase

group, is synthesized in a two-reaction pathway (Fig. 28-4,

left). In the first reaction, aspartate’s amino group is linked

to IMP in a reaction driven by the hydrolysis of GTP to

GDP  P

to yield adenylosuccinate. In the second reac-

tion, adenylosuccinate lyase eliminates fumarate from

adenylosuccinate to form AMP.This enzyme also catalyzes

Reaction 9 of the IMP pathway (Fig. 28-2).

GMP is also synthesized from IMP in a two-reaction

pathway (Fig. 28-4, right). In the first reaction, IMP dehy-

drogenase catalyzes the NAD



-dependent oxidation of

IMP to form xanthosine monophosphate (XMP; the ri-

bonucleotide of the base xanthine). XMP is then converted

to GMP by the replacement of its 2-keto group with gluta-

mine’s amide nitrogen in a reaction driven by the hydroly-

sis of ATP to AMP  PP

(and subsequently to 2 P

IMP dehydrogenase, a homotetramer of 514-residue

subunits, was incubated with IMP, NAD



, and the fungally

produced inhibitor mycophenolic acid (MPA).

The X-ray structure of the resulting complex, determined

by Keith Wilson, reveals that the enzyme had bound MPA

together with a reaction intermediate in which IMP atom

Mycophenolic acid (MPA)

OCH

1112 Chapter 28. Nucleotide Metabolism

Figure 28-4 IMP is converted to AMP or GMP in separate

two-reaction pathways. The X-ray structures for all of the

enzymes catalyzing these reactions are shown to the outside of

the corresponding reaction arrow. The X-ray structures for these

homooligomers are shown as described in the legend to Fig. 28-2.

Adenylosuccinate synthetase from E. coli, determined by

Herbert Fromm and Richard Honzatko, Iowa State University, is

a C

dimer in complex with IMP (green), GDP (red), and

hadacidin (magenta; a competitive inhibitor of aspartate); PDBid

1GIM.Adenylosuccinate lyase, from Thermatoga maritima,

determined by Todd Yeates, UCLA, is a D

tetramer; PDBid

1C3U. IMP dehydrogenase from Chinese hamsters, determined

by Keith Wilson,Vertex Pharmaceuticals, Cambridge,

Massachusetts, is a C

tetramer in complex with oxidized IMP

(red) and MPA (purple); PDBid 1JR1. GMP synthetase from

E. coli, determined by Janet Smith, Purdue University, is a D

tetramer in complex with AMP (red), pyrophosphate (blue), and

citrate (purple); PDBid 1GPM.

JWCL281_c28_1107-1142.qxd 4/22/10 9:16 AM Page 1112

C2 had become covalently linked to the Cys 331 S atom and

then dehydrogenated by NAD



to yield a thioimidate ester:

The mutagenic replacement of Cys 331 by Ala inactivates

the enzyme.These observations strongly support a catalytic

mechanism in which the Cys 331 thiol group nucleophili-

cally attacks IMP’s C2 atom, followed by hydride transfer

to NAD



to yield the above covalently bound intermedi-

ate, which is subsequently hydrolyzed to yield XMP. The

MPA binds to the enzyme with its bicyclic ring stacked on

the purine ring (as would be expected for NAD



’s nicoti-

namide ring) and with its phenolic hydroxyl group in the

proposed hydrolytic water site. This blocks the hydrolysis

of the thioimidate ester, thereby inactivating the enzyme.

IMP dehydrogenase activity is essential to the immune

response (Section 35-2) because it is required by the im-

mune system cells known as B and T lymphocytes to gener-

ate the guanosine nucleotides they need to proliferate.

Moreover, certain cancer cells have increased IMP dehy-

drogenase activity. Hence,IMP dehydrogenase is a target for

both immunosuppressive therapy and cancer chemotherapy.

Indeed, MPA is in clinical use to prevent the rejection of

transplanted kidneys.

a. Nucleoside Diphosphates and Triphosphates Are

Synthesized by the Phosphorylation of Nucleoside

Monophosphates

In order to participate in nucleic acid synthesis, nucleo-

side monophosphates must first be converted to the corre-

sponding nucleoside triphosphates. In the first of the two

sequential phosphorylation reactions that do so, nucleoside

diphosphates are synthesized from the corresponding

nucleoside monophosphates by base-specific nucleoside

monophosphate kinases. For example, adenylate kinase

(Section 17-4Fd) catalyzes the phosphorylation of AMP

to ADP:

Similarly, GDP is produced by a guanine-specific enzyme:

These nucleoside monophosphate kinases do not discrimi-

nate between ribose and deoxyribose in the substrate.

Nucleoside diphosphates are converted to the corre-

sponding triphosphates by nucleoside diphosphate kinase;

for instance,

Although this reaction is written with ATP as the phospho-

ryl donor and GDP as the acceptor, nucleoside diphos-

phate kinase is nonspecific as to the bases on either of its

ATP  GDP Δ ADP  GTP

GMP  ATP Δ GDP  ADP

AMP  ATP Δ 2ADP

Ribose-5-P

Enzyme–product thioimidate ester

Enzyme

substrates and as to whether their sugar residues are ribose

or deoxyribose. The reaction occurs via a Ping Pong mecha-

nism in which the substrate NTP phosphorylates an enzyme

His residue, which in turn, phosphorylates the substrate NDP.

The phosphoglycerate mutase reaction of glycolysis also has

a phospho-His intermediate (Section 17-2H).The nucleoside

diphosphate kinase reaction, as might be expected from the

nearly identical structures of its substrates and products,

normally operates close to equilibrium (G ⬇ 0). ADP is,

of course, also converted to ATP by a variety of energy-

releasing reactions such as those of glycolysis and oxidative

phosphorylation. Indeed, it is these reactions that ultimately

drive the foregoing kinase reactions.

C. Regulation of Purine Nucleotide Biosynthesis

The pathways involved in nucleic acid metabolism are

tightly regulated, as is evidenced, for example, by the in-

creased rates of nucleotide synthesis during cell prolifera-

tion. In fact, the pathways synthesizing IMP,ATP, and GTP

are individually regulated in most cells so as not only to

control the total amounts of purine nucleotides produced

but also to coordinate the relative amounts of ATP and

GTP. This control network is diagrammed in Fig. 28-5.

The IMP pathway is regulated at its first two reactions:

those catalyzing the synthesis of PRPP and 5-phosphoribo-

Section 28-1. Synthesis of Purine Ribonucleotides 1113

Figure 28-5 Control network for the purine biosynthesis

pathway. Red octagons and green dots indicate control points.

Feedback inhibition is indicated by dashed red arrows and

feedforward activation is represented by dashed green arrows.

See the Animated Figures

Ribose-5-phosphate

Inhibition

PRPP

5-Phosphoribosylamine

IMP

Adenylosuccinate

AMP

ADP

ATP

GMP

GDP

GTP

XMP

Activation

JWCL281_c28_1107-1142.qxd 4/22/10 9:16 AM Page 1113

sylamine. We have already seen that ribose phosphate py-

rophosphokinase, the enzyme catalyzing Reaction 1 of the

IMP pathway, is inhibited by both ADP and GDP (Section

28-1A). Amidophosphoribosyltransferase, the enzyme cat-

alyzing the first committed step of the IMP pathway (Reac-

tion 2), is likewise subject to feedback inhibition. In this

case, however, the enzyme binds ATP, ADP, and AMP at

one inhibitory site and GTP, GDP, and GMP at another.

The rate of IMP production is consequently independently

but synergistically controlled by the levels of adenine nu-

cleotides and guanine nucleotides. Moreover, amidophos-

phoribosyltransferase is allosterically stimulated by PRPP

(feedforward activation).

A second level of regulation occurs immediately below

the branch point leading from IMP to AMP and GMP (Fig.

28-4). AMP and GMP are each competitive inhibitors of

IMP in their own synthesis, so that excessive buildup of

these products is impeded. In addition, the synthesis rates

of adenine and guanine nucleotides are coordinated be-

cause GTP powers the synthesis of AMP from IMP,

whereas ATP powers the synthesis of GMP from IMP. This

reciprocity serves to balance the production of AMP and

GMP (which are required in roughly equal amounts in

nucleic acid biosynthesis): The rate of synthesis of GMP

increases with [ATP], whereas that of AMP increases with

[GTP].

D. Salvage of Purines

Most cells have an active turnover of many of their nucleic

acids (particularly some types of RNA) which, through

degradative processes described in Section 28-4A, result in

the release of adenine, guanine, and hypoxanthine. These

free purines are reconverted to their corresponding nu-

cleotides through salvage pathways. In contrast to the

de novo purine nucleotide synthesis pathway, which is vir-

tually identical in all cells, salvage pathways are diverse in

character and distribution. In mammals, purines are, for the

most part, salvaged by two different enzymes. Adenine

phosphoribosyltransferase (APRT) mediates AMP forma-

tion through the transfer of adenine to PRPP with the

release of PP

Hypoxanthine–guanine phosphoribosyltransferase (HGPRT)

catalyzes the analogous reaction for both hypoxanthine

and guanine:

a. Lesch–Nyhan Syndrome Results from

HGPRT Deficiency

The symptoms of Lesch–Nyhan syndrome, which is

caused by a severe HGPRT deficiency, indicate that purine

salvage reactions have functions other than conservation

of the energy required for de novo purine biosynthesis.This

sex-linked congenital defect (affects almost only males) re-

sults in excessive uric acid production (uric acid is a purine

Guanine ⫹ PRPP Δ GMP ⫹ PP

Hypoxanthine ⫹ PRPP Δ IMP ⫹ PP

Adenine ⫹ PRPP Δ AMP ⫹ PP

degradation product; Section 28-4A) and neurological ab-

normalities such as spasticity, mental retardation, and

highly aggressive and destructive behavior, including a

bizarre compulsion toward self-mutilation. For example,

many children with Lesch–Nyhan syndrome have such an

irresistible urge to bite their lips and fingers that they must

be restrained. If the restraints are removed, communicative

patients will plead that the restraints be replaced even as

they attempt to injure themselves.

The excessive uric acid production in patients with

Lesch–Nyhan syndrome is readily explained. The lack of

HGPRT activity leads to an accumulation of the PRPP that

would normally be used in the salvage of hypoxanthine and

guanine.The excess PRPP activates amidophosphoribosyl-

transferase (which catalyzes Reaction 2 of the IMP biosyn-

thesis pathway; Fig. 28-2), thereby greatly increasing the

rate of synthesis of purine nucleotides and consequently

that of their degradation product, uric acid.Yet the physio-

logical basis of the associated neurological abnormalities

remains obscure.That a defect in a single enzyme can cause

such profound but well-defined behavioral changes never-

theless has important psychiatric implications.

2 SYNTHESIS OF PYRIMIDINE

RIBONUCLEOTIDES

The biosynthesis of pyrimidines is a simpler process than

that of purines. Isotopic labeling experiments have shown

that atoms N1, C4, C5, and C6 of the pyrimidine ring are all

derived from aspartic acid, C2 arises from HCO

⫺

, and N3

is contributed by glutamine (Fig. 28-6). In this section we

discuss the pathways for pyrimidine ribonucleotide biosyn-

thesis and how these processes are regulated.

A. Synthesis of UMP

The major breakthrough in the determination of the path-

way for the de novo biosynthesis of pyrimidine ribonu-

cleotides was the observation that mutants of the bread

mold Neurospora crassa, which are unable to synthesize

pyrimidines and therefore require both cytosine and uracil

in their growth medium, grow normally when supplied in-

stead with the pyrimidine orotic acid (uracil-6-carboxylic

acid).

This observation led to the elucidation of the following six-

reaction pathway for the biosynthesis of UMP (Fig. 28-7).

Note that, in contrast to the case for purine nucleotides, the

pyrimidine ring is coupled to the ribose-5-phosphate moi-

ety after the ring has been synthesized.

Orotic acid (uracil-6-carboxylic acid)

COOH

1114 Chapter 28. Nucleotide Metabolism

JWCL281_c28_1107-1142.qxd 8/9/10 9:46 AM Page 1114

1. Synthesis of carbamoyl phosphate. The first reaction

of pyrimidine biosynthesis is the synthesis of carbamoyl

phosphate from HCO



and the amide nitrogen of glutamine

by the cytosolic enzyme carbamoyl phosphate synthetase II

(CPS II). This reaction is unusual in that it does not use

biotin and consumes two molecules of ATP: One provides

a phosphate group and the other energizes the reaction.

We have previously discussed the synthesis of carbamoyl

phosphate in connection with the formation of arginine

(Section 26-2A).The carbamoyl phosphate that is used to

Section 28-2. Synthesis of Pyrimidine Ribonucleotides 1115

Figure 28-6 The biosynthetic origins of pyrimidine ring atoms.

Figure 28-7 Metabolic pathway for the de novo synthesis of

UMP. The pathway consists of six enzymatically catalyzed

reactions. Note that, in contrast to the case for purine

biosynthesis (Fig. 28-2), the pyrimidine ring is formed before its

attachment to a ribose ring.The X-ray structures for the enzymes

are drawn as described in the legend to Fig. 28-2. Enzyme 1, from

E. coli, determined by Hazel Holden, University of Wisconsin, is

an 



heterooctamer with D

symmetry, whose large subunits

each bind two ADPNPs (red) and one ornithine (purple); PDBid

1D3H. Enzyme 2, from E. coli, determined by William Lipscomb,

Harvard University, is a c

heterododecamer with D

symmetry,

whose regulatory (r) subunits each bind a CTP (green); PDBid

Glutamine amide Aspartate

HCO

–

5AT1. Enzyme 3 from E. coli, determined by Hazel Holden,

University of Wisconsin, is a C

dimer that binds carbamoyl

aspartate (purple) in one subunit and orotate (red) in the other;

PDBid 1J79. Enzyme 4, from humans, determined by Jon Clardy,

Cornell University, is a monomer that binds orotate (yellow),

FMN (magenta), and A77 1726 (green); PDBid 1D3H. Enzyme 5,

from Salmonella typhimurium, determined by James Sacchettini,

Albert Einstein College of Medicine, is a C

dimer that binds

orotate (orange) and PRPP (purple); PDBid 1OPR. Enzyme 6,

from B. subtilis, determined by Steven Ealick, Cornell University,

is a C

dimer that binds UMP (green); PDBid 1DBT. See the

Animated Figures

–

2 ATP +

HCO

–

Glutamine

2 ADP +

Glutamate

carbamoyl phosphate

synthetase II

Carbamoyl phosphate

Aspartate

COO

–

Carbamoyl aspartate

dihydroorotase

COO

–

Dihydroorotate

dihydroorotate

dehydrogenase

Quinone

Reduced

quinone

PRPP

Orotate

orotate

phosphoribosyl

transferase

COO

–

COO

–

Orotidine-5-monophosphate (OMP)

OH OH

–

OMP decarboxylase

Uridine monophosphate (UMP)

OH OH

–

aspartate

transcarbamoylase

(ATCase)

JWCL281_c28_1107-1142.qxd 6/8/10 10:39 AM Page 1115

synthesize arginine via the urea cycle is synthesized by a

separate mitochondrial enzyme, carbamoyl phosphate syn-

thetase I (CPS I), which uses ammonia as its nitrogen

source. Prokaryotes only have one carbamoyl phosphate

synthetase, which supplies both pyrimidine and arginine

biosynthesis and utilizes glutamine. This latter enzyme, as

we have seen, contains three different active sites that are

connected by a remarkable 96-Å-long tunnel through

which intermediate products diffuse (Fig. 26-9).

The pyrimidine biosynthetic pathway is a target for

antiparasitic drugs. For example, the parasitic protozoan

Toxoplasma gondii, which infects most mammals, causes

toxoplasmosis, a disease whose complications include

blindness, neurological dysfunction, and death in immuno-

compromised individuals (e.g., those with AIDS). Most

parasites have evolved to take advantage of nutrients sup-

plied by their hosts. However, T. gondii is unable to meet its

needs exclusively through nucleotide salvage pathways and

retains the ability to synthesize uracil de novo. Drugs that

target the parasite’s carbamoyl phosphate synthetase II

(an enzyme whose structure and kinetics distinguish it

from its mammalian counterpart) could therefore prevent

T. gondii growth. Moreover, there is evidence that T. gondii

strains that have been engineered to lack carbamoyl phos-

phate synthetase II are avirulent and could be useful as

vaccines in humans and livestock.

2. Synthesis of carbamoyl aspartate. Condensation of

carbamoyl phosphate with aspartate to form carbamoyl

aspartate is catalyzed by aspartate transcarbamylase

(ATCase). This reaction, the pathway’s flux-generating

step, occurs without need of ATP because carbamoyl phos-

phate is intrinsically activated. The structure and regula-

tion of E. coli ATCase is discussed in Section 13-4.

3. Ring closure to form dihydroorotate. The third reac-

tion of the pathway was elucidated by Arthur Kornberg

following his observation that microorganisms made to uti-

lize orotic acid as a carbon source first reduce it to dihy-

droorotate. The reaction forming the pyrimidine ring

yields dihydroorotate in an intramolecular condensation

catalyzed by the zinc metalloenzyme dihydroorotase.

4. Oxidation of dihydroorotate. Dihydroorotate is irre-

versibly oxidized to orotate by dihydroorotate dehydroge-

nase (DHODH). The eukaryotic enzyme, which contains

FMN, is an integral membrane protein that is located on

the outer surface of the inner mitochondrial membrane,

where ubiquinone supplies its oxidizing power. The other

five enzymes of pyrimidine nucleotide biosynthesis are

cytosolic in animal cells. Many bacterial dihydroorotate de-

hydrogenases are NAD



-linked flavoproteins that contain

FMN, FAD, and a [2Fe–2S] cluster. These enzymes nor-

mally function degradatively, that is, in the direction oro-

tate S dihydroorotate, thereby permitting these bacteria

to metabolize orotate and accounting for Kornberg’s ob-

servation. The reaction mediated by eukaryotic DHODH

involves two redox steps, as is indicated in Fig. 28-8. The

X-ray structure of human DHODH in complex with oro-

tate, determined by Jon Clardy, reveals that the pyrimidine

ring of orotate is stacked over the FMN’s flavin ring with

the orotate C6 and FMN N5 separated by 3.6 Å, a distance

that is compatible with direct hydride transfer between

these two centers. A tunnel leads from the opposite side of

the flavin ring to a hydrophobic region on the enzyme sur-

face. The enzyme presumably binds to the mitochondrial

membrane surface via this hydrophobic patch, thereby per-

mitting ubiquinone, which readily diffuses within the mito-

chondrial membrane, to approach and reoxidize the en-

zyme’s bound FMNH

. In the X-ray structure, this tunnel

contains a tightly bound molecule named A77 1726, which

is the primary metabolite of leflunomide (trade name

Arava),

a compound that is in clinical use for the treatment of

rheumatoid arthritis. A77 1726 attenuates this autoimmune

disease by blocking pyrimidine biosynthesis in T lympho-

cytes, thereby reducing their inappropriate proliferation.

However,A77 1726 does not inhibit bacterial DHODHs.

5. Acquisition of the ribose phosphate moiety. Orotate

reacts with PRPP to yield orotidine-5-monophosphate

(OMP) in a reaction catalyzed by orotate phosphoribosyl-

transferase and driven by hydrolysis of the eliminated PP

This reaction fixes the anomeric form of pyrimidine nu-

cleotides in the  configuration. Orotate phosphoribosyl-

transferase also acts to salvage other pyrimidine bases,

such as uracil and cytosine, by converting them to their cor-

responding nucleotides. Although the various phosphori-

bosyltransferases, including HGPRT, exhibit little se-

quence similarity, their X-ray structures indicate that they

contain a common structural core that resembles the dinu-

cleotide binding fold (Section 8-3Bi) but lacks one of its

 strands.

6. Decarboxylation to form UMP. The final reaction of

the pathway is the decarboxylation of OMP by OMP de-

carboxylase (ODCase) to form UMP. ODCase enhances

the rate (k

cat

) of OMP decarboxylation by a factor of

2  10

over that of the uncatalyzed reaction, making it the

most catalytically proficient enzyme known. Yet ODCase

has no cofactors to help stabilize the reaction’s putative

carbanion intermediate. How is it able to do so? The X-ray

structure, by Steven Ealick, of ODCase from B. subtilis in

complex with UMP indicates that a bound OMP’s C6

A77 1726

Leflunomide

1116 Chapter 28. Nucleotide Metabolism

JWCL281_c28_1107-1142.qxd 4/22/10 9:16 AM Page 1116

carboxyl group that is coplanar with its pyrimidine ring

would be in close proximity to the side chains of both Asp

60 and Lys 62. Ealick has therefore proposed a mechanism

(Fig. 28-9) in which the electrostatic interactions between

the closely spaced carboxyl groups of OMP and Asp 60

destabilize OMP’s ground state. This destabilization would

be reduced in the transition state by the shift of OMP’s neg-

ative charge from its carboxyl group toward C6, where it

would be stabilized by the adjacent positively charged side

chain of Lys 62. This side chain is also proposed to proton-

ate the fragmenting C¬C bond when it becomes suffi-

ciently basic to accept the proton, thus avoiding the

formation of a high-energy carbanion intermediate. The

unfavorable electrostatic interaction between OMP and

Asp 60 occurs because the enzyme tightly binds OMP

through extensive interactions with its other functional

groups. Indeed, the removal of OMP’s phosphate group,

which is quite distant from the C6 carboxyl group, decreases

the catalytic reaction’s k

cat

by a factor of 7  10

, thus

providing a striking example of how binding energy can be

applied to catalysis (preferential transition state binding).

In bacteria, the six enzyme activities mediating UMP

biosynthesis occur on independent proteins (Fig. 28-7). In

animals, however, as Mary Ellen Jones demonstrated, the

first three enzymatic activities of the pathway, carbamoyl

Section 28-2. Synthesis of Pyrimidine Ribonucleotides 1117

Figure 28-8 Reactions catalyzed by eukaryotic dihydroorotate

dehydrogenase. The reaction is initiated by the enzyme-mediated

abstraction of a proton from C5 of dihydroorotate followed by

the direct hydride transfer from C6 of dihydroorotate to N5 of

FMN to yield orotate and FMNH



, which may then be

Figure 28-9 Proposed catalytic mechanism for OMP decarboxylase. [After Appleby, T.C.,

Kinsland, C., Begley, T.P., and Ealick, S.E., Proc. Natl. Acad. Sci. 97, 2005 (2000).]

Dihydroorotate

FMN

Coenzyme QH

COO

Orotate

COO

CO CH

(CH

)

CH C

FMNH

Coenzyme Q

CO CH

(CH

)

CH C

–

N+

Lys 62

Asp 60

–

Lys 62

O H

Asp 60

–

‡

N␦+

Lys 62

Asp 60

␦–

protonated to yield FMNH

.The FMNH

(or FMNH



) then

reacts with coenzyme Q acquired from the inner mitochondrial

membrane to regenerate the enzyme in its FMN form and yield

coenzyme QH

, which then re-enters the inner mitochondrial

membrane.

JWCL281_c28_1107-1142.qxd 6/8/10 10:39 AM Page 1117

phosphate synthetase II, ATCase, and dihydroorotase,

occur on a single 2225-residue polypeptide chain known as

CAD, which forms 1400-kD homohexamers. Similarly,

Reactions 5 and 6 of the animal pyrimidine pathway are

catalyzed by a single 480-residue polypeptide named UMP

synthase that forms 102-kD homodimers.

B. Synthesis of UTP and CTP

The synthesis of UTP from UMP is analogous to the syn-

thesis of purine nucleotide triphosphates (Section 28-1B).

The process occurs by the sequential actions of a nucleo-

side monophosphate kinase and nucleoside diphosphate

kinase:

CTP is formed by amination of UTP by CTP synthetase

(Fig. 28-10). In animals, the amino group is donated by

glutamine, whereas in bacteria it is supplied directly by

ammonia.

C. Regulation of Pyrimidine

Nucleotide Biosynthesis

In bacteria, the pyrimidine biosynthesis pathway is prima-

rily regulated at Reaction 2, the ATCase reaction

(Fig. 28-11a). In E. coli, control is exerted there through

the allosteric stimulation of ATCase by ATP and its inhibi-

tion by CTP (Section 13-4). In many bacteria, however,

UTP is the major ATCase inhibitor.

In animals, ATCase is not a regulatory enzyme. Rather,

pyrimidine biosynthesis is controlled by the activity of

carbamoyl phosphate synthetase II, which is inhibited by

UDP and UTP and activated by ATP and PRPP (Fig.

28-11b). A second level of control in the mammalian path-

way occurs at OMP decarboxylase, for which UMP and to

a lesser extent CMP are competitive inhibitors. In all or-

ganisms, the rate of OMP production varies with the avail-

ability of its precursor, PRPP. The PRPP level, it will be re-

called, depends on the activity of ribose phosphate

pyrophosphokinase, which is inhibited by ADP and GDP

(Section 28-1A).

UDP  ATP Δ UTP  ADP

UMP  ATP Δ UDP  ADP

a. Orotic Aciduria Results from an

Inherited Enzyme Deficiency

Orotic aciduria, an inherited human disease, is charac-

terized by the excretion of large amounts of orotic acid in

the urine, retarded growth, and severe anemia. It results

from a deficiency in the bifunctional enzyme catalyzing

1118 Chapter 28. Nucleotide Metabolism

Figure 28-10 Synthesis of CTP from UTP.

Figure 28-11 Regulation of pyrimidine biosynthesis. The

control networks are shown for (a) E. coli and (b) animals. Red

octagons and green dots indicate control points. Feedback

inhibition is represented by dashed red arrows and activation is

indicated by dashed green arrows.

See the Animated Figures

–

POP

–

O OP

OHOH

–

POP

–

O OP

OHOH

UTP

Glutamine

+ ATP + H

Glutamate

+ ADP + P

CTP synthetase

HH HH

CTP

pyrimidine

HCO

(a) (b)

E. coli

biosynthesis

Animal pyrimidine

biosynthesis

–

+ Glutamine +

Carbamoyl phosphate

Carbamoyl aspartate

Dihydroorotate

Orotate

OMP

UMP

UTP

CTP

PRPP

HCO

–

+ Glutamine +

Carbamoyl phosphate

Carbamoyl aspartate

Dihydroorotate

Orotate

OMP

UMP

UTP

CTP

PRPP

Inhibition

ATP

Activation

UDP

Inhibition

ATP

Activation

JWCL281_c28_1107-1142.qxd 4/22/10 9:16 AM Page 1118

Reactions 5 and 6 of pyrimidine nucleotide biosynthesis.

Consideration of the biochemistry of this situation led to

its effective treatment: the administration of uridine and/or

cytidine. The UMP formed through the phosphorylation of

these nucleosides, besides replacing that normally synthe-

sized, inhibits carbamoyl phosphate synthetase II so as to

attenuate the rate of orotic acid synthesis. Few other ge-

netic deficiencies in pyrimidine nucleotide biosynthesis are

known in humans, presumably because most such defects

are lethal in utero.

3 FORMATION OF

DEOXYRIBONUCLEOTIDES

DNA differs chemically from RNA in two major respects:

(1) its nucleotides contain 2¿-deoxyribose residues rather

than ribose residues, and (2) it contains the base thymine

(5-methyluracil) rather than uracil. In this section we con-

sider the biosynthesis of these DNA components.

A. Production of Deoxyribose Residues

Deoxyribonucleotides are synthesized from their correspon-

ding ribonucleotides by the reduction of their C2¿ position

rather than by their de novo synthesis from deoxyribose-

containing precursors.

This pathway was established through Irwin Rose’s study

of how rats metabolize cytidine that is

C-labeled in both

its base and ribose components.The dCMP recovered from

the rats’ DNA had the same labeling ratio in its cytosine

and deoxyribose residues as had the original cytidine, indi-

cating that the DNA’s components remained linked during

DNA synthesis. If the cytosine and the ribose residues had

become separated, dilution of the labeled cytosine and ri-

bose residues with unlabeled residues, which are present in

rat tissues in different amounts, would have altered this

ratio.

The enzymes that catalyze the formation of deoxyribonu-

cleotides by the reduction of the corresponding ribonu-

cleotides are named ribonucleotide reductases (RNRs).

Three classes of RNRs are known that differ in their

NDP

dNDP

OHOH

–

Base

HOH

–

Base

substrates (NDP or NTP), the cofactors they employ, and in

the way they obtain reducing equivalents (see below).

Class I and II RNRs are widely distributed among prokary-

otes; some species have a Class I RNR, whereas other,

sometimes related species have a Class II RNR. However,

all eukaryotes except a few unicellular species have Class I

RNRs. Class III RNRs occur in prokaryotes that can grow

anaerobically. (Class III RNRs are O

-sensitive whereas

Class I RNRs require O

for activation; see below.) In fact,

E. coli, which can grow both aerobically and anaerobically,

expresses a Class I and a Class III RNR. In what follows,

we shall mainly discuss the mechanism of Class I RNRs but

end with a consideration of the evolutionary relationships

among the different classes of RNRs.

a. Class I Ribonucleotide Reductase:

Structure and Mechanism

The E. coli Class I RNR, as Peter Reichard demon-

strated, is mainly present in vitro as a heterotetramer that

can be decomposed to two catalytically inactive homo-

dimers, R1

(761-residue subunits) and R2

(375-residue sub-

units), which together form the enzyme’s two active sites

(Fig. 28-12a). Each R1 subunit contains a substrate binding

site as well as three independent effector binding sites that

control both the enzyme’s catalytic activity and its sub-

strate specificity (see below). R1’s catalytic residues in-

clude several redox-active thiol groups.

The X-ray structure of R2

(Fig. 28-12b), determined by

Hans Eklund, reveals that each of its subunits contains a

novel binuclear Fe(III) prosthetic group whose two Fe(III)

ions are bridged by both an O

2

ion (a -oxo bridge) and

the carboxyl group of Glu 115 (Fig. 28-12c). Each Fe(III) is

further liganded by two carboxyl O atoms from Asp or Glu

residues, a His N



atom, and a water molecule. The Fe(III)

complex interacts with Tyr 122 to form, as EPR measure-

ments indicate, an unusual tyrosyl free radical (TyrO) that

is 5 Å from the closest Fe atom and is buried 10 Å beneath

the surface of the protein, where it is out of contact with

solvent and any oxidizable side chain [tyrosyl radicals have

also been observed in cytochrome c oxidase (Section 22-

2C5c) and in Photosystem II (Section 24-2Cd)].

The E. coli RNR is inhibited by hydroxyurea, which

specifically quenches (destroys) the tyrosyl radical, and by

8-hydroxyquinoline, which chelates Fe

3

ions.

Mammalian RNRs have similar characteristics to the

E. coli enzyme. Indeed, hydroxyurea is in clinical use as an

antitumor agent.

If E. coli RNR is incubated with [3¿-

H]UDP, a small

but reproducible fraction of the

H is released as

This observation, together with kinetic, spectroscopic, and

site-directed mutagenesis studies, led JoAnne Stubbe to

OHNHC

Hydroxyurea 8-Hydroxyquinoline

Section 28-3. Formation of Deoxyribonucleotides 1119

JWCL281_c28_1107-1142.qxd 6/8/10 10:39 AM Page 1119

R2 dimer

R1 dimer

(a)

Substrate-binding site

(ATP, GDP, UDP, CDP)

Specificity site

(ATP, dATP,

dGTP, dTTP)

Activity site

(ATP, dATP)

Hexamerization

site (ATP)

Allosteric

sites

Tyr

•

Tyr

O•

Tyr 122

Asp 84

Glu 115

2–

–

Fe 1

(c)

His 118

Glu 204

Glu 238

–

Fe 2

His 241

1120 Chapter 28. Nucleotide Metabolism

Figure 28-12 (Opposite) Class I ribonucleotide reductase from

E. coli. (a) A schematic diagram of its quaternary structure.The

enzyme consists of two pairs of identical subunits, R1

and R2

Each R2 subunit contains a binuclear Fe(III) complex that

generates a phenoxy radical at its Tyr 122. The R1 subunits each

contain three different allosteric effector sites and five

catalytically important Cys residues.The enzyme’s two active

sites are located near the interface between neighboring R1 and

R2 subunits. (b) The X-ray structure of R2

as viewed

perpendicular to its 2-fold axis with the dimer’s longest

dimension in the horizontal plane. One subunit of the

homodimeric protein is shown in blue and the other in yellow.

The Fe(III) ions of its binuclear Fe complexes are represented by

orange spheres and the radical-harboring Tyr 122 side chains are

drawn in space-filling form with their C and O atoms green and

red. Note that each subunit consists mainly of a bundle of eight

unusually long helices. (c) The binuclear Fe(III) complex of R2.

Each Fe(III) ion is octahedrally coordinated by a His N

␦

atom

and five O atoms, including those of the O

2⫺

ion and the Glu

carboxyl group that bridge the two Fe(III) ions. (d) The X-ray

structure of the R1 dimer, each subunit of which is in complex

with the 20-residue C-terminal peptide of R2 together with GDP

in the active site and dTTP in the specificity site. The ATP analog

AMPPNP bound in the activity site of the closely similar

complex of R1 with the 20-residue peptide and AMPPNP has

been superimposed on this structure. The structure is viewed

along its 2-fold axis with its two subunits lavender and light

green, the two R2 peptides cyan and magenta, and the GDP,

dTTP, and ATP shown in space-filling form colored according to

atom type (C green, N blue, O red, and P gold). [Parts b and d

based on X-ray structures by Hans Eklund, Swedish University

of Agricultural Sciences, Uppsala, Sweden. PDBids (b) 1RIB and

(d) 3R1R and 4R1R.]

See the Animated Figures and Interactive

Exercise 28

(b)

(d)

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