Harper’s Illustrated Biochemistry - Part 5 pptx

proto-Formation of Heme Involves Incorporation of Iron Into Protoporphyrin The final step in heme synthesis involves the tion of ferrous iron into protoporphyrin in a reaction incorpora-

PORPHYRINS & BILE PIGMENTS / 271

C 2

C H H 1

N

δ HC HC 8

HC HC 7 C

C NH IV

C C

CH

5 H H 6

N

CH

CH 3

N

Porphin

(C20H14N4)

Pyrrole

Figure 32–1. The porphin molecule Rings are

la-beled I, II, III, and IV Substituent positions on the rings

are labeled 1, 2, 3, 4, 5, 6, 7, and 8 The methenyl

bridges ( HC ) are labeled α, β, γ, and δ.

4 7

2 1

5 6

I

II IV

III

P P

P A

A P

I

II IV

III

Figure 32–2. Uroporphyrin III A (acetate) =

CH 2 COOH; P (propionate) = CH 2 CH2COOH.

droxymethylbilane (HMB) The reaction is catalyzed by

uroporphyrinogen I synthase, also named PBG

deami-nase or HMB synthase HMB cyclizes spontaneously to

form uroporphyrinogen I (left-hand side of Figure

32–6) or is converted to uroporphyrinogen III by the

action of uroporphyrinogen III synthase (right-hand side

of Figure 32–6) Under normal conditions, the

uropor-phyrinogen formed is almost exclusively the III isomer,

but in certain of the porphyrias (discussed below), the

type I isomers of porphyrinogens are formed in excess

Note that both of these uroporphyrinogens havethe pyrrole rings connected by methylene bridges

(CH2 ), which do not form a conjugated ring tem Thus, these compounds are colorless (as are allporphyrinogens) However, the porphyrinogens arereadily auto-oxidized to their respective colored por-phyrins These oxidations are catalyzed by light and bythe porphyrins that are formed

sys-Uroporphyrinogen III is converted to phyrinogen III by decarboxylation of all of the acetate(A) groups, which changes them to methyl (M) sub-

copropor-stituents The reaction is catalyzed by gen decarboxylase, which is also capable of converting

uroporphyrino-uroporphyrinogen I to coproporphyrinogen I (Figure32–7) Coproporphyrinogen III then enters the mito-

chondria, where it is converted to protoporphyrinogen III and then to protoporphyrin III Several steps are

involved in this conversion The mitochondrial enzyme

coproporphyrinogen oxidase catalyzes the

decarboxy-lation and oxidation of two propionic side chains toform protoporphyrinogen This enzyme is able to actonly on type III coproporphyrinogen, which would ex-plain why type I protoporphyrins do not generally occur

in nature The oxidation of protoporphyrinogen to toporphyrin is catalyzed by another mitochondrial en-

pro-zyme, protoporphyrinogen oxidase In mammalian

liver, the conversion of coproporphyrinogen to porphyrin requires molecular oxygen

proto-Formation of Heme Involves Incorporation

of Iron Into Protoporphyrin

The final step in heme synthesis involves the tion of ferrous iron into protoporphyrin in a reaction

incorpora-catalyzed by ferrochelatase (heme synthase), another

mitochondrial enzyme (Figure 32–4)

A summary of the steps in the biosynthesis of theporphyrin derivatives from PBG is given in Figure32–8 The last three enzymes in the pathway and ALAsynthase are located in the mitochondrion, whereas theother enzymes are cytosolic Both erythroid and non-erythroid (“housekeeping”) forms of the first four en-zymes are found Heme biosynthesis occurs in mostmammalian cells with the exception of mature erythro-cytes, which do not contain mitochondria However,

Table 32–1 Examples of some important human

and animal hemoproteins.1

Hemoglobin Transport of oxygen in blood

Myoglobin Storage of oxygen in muscle

Cytochrome c Involvement in electron transport chain

Cytochrome P450 Hydroxylation of xenobiotics

Catalase Degradation of hydrogen peroxide

Tryptophan Oxidation of trypotophan

pyrrolase

1 The functions of the above proteins are described in various

chapters of this text.

272 / CHAPTER 32

A

P

P A

A P

Coproporphyrin I Coproporphyrin III

Uroporphyrins were first found in the urine, but they are not restricted to urine.

Coproporphyrins were first isolated from feces, but they are also found in urine.

M P

M P

A P

A

P

Figure 32–3. Uroporphyrins and coproporphyrins A (acetate); P (propionate); M (methyl) = CH 3 ; V (vinyl) = CHCH 2

approximately 85% of heme synthesis occurs in

eryth-roid precursor cells in the bone marrow and the

major-ity of the remainder in hepatocytes.

The porphyrinogens described above are colorless,

containing six extra hydrogen atoms as compared with

the corresponding colored porphyrins These reduced

porphyrins (the porphyrinogens) and not the

corre-sponding porphyrins are the actual intermediates in the

biosynthesis of protoporphyrin and of heme

ALA Synthase Is the Key Regulatory

Enzyme in Hepatic Biosynthesis of Heme

ALA synthase occurs in both hepatic (ALAS1) and

ery-throid (ALAS2) forms The rate-limiting reaction in the

synthesis of heme in liver is that catalyzed by ALAS1

(Figure 32–5), a regulatory enzyme It appears that

heme, probably acting through an aporepressor

mole-cule, acts as a negative regulator of the synthesis of

ALAS1 This repression-derepression mechanism is picted diagrammatically in Figure 32–9 Thus, the rate

de-of synthesis de-of ALAS1 increases greatly in the absence

of heme and is diminished in its presence The turnoverrate of ALAS1 in rat liver is normally rapid (half-lifeabout 1 hour), a common feature of an enzyme catalyz-ing a rate-limiting reaction Heme also affects transla-tion of the enzyme and its transfer from the cytosol tothe mitochondrion

Many drugs when administered to humans can sult in a marked increase in ALAS1 Most of thesedrugs are metabolized by a system in the liver that uti-

re-lizes a specific hemoprotein, cytochrome P450 (see

Chapter 53) During their metabolism, the utilization

of heme by cytochrome P450 is greatly increased,which in turn diminishes the intracellular heme con-centration This latter event effects a derepression ofALAS1 with a corresponding increased rate of hemesynthesis to meet the needs of the cells

V P

V M

M P

V P

V M

M P

Fe2+

Protoporphyrin III (IX)

(parent porphyrin of heme)

PORPHYRINS & BILE PIGMENTS / 273

C COOH

NH2H

NH2H

NH2H

Two molecules of δ-aminolevulinate (first precursor pyrrole)Porphobilinogen

CH2

CH2

H NH

C O C

COOH

C C C CH

CH2 N H

CH2

CH2

COOH

CH2COOH

NH2

ALA DEHYDRATASE 2H2O

ALA SYNTHASE

CO2

δ-Aminolevulinate (ALA) α-Amino-β-ketoadipate

Pyridoxal phosphate

Figure 32–5. Biosynthesis of porphobilinogen ALA synthase occurs in the dria, whereas ALA dehydratase is present in the cytosol.

mitochon-Several factors affect drug-mediated derepression ofALAS1 in liver—eg, the administration of glucose can

prevent it, as can the administration of hematin (an

ox-idized form of heme)

The importance of some of these regulatory nisms is further discussed below when the porphyrias

mecha-are described

Regulation of the erythroid form of ALAS (ALAS2)

differs from that of ALAS1 For instance, it is not

in-duced by the drugs that affect ALAS1, and it does not

undergo feedback regulation by heme

PORPHYRINS ARE COLORED

& FLUORESCE

The various porphyrinogens are colorless, whereas the

various porphyrins are all colored In the study of

por-phyrins or porphyrin derivatives, the characteristic

ab-sorption spectrum that each exhibits—in both the visible

and the ultraviolet regions of the spectrum—is of great

value An example is the absorption curve for a solution

of porphyrin in 5% hydrochloric acid (Figure 32–10)

Note particularly the sharp absorption band near 400

nm This is a distinguishing feature of the porphin ring

and is characteristic of all porphyrins regardless of the

side chains present This band is termed the Soret band

after its discoverer, the French physicist Charles Soret When porphyrins dissolved in strong mineral acids

or in organic solvents are illuminated by ultraviolet

light, they emit a strong red fluorescence This

fluores-cence is so characteristic that it is often used to detectsmall amounts of free porphyrins The double bondsjoining the pyrrole rings in the porphyrins are responsi-ble for the characteristic absorption and fluorescence ofthese compounds; these double bonds are absent in theporphyrinogens

An interesting application of the photodynamicproperties of porphyrins is their possible use in thetreatment of certain types of cancer, a procedure called

cancer phototherapy Tumors often take up more

por-phyrins than do normal tissues Thus, phyrin or other related compounds are administered to

hematopor-a phematopor-atient with hematopor-an hematopor-approprihematopor-ate tumor The tumor is thenexposed to an argon laser, which excites the porphyrins,producing cytotoxic effects

Spectrophotometry Is Used to Test for Porphyrins & Their Precursors

Coproporphyrins and uroporphyrins are of clinical terest because they are excreted in increased amounts in

in-274 / CHAPTER 32

HOOC COOH

CH2

CH2H2C

CH C

H N

H2C

NH2Four molecules of porphobilinogen

NH34

Hydroxymethylbilane (linear tetrapyrrole)

C C

C C

H N

P C C C C H

C C

H N

H2 C

C C

C C

H N

C C C C H

H2 C

P C C C C H

A P

A

Type III uroporphyrinogen Type I

uroporphyrinogen

P A

UROPORPHYRINOGEN I SYNTHASE

UROPORPHYRINOGEN III SYNTHASE SPONTANEOUS

CYCLIZATION

Figure 32–6. Conversion of porphobilinogen to

uro-porphyrinogens Uroporphyrinogen synthase I is also

called porphobilinogen (PBG) deaminase or

hydroxy-methylbilane (HMB) synthase.

the porphyrias These compounds, when present in

urine or feces, can be separated from each other by

ex-traction with appropriate solvent mixtures They can

then be identified and quantified using

spectrophoto-metric methods

ALA and PBG can also be measured in urine by

ap-propriate colorimetric tests

THE PORPHYRIAS ARE GENETIC

DISORDERS OF HEME METABOLISM

The porphyrias are a group of disorders due to

abnor-malities in the pathway of biosynthesis of heme; they

can be genetic or acquired They are not prevalent, but

it is important to consider them in certain

circum-stances (eg, in the differential diagnosis of abdominal

pain and of a variety of neuropsychiatric findings); erwise, patients will be subjected to inappropriate treat-ments It has been speculated that King George III had

oth-a type of porphyrioth-a, which moth-ay oth-account for his periodicconfinements in Windsor Castle and perhaps for some

of his views regarding American colonists Also, the

photosensitivity (favoring nocturnal activities) and vere disfigurement exhibited by some victims of con-

se-genital erythropoietic porphyria have led to the tion that these individuals may have been theprototypes of so-called werewolves No evidence to sup-port this notion has been adduced

sugges-Biochemistry Underlies the Causes, Diagnoses, & Treatments

of the Porphyrias

Six major types of porphyria have been described, sulting from depressions in the activities of enzymes 3through 8 shown in Figure 32–9 (see also Table 32–2).Assay of the activity of one or more of these enzymesusing an appropriate source (eg, red blood cells) is thusimportant in making a definitive diagnosis in a sus-pected case of porphyria Individuals with low activities

re-of enzyme 1 (ALAS2) develop anemia, not porphyria(see Table 32–2) Patients with low activities of enzyme

2 (ALA dehydratase) have been reported, but veryrarely; the resulting condition is called ALA dehy-dratase-deficient porphyria

In general, the porphyrias described are inherited in

an autosomal dominant manner, with the exception ofcongenital erythropoietic porphyria, which is inherited

in a recessive mode The precise abnormalities in thegenes directing synthesis of the enzymes involved inheme biosynthesis have been determined in some in-stances Thus, the use of appropriate gene probes hasmade possible the prenatal diagnosis of some of theporphyrias

As is true of most inborn errors, the signs and toms of porphyria result from either a deficiency ofmetabolic products beyond the enzymatic block orfrom an accumulation of metabolites behind the block

symp-If the enzyme lesion occurs early in the pathwayprior to the formation of porphyrinogens (eg, enzyme 3

of Figure 32–9, which is affected in acute intermittentporphyria), ALA and PBG will accumulate in body tis-sues and fluids (Figure 32–11) Clinically, patientscomplain of abdominal pain and neuropsychiatricsymptoms The precise biochemical cause of thesesymptoms has not been determined but may relate toelevated levels of ALA or PBG or to a deficiency ofheme

On the other hand, enzyme blocks later in the way result in the accumulation of the porphyrinogens

path-PORPHYRINS & BILE PIGMENTS / 275

Uroporphyrinogen I

Uroporphyrinogen III

P P

P P

P P A A

Coproporphyrinogen I

Coproporphyrinogen III

P P

P P

P P M M

UROPORPHYRINOGEN DECARBOXYLASE

4CO2

4CO2

Figure 32–7. Decarboxylation of

uropor-phyrinogens to coproporuropor-phyrinogens in

cy-tosol (A, acetyl; M, methyl; P, propionyl.)

Porphobilinogen

Hydroxymethylbilane

SPONTANEOUS

Uroporphyrinogen I

4CO24CO2

Coproporphyrinogen III

Light

6H

Coproporphyrin I

Uroporphyrinogen III

Protoporphyrin III

Coproporphyrinogen I Coproporphyrin

III

Light

6H

Uroporphyrin III

PROTOPORPHYRINOGEN OXIDASE

FERROCHELATASE

UROPORPHYRINOGEN I SYNTHASE

UROPORPHYRINOGEN III SYNTHASE

UROPORPHYRINOGEN DECARBOXYLASE

COPROPORPHYRINOGEN OXIDASE

Figure 32–8. Steps in the biosynthesis of the porphyrin derivatives from porphobilinogen phyrinogen I synthase is also called porphobilinogen deaminase or hydroxymethylbilane synthase.

Uropor-276 / CHAPTER 32

Protoporphyrinogen III

Coproporphyrinogen III

Protoporphyrin III Heme

Aporepressor

6 COPROPORPHYRINOGEN OXIDASE

7 PROTOPORPHYRINOGEN OXIDASE

8 FERROCHELATASE

5 UROPORPHYRINOGEN DECARBOXYLASE

4 UROPORPHYRINOGEN III SYNTHASE

3 UROPORPHYRINOGEN I SYNTHASE

2 ALA DEHYDRATASE

1 ALA SYNTHASE

Figure 32–9. Intermediates, enzymes, and regulation of heme thesis The enzyme numbers are those referred to in column 1 of Table 32–2 Enzymes 1, 6, 7, and 8 are located in mitochondria, the others in the cytosol Mutations in the gene encoding enzyme 1 causes X-linked sideroblastic anemia Mutations in the genes encoding enzymes 2–8 cause the porphyrias, though only a few cases due to deficiency of en- zyme 2 have been reported Regulation of hepatic heme synthesis oc- curs at ALA synthase (ALAS1) by a repression-derepression mecha- nism mediated by heme and its hypothetical aporepressor The dotted lines indicate the negative (− ) regulation by repression En- zyme 3 is also called porphobilinogen deaminase or hydroxymethyl- bilane synthase.

syn-PORPHYRINS & BILE PIGMENTS / 277

300 1 2 3

4 5

400 500 Wavelength (nm)

Accumulation of ALA and PBG and/or decrease in heme in cells and body fluids

Accumulation of porphyrinogens in skin and tissues

Neuropsychiatric signs and symptoms

Spontaneous oxidation

of porphyrinogens to porphyrins

Figure 32–11. Biochemical causes of the major signs and symptoms of the porphyrias.

Table 32–2 Summary of major findings in the porphyrias.1

Enzyme Involved 2 Type, Class, and MIM Number Major Signs and Symptoms Results of Laboratory Tests

1 ALA synthase X-linked sideroblastic anemia 3 Anemia Red cell counts and hemoglobin

201300)

2 ALA dehydratase ALA dehydratase deficiency Abdominal pain, neuropsychiatric Urinary δ-aminolevulinic acid

(hepatic) (MIM 125270) symptoms

3 Uroporphyrinogen I Acute intermittent porphyria Abdominal pain, neuropsychiatric Urinary porphobilinogen positive, synthase 4 (hepatic) (MIM 176000) symptoms uroporphyrin positive

4 Uroporphyrinogen III Congenital erythropoietic No photosensitivity Uroporphyrin positive,

263700)

5 Uroporphyrinogen Porphyria cutanea tarda (he- Photosensitivity Uroporphyrin positive,

6 Coproporphyrinogen Hereditary coproporphyria Photosensitivity, abdominal pain, Urinary porphobilinogen oxidase (hepatic) (MIM 121300) neuropsychiatric symptoms tive, urinary uroporphyrin

posi-positive, fecal phyrin positive

protopor-7 Protoporphyrinogen Variegate porphyria (hepatic) Photosensitivity, abdominal pain, Urinary porphobilinogen oxidase (MIM 176200) neuropsychiatric symptoms tive, fecal protoporphyrin

posi-positive

8 Ferrochelatase Protoporphyria (erythropoietic) Photosensitivity Fecal protoporphyrin

positive

1 Only the biochemical findings in the active stages of these diseases are listed Certain biochemical abnormalities are detectable in the tent stages of some of the above conditions Conditions 3, 5, and 8 are generally the most prevalent porphyrias.

la-2 The numbering of the enzymes in this table corresponds to that used in Figure 32-9.

3 X-linked sideroblastic anemia is not a porphyria but is included here because δ−aminolevulinic acid synthase is involved.

4 This enzyme is also called porphobilinogen deaminase or hydroxymethylbilane synthase.

278 / CHAPTER 32

indicated in Figures 32–9 and 32–11 Their oxidation

products, the corresponding porphyrin derivatives,

cause photosensitivity, a reaction to visible light of

about 400 nm The porphyrins, when exposed to light

of this wavelength, are thought to become “excited”

and then react with molecular oxygen to form oxygen

radicals These latter species injure lysosomes and other

organelles Damaged lysosomes release their degradative

enzymes, causing variable degrees of skin damage,

in-cluding scarring

The porphyrias can be classified on the basis of the

organs or cells that are most affected These are

gener-ally organs or cells in which synthesis of heme is

partic-ularly active The bone marrow synthesizes considerable

hemoglobin, and the liver is active in the synthesis of

another hemoprotein, cytochrome P450 Thus, one

classification of the porphyrias is to designate them as

predominantly either erythropoietic or hepatic; the

types of porphyrias that fall into these two classes are so

characterized in Table 32–2 Porphyrias can also be

classified as acute or cutaneous on the basis of their

clinical features Why do specific types of porphyria

af-fect certain organs more markedly than others? A

par-tial answer is that the levels of metabolites that cause

damage (eg, ALA, PBG, specific porphyrins, or lack of

heme) can vary markedly in different organs or cells

de-pending upon the differing activities of their

heme-forming enzymes

As described above, ALAS1 is the key regulatory

en-zyme of the heme biosynthetic pathway in liver A large

number of drugs (eg, barbiturates, griseofulvin) induce

the enzyme Most of these drugs do so by inducing

cy-tochrome P450 (see Chapter 53), which uses up heme

and thus derepresses (induces) ALAS1 In patients with

porphyria, increased activities of ALAS1 result in

in-creased levels of potentially harmful heme precursors

prior to the metabolic block Thus, taking drugs that

cause induction of cytochrome P450 (so-called

micro-somal inducers) can precipitate attacks of porphyria

The diagnosis of a specific type of porphyria can

generally be established by consideration of the clinical

and family history, the physical examination, and

ap-propriate laboratory tests The major findings in the six

principal types of porphyria are listed in Table 32–2

High levels of lead can affect heme metabolism by

combining with SH groups in enzymes such as

fer-rochelatase and ALA dehydratase This affects

por-phyrin metabolism Elevated levels of protoporpor-phyrin

are found in red blood cells, and elevated levels of ALA

and of coproporphyrin are found in urine

It is hoped that treatment of the porphyrias at the

gene level will become possible In the meantime,

treat-ment is essentially symptomatic It is important for

pa-tients to avoid drugs that cause induction of

cyto-chrome P450 Ingestion of large amounts of drates (glucose loading) or administration of hematin (ahydroxide of heme) may repress ALAS1, resulting in di-minished production of harmful heme precursors Pa-tients exhibiting photosensitivity may benefit from ad-ministration of β-carotene; this compound appears tolessen production of free radicals, thus diminishingphotosensitivity Sunscreens that filter out visible lightcan also be helpful to such patients

carbohy-CATABOLISM OF HEME PRODUCES BILIRUBIN

Under physiologic conditions in the human adult, 1–2

× 108erythrocytes are destroyed per hour Thus, in 1day, a 70-kg human turns over approximately 6 g of he-moglobin When hemoglobin is destroyed in the body,

globin is degraded to its constituent amino acids, which are reused, and the iron of heme enters the iron pool, also for reuse The iron-free porphyrin portion of

heme is also degraded, mainly in the reticuloendothelialcells of the liver, spleen, and bone marrow

The catabolism of heme from all of the heme teins appears to be carried out in the microsomal frac-

pro-tions of cells by a complex enzyme system called heme oxygenase By the time the heme derived from heme

proteins reaches the oxygenase system, the iron has ally been oxidized to the ferric form, constituting

usu-hemin The heme oxygenase system is

substrate-in-ducible As depicted in Figure 32–12, the hemin is duced to heme with NADPH, and, with the aid ofmore NADPH, oxygen is added to the α-methenylbridge between pyrroles I and II of the porphyrin Theferrous iron is again oxidized to the ferric form With

re-the furre-ther addition of oxygen, ferric ion is released, carbon monoxide is produced, and an equimolar quantity of biliverdin results from the splitting of the

tetrapyrrole ring

In birds and amphibia, the green biliverdin IX is

ex-creted; in mammals, a soluble enzyme called biliverdin reductase reduces the methenyl bridge between pyrrole

III and pyrrole IV to a methylene group to produce

bilirubin, a yellow pigment (Figure 32–12).

It is estimated that 1 g of hemoglobin yields 35 mg

of bilirubin The daily bilirubin formation in humanadults is approximately 250–350 mg, deriving mainlyfrom hemoglobin but also from ineffective erythro-poiesis and from various other heme proteins such ascytochrome P450

The chemical conversion of heme to bilirubin byreticuloendothelial cells can be observed in vivo as thepurple color of the heme in a hematoma is slowly con-verted to the yellow pigment of bilirubin

PORPHYRINS & BILE PIGMENTS / 279

HN

HN H H HN

HN

P P O

CO (exhaled)

α

Figure 32–12. Schematic representation of the microsomal heme oxygenase system (Modified from

Schmid R, McDonough AF in: The Porphyrins Dolphin D [editor] Academic Press, 1978.)

280 / CHAPTER 32

Bilirubin formed in peripheral tissues is transported

to the liver by plasma albumin The further metabolism

of bilirubin occurs primarily in the liver It can be

di-vided into three processes: (1) uptake of bilirubin by

liver parenchymal cells, (2) conjugation of bilirubin

with glucuronate in the endoplasmic reticulum, and (3)

secretion of conjugated bilirubin into the bile Each of

these processes will be considered separately

THE LIVER TAKES UP BILIRUBIN

Bilirubin is only sparingly soluble in water, but its

solu-bility in plasma is increased by noncovalent binding to

albumin Each molecule of albumin appears to have

one high-affinity site and one low-affinity site for

bilirubin In 100 mL of plasma, approximately 25 mg

of bilirubin can be tightly bound to albumin at its

high-affinity site Bilirubin in excess of this quantity can be

bound only loosely and thus can easily be detached and

diffuse into tissues A number of compounds such as

antibiotics and other drugs compete with bilirubin for

the high-affinity binding site on albumin Thus, these

compounds can displace bilirubin from albumin and

have significant clinical effects

In the liver, the bilirubin is removed from albumin

and taken up at the sinusoidal surface of the

hepato-cytes by a carrier-mediated saturable system This

facil-itated transport system has a very large capacity, so

that even under pathologic conditions the system does

not appear to be rate-limiting in the metabolism of

bilirubin

Since this facilitated transport system allows the

equilibrium of bilirubin across the sinusoidal

mem-brane of the hepatocyte, the net uptake of bilirubin will

be dependent upon the removal of bilirubin via

subse-quent metabolic pathways

Once bilirubin enters the hepatocytes, it can bind to

certain cytosolic proteins, which help to keep it

solubi-lized prior to conjugation Ligandin (a family of

glu-tathione S-transferases) and protein Y are the involved

proteins They may also help to prevent efflux of

biliru-bin back into the blood stream

Conjugation of Bilirubin With Glucuronic

Acid Occurs in the Liver

Bilirubin is nonpolar and would persist in cells (eg,

bound to lipids) if not rendered water-soluble

Hepato-cytes convert bilirubin to a polar form, which is readily

excreted in the bile, by adding glucuronic acid

mole-cules to it This process is called conjugation and can

employ polar molecules other than glucuronic acid (eg,

sulfate) Many steroid hormones and drugs are also

converted to water-soluble derivatives by conjugation inpreparation for excretion (see Chapter 53)

The conjugation of bilirubin is catalyzed by a cific glucuronosyltransferase The enzyme is mainlylocated in the endoplasmic reticulum, uses UDP-glucuronic acid as the glucuronosyl donor, and is re-ferred to as bilirubin-UGT Bilirubin monoglucuronide

spe-is an intermediate and spe-is subsequently converted to thediglucuronide (Figures 32–13 and 32–14) Most of thebilirubin excreted in the bile of mammals is in the form

of bilirubin diglucuronide However, when bilirubinconjugates exist abnormally in human plasma (eg, inobstructive jaundice), they are predominantly mono-

glucuronides Bilirubin-UGT activity can be induced

by a number of clinically useful drugs, including nobarbital More information about glucuronosylation

phe-is presented below in the dphe-iscussion of inherited dphe-isor-ders of bilirubin conjugation

disor-Bilirubin Is Secreted Into Bile

Secretion of conjugated bilirubin into the bile occurs by

an active transport mechanism, which is probably limiting for the entire process of hepatic bilirubin me-tabolism The protein involved is MRP-2 (multidrugresistance-like protein 2), also called multispecific or-ganic anion transporter (MOAT) It is located in theplasma membrane of the bile canalicular membraneand handles a number of organic anions It is a member

rate-of the family rate-of ATP-binding cassette (ABC) porters The hepatic transport of conjugated bilirubininto the bile is inducible by those same drugs that arecapable of inducing the conjugation of bilirubin Thus,the conjugation and excretion systems for bilirubin be-have as a coordinated functional unit

trans-Figure 32–15 summarizes the three major processesinvolved in the transfer of bilirubin from blood to bile.Sites that are affected in a number of conditions caus-ing jaundice (see below) are also indicated

V M M M

V

H2C C

O – OOC(CH2O)4C

Figure 32–13. Structure of bilirubin diglucuronide (conjugated, “direct-reacting” bilirubin) Glucuronic acid is attached via ester linkage to the two propionic acid groups of bilirubin to form an acylglucuronide.

PORPHYRINS & BILE PIGMENTS / 281

UDP-GLUCOSE DEHYDROGENASE

TRANSFERASE

TRANSFERASE

UDP-GLUCURONOSYL-2NAD+ 2NADH + 2H+

UDP-Glucuronic acid UDP-Glucose

Bilirubin monoglucuronide

+ UDP

UDP-Glucuronic acid + Bilirubin

Bilirubin diglucuronide + UDP

UDP-Glucuronic acid + Bilirubin monoglucuronide

Figure 32–14. Conjugation of bilirubin

with glucuronic acid The glucuronate donor,

glucuronic acid, is formed from

UDP-glucose as depicted The

UDP-glucuronosyl-transferase is also called bilirubin-UGT.

Conjugated Bilirubin Is Reduced to Urobilinogen by Intestinal Bacteria

As the conjugated bilirubin reaches the terminal ileumand the large intestine, the glucuronides are removed by

specific bacterial enzymes (-glucuronidases), and the

pigment is subsequently reduced by the fecal flora to a

group of colorless tetrapyrrolic compounds called bilinogens (Figure 32–16) In the terminal ileum and

uro-large intestine, a small fraction of the urobilinogens is absorbed and reexcreted through the liver to constitute

re-the enterohepatic urobilinogen cycle Under abnormal

conditions, particularly when excessive bile pigment isformed or liver disease interferes with this intrahepaticcycle, urobilinogen may also be excreted in the urine.Normally, most of the colorless urobilinogensformed in the colon by the fecal flora are oxidized there

to urobilins (colored compounds) and are excreted inthe feces (Figure 32–16) Darkening of feces uponstanding in air is due to the oxidation of residual uro-bilinogens to urobilins

HYPERBILIRUBINEMIA CAUSES JAUNDICE

When bilirubin in the blood exceeds 1 mg/dL (17.1µmol/L), hyperbilirubinemia exists Hyperbilirubine-mia may be due to the production of more bilirubinthan the normal liver can excrete, or it may result fromthe failure of a damaged liver to excrete bilirubin pro-duced in normal amounts In the absence of hepaticdamage, obstruction of the excretory ducts of theliver—by preventing the excretion of bilirubin—willalso cause hyperbilirubinemia In all these situations,bilirubin accumulates in the blood, and when it reaches

a certain concentration (approximately 2–2.5 mg/dL),

2 CONJUGATION Neonatal jaundice

“Toxic” jaundice Crigler-Najjar syndrome Gilbert syndrome

3 SECRETION Dubin-Johnson syndrome

1 UPTAKE

Bilirubin diglucuronide

BILE DUCTULE

Figure 32–15. Diagrammatic representation of the

three major processes (uptake, conjugation, and

secre-tion) involved in the transfer of bilirubin from blood to

bile Certain proteins of hepatocytes, such as ligandin (a

family of glutathione S-transferase) and Y protein, bind

intracellular bilirubin and may prevent its efflux into the

blood stream The process affected in a number of

con-ditions causing jaundice is also shown.

OH N

E M

M P

H H

H H2C

NH

H N

OH N

E M

M P

H H H H H2C

N

H N

OH N

E M

M P

Figure 32–16. Structure of some bile pigments.

it diffuses into the tissues, which then become yellow

That condition is called jaundice or icterus.

In clinical studies of jaundice, measurement of

bilirubin in the serum is of great value A method for

quantitatively assaying the bilirubin content of the

serum was first devised by van den Bergh by application

of Ehrlich’s test for bilirubin in urine The Ehrlich

reac-tion is based on the coupling of diazotized sulfanilic

acid (Ehrlich’s diazo reagent) and bilirubin to produce

a reddish-purple azo compound In the original

proce-dure as described by Ehrlich, methanol was used to

provide a solution in which both bilirubin and the

diazo regent were soluble Van den Bergh inadvertently

omitted the methanol on an occasion when assay of bile

pigment in human bile was being attempted To his

surprise, normal development of the color occurred

“di-rectly.” This form of bilirubin that would react without

the addition of methanol was thus termed

“direct-reacting.” It was then found that this same direct

reac-tion would also occur in serum from cases of jaundice

due to biliary obstruction However, it was still

neces-sary to add methanol to detect bilirubin in normal

serum or that which was present in excess in serum

from cases of hemolytic jaundice where no evidence of

obstruction was to be found To that form of bilirubin

which could be measured only after the addition of

methanol, the term “indirect-reacting” was applied.

It was subsequently discovered that the indirect

bilirubin is “free” (unconjugated) bilirubin en route to

the liver from the reticuloendothelial tissues, where the

bilirubin was originally produced by the breakdown of

heme porphyrins Since this bilirubin is not

water-solu-ble, it requires methanol to initiate coupling with the

diazo reagent In the liver, the free bilirubin becomes

conjugated with glucuronic acid, and the conjugate,

bilirubin glucuronide, can then be excreted into the

bile Furthermore, conjugated bilirubin, being

water-soluble, can react directly with the diazo reagent, sothat the “direct bilirubin” of van den Bergh is actually abilirubin conjugate (bilirubin glucuronide)

Depending on the type of bilirubin present inplasma—ie, unconjugated or conjugated—hyperbiliru-

binemia may be classified as retention binemia, due to overproduction, or regurgitation hy- perbilirubinemia, due to reflux into the bloodstream

hyperbiliru-because of biliary obstruction

Because of its hydrophobicity, only unconjugatedbilirubin can cross the blood-brain barrier into the cen-tral nervous system; thus, encephalopathy due to hyper-

bilirubinemia (kernicterus) can occur only in

connec-tion with unconjugated bilirubin, as found in retenconnec-tionhyperbilirubinemia On the other hand, because of itswater-solubility, only conjugated bilirubin can appear

in urine Accordingly, choluric jaundice (choluria is

the presence of bile pigments in the urine) occurs only

in regurgitation hyperbilirubinemia, and acholuric jaundice occurs only in the presence of an excess of un-

unconju-B N EONATAL “P HYSIOLOGIC J AUNDICE ”

This transient condition is the most common cause ofunconjugated hyperbilirubinemia It results from an ac-

PORPHYRINS & BILE PIGMENTS / 283

celerated hemolysis around the time of birth and an

im-mature hepatic system for the uptake, conjugation, and

secretion of bilirubin Not only is the bilirubin-UGT

activity reduced, but there probably is reduced synthesis

of the substrate for that enzyme, UDP-glucuronic acid

Since the increased amount of bilirubin is

unconju-gated, it is capable of penetrating the blood-brain

bar-rier when its concentration in plasma exceeds that

which can be tightly bound by albumin (20–25

mg/dL) This can result in a hyperbilirubinemic toxic

encephalopathy, or kernicterus, which can cause

men-tal retardation Because of the recognized inducibility

of this bilirubin-metabolizing system, phenobarbital

has been administered to jaundiced neonates and is

ef-fective in this disorder In addition, exposure to blue

light (phototherapy) promotes the hepatic excretion of

unconjugated bilirubin by converting some of the

bilirubin to other derivatives such as maleimide

frag-ments and geometric isomers that are excreted in the

bile

C C RIGLER -N AJJAR S YNDROME , T YPE I;

C ONGENITAL N ONHEMOLYTIC J AUNDICE

Type I Crigler-Najjar syndrome is a rare autosomal

re-cessive disorder It is characterized by severe congenital

jaundice (serum bilirubin usually exceeds 20 mg/dL)

due to mutations in the gene encoding bilirubin-UGT

activity in hepatic tissues The disease is often fatal

within the first 15 months of life Children with this

condition have been treated with phototherapy,

result-ing in some reduction in plasma bilirubin levels

Phe-nobarbital has no effect on the formation of bilirubin

glucuronides in patients with type I Crigler-Najjar

syn-drome A liver transplant may be curative

D C RIGLER -N AJJAR S YNDROME , T YPE II

This rare inherited disorder also results from mutations

in the gene encoding bilirubin-UGT, but some activity

of the enzyme is retained and the condition has a more

benign course than type I Serum bilirubin

concentra-tions usually do not exceed 20 mg/dL Patients with

this condition can respond to treatment with large

doses of phenobarbital

E G ILBERT S YNDROME

Again, this is caused by mutations in the gene encoding

bilirubin-UGT, but approximately 30% of the

en-zyme’s activity is preserved and the condition is entirely

harmless

F T OXIC H YPERBILIRUBINEMIA

Unconjugated hyperbilirubinemia can result from

toxin-induced liver dysfunction such as that caused by

chloroform, arsphenamines, carbon tetrachloride,

ace-taminophen, hepatitis virus, cirrhosis, and Amanita

mushroom poisoning These acquired disorders are due

to hepatic parenchymal cell damage, which impairsconjugation

Obstruction in the Biliary Tree Is the Commonest Cause of Conjugated Hyperbilirubinemia

A O BSTRUCTION OF THE B ILIARY T REE

Conjugated hyperbilirubinemia commonly results fromblockage of the hepatic or common bile ducts, mostoften due to a gallstone or to cancer of the head of thepancreas Because of the obstruction, bilirubin diglu-curonide cannot be excreted It thus regurgitates intothe hepatic veins and lymphatics, and conjugatedbilirubin appears in the blood and urine (choluric jaun-dice)

The term cholestatic jaundice is used to include all

cases of extrahepatic obstructive jaundice It also coversthose cases of jaundice that exhibit conjugated hyper-bilirubinemia due to micro-obstruction of intrahepaticbiliary ductules by swollen, damaged hepatocytes (eg, asmay occur in infectious hepatitis)

B D UBIN -J OHNSON S YNDROME

This benign autosomal recessive disorder consists ofconjugated hyperbilirubinemia in childhood or duringadult life The hyperbilirubinemia is caused by muta-tions in the gene encoding MRP-2 (see above), the pro-tein involved in the secretion of conjugated bilirubininto bile The centrilobular hepatocytes contain an ab-normal black pigment that may be derived from epi-nephrine

C R OTOR S YNDROME

This is a rare benign condition characterized by chronicconjugated hyperbilirubinemia and normal liver histol-ogy Its precise cause has not been identified, but it isthought to be due to an abnormality in hepatic storage

Some Conjugated Bilirubin Can Bind Covalently to Albumin

When levels of conjugated bilirubin remain high inplasma, a fraction can bind covalently to albumin (deltabilirubin) Because it is bound covalently to albumin,this fraction has a longer half-life in plasma than doesconventional conjugated bilirubin Thus, it remains ele-vated during the recovery phase of obstructive jaundiceafter the remainder of the conjugated bilirubin has de-clined to normal levels; this explains why some patientscontinue to appear jaundiced after conjugated bilirubinlevels have returned to normal

284 / CHAPTER 32

Urobilinogen & Bilirubin in Urine

Are Clinical Indicators

Normally, there are mere traces of urobilinogen in the

urine In complete obstruction of the bile duct, no

urobilinogen is found in the urine, since bilirubin has

no access to the intestine, where it can be converted to

urobilinogen In this case, the presence of bilirubin

(conjugated) in the urine without urobilinogen suggests

obstructive jaundice, either intrahepatic or posthepatic

In jaundice secondary to hemolysis, the increased

production of bilirubin leads to increased production of

urobilinogen, which appears in the urine in large

amounts Bilirubin is not usually found in the urine in

hemolytic jaundice (because unconjugated bilirubin

does not pass into the urine), so that the combination

of increased urobilinogen and absence of bilirubin is

suggestive of hemolytic jaundice Increased blood

de-struction from any cause brings about an increase in

urine urobilinogen

Table 32–3 summarizes laboratory results obtained

on patients with three different causes of

jaundice—he-molytic anemia (a prehepatic cause), hepatitis (a hepatic

cause), and obstruction of the common bile duct (a

posthepatic cause) Laboratory tests on blood

(evalua-tion of the possibility of a hemolytic anemia and

mea-surement of prothrombin time) and on serum (eg,

elec-trophoresis of proteins; activities of the enzymes ALT,

AST, and alkaline phosphatase) are also important in

helping to distinguish between prehepatic, hepatic, and

posthepatic causes of jaundice

SUMMARY

• Hemoproteins, such as hemoglobin and the

cy-tochromes, contain heme Heme is an

iron-por-phyrin compound (Fe2 +-protoporphyrin IX) in

which four pyrrole rings are joined by methenylbridges The eight side groups (methyl, vinyl, andpropionyl substituents) on the four pyrrole rings ofheme are arranged in a specific sequence

• Biosynthesis of the heme ring occurs in mitochondriaand cytosol via eight enzymatic steps It commenceswith formation of δ-aminolevulinate (ALA) fromsuccinyl-CoA and glycine in a reaction catalyzed byALA synthase, the regulatory enzyme of the pathway

• Genetically determined abnormalities of seven of theeight enzymes involved in heme biosynthesis result inthe inherited porphyrias Red blood cells and liverare the major sites of metabolic expression of the por-phyrias Photosensitivity and neurologic problemsare common complaints Intake of certain com-pounds (such as lead) can cause acquired porphyrias.Increased amounts of porphyrins or their precursorscan be detected in blood and urine, facilitating diag-nosis

• Catabolism of the heme ring is initiated by the zyme heme oxygenase, producing a linear tetrapyr-role

en-• Biliverdin is an early product of catabolism and onreduction yields bilirubin The latter is transported

by albumin from peripheral tissues to the liver, where

it is taken up by hepatocytes The iron of heme andthe amino acids of globin are conserved and reuti-lized

• In the liver, bilirubin is made water-soluble by gation with two molecules of glucuronic acid and issecreted into the bile The action of bacterial en-zymes in the gut produces urobilinogen and urobilin,which are excreted in the feces and urine

conju-• Jaundice is due to elevation of the level of bilirubin

in the blood The causes of jaundice can be classified

Table 32–3 Laboratory results in normal patients and patients with three different causes of jaundice.

Condition Serum Bilirubin Urine Urobilinogen Urine Bilirubin Fecal Urobilinogen

Normal Direct: 0.1–0.4 mg/dL 0–4 mg/24 h Absent 40–280 mg/24 h

Indirect: 0.2–0.7 mg/dL Hemolytic anemia ↑ Indirect Increased Absent Increased

Hepatitis ↑ Direct and indirect Decreased if micro- Present if micro- Decreased

obstruction is obstruction occurs present

Obstructive jaundice 1 ↑ Direct Absent Present Trace to absent

1 The commonest causes of obstructive (posthepatic) jaundice are cancer of the head of the pancreas and a gallstone lodged in the mon bile duct The presence of bilirubin in the urine is sometimes referred to as choluria—therefore, hepatitis and obstruction of the common bile duct cause choluric jaundice, whereas the jaundice of hemolytic anemia is referred to as acholuric The laboratory results in patients with hepatitis are variable, depending on the extent of damage to parenchymal cells and the extent of micro-obstruction to bile ductules Serum levels of ALT and AST are usually markedly elevated in hepatitis, whereas serum levels of alkaline phosphatase are ele- vated in obstructive liver disease.

com-PORPHYRINS & BILE PIGMENTS / 285

as prehepatic (eg, hemolytic anemias), hepatic (eg,

hepatitis), and posthepatic (eg, obstruction of the

common bile duct) Measurements of plasma total

and nonconjugated bilirubin, of urinary

urobilino-gen and bilirubin, and of certain serum enzymes as

well as inspection of stool samples help distinguish

between these causes

REFERENCES

Anderson KE et al: Disorders of heme biosynthesis: X-linked

sid-eroblastic anemia and the porphyrias In: The Metabolic and

Molecular Bases of Inherited Disease, 8th ed Scriver CR et al

(editors) McGraw-Hill, 2001.

Berk PD, Wolkoff AW: Bilirubin metabolism and the

hyperbiliru-binemias In: Harrison’s Principles of Internal Medicine, 15th

ed Braunwald E et al (editors) McGraw-Hill, 2001 Chowdhury JR et al: Hereditary jaundice and disorders of bilirubin

metabolism In: The Metabolic and Molecular Bases of

Inher-ited Disease, 8th ed Scriver CR et al (editors) McGraw-Hill,

2001.

Desnick RJ: The porphyrias In: Harrison’s Principles of Internal

Medicine, 15th ed Braunwald E et al (editors) McGraw-Hill,

2001.

Elder GH: Haem synthesis and the porphyrias In: Scientific

Foun-dations of Biochemistry in Clinical Practice, 2nd ed Williams

DL, Marks V (editors) Butterworth-Heinemann, 1994.

C C C

N CH N

1 2

8

9 4 5

N H

HC

C CH CH 3

2 4

6 5

Nucleotides—the monomer units or building blocks of

nucleic acids—serve multiple additional functions They

form a part of many coenzymes and serve as donors of

phosphoryl groups (eg, ATP or GTP), of sugars (eg,

UDP- or GDP-sugars), or of lipid (eg,

CDP-acylglyc-erol) Regulatory nucleotides include the second

mes-sengers cAMP and cGMP, the control by ADP of

ox-idative phosphorylation, and allosteric regulation of

enzyme activity by ATP, AMP, and CTP Synthetic

purine and pyrimidine analogs that contain halogens,

thiols, or additional nitrogen are employed for

chemo-therapy of cancer and AIDS and as suppressors of the

immune response during organ transplantation

PURINES, PYRIMIDINES, NUCLEOSIDES,

& NUCLEOTIDES

Purines and pyrimidines are nitrogen-containing

hete-rocycles, cyclic compounds whose rings contain both

carbon and other elements (hetero atoms) Note that

the smaller pyrimidine has the longer name and the

larger purine the shorter name and that their six-atom

rings are numbered in opposite directions (Figure

33–1) The planar character of purines and pyrimidines

facilitates their close association, or “stacking,” which

stabilizes double-stranded DNA (Chapter 36) The oxo

and amino groups of purines and pyrimidines exhibit

keto-enol and amine-imine tautomerism (Figure 33–2),

but physiologic conditions strongly favor the amino

and oxo forms

Nucleosides & Nucleotides Nucleosides are derivatives of purines and pyrimidines

that have a sugar linked to a ring nitrogen Numerals

sugar from those of the heterocyclic base The sugar in

ribonucleosides is D-ribose, and in

deoxyribonucleo-sides it is 2-deoxy-D-ribose The sugar is linked to theheterocyclic base via a
-N-glycosidic bond, almost al-ways to N-1 of a pyrimidine or to N-9 of a purine (Fig-ure 33–3)

NUCLEOTIDES / 287

Mononucleotides are nucleosides with a phosphoryl

group esterified to a hydroxyl group of the sugar The

dAMP thus represent nucleotides with a phosphoryl

group on C-5 of the pentose Additional phosphoryl

groups linked by acid anhydride bonds to the

phos-phoryl group of a mononucleotide form nucleoside

diphosphates and triphosphates (Figure 33–4).

Steric hindrance by the base restricts rotation about

nu-cleotides Both therefore exist as syn or anti conformers(Figure 33–5) While both conformers occur in nature,anti conformers predominate Table 33–1 lists themajor purines and pyrimidines and their nucleosideand nucleotide derivatives Single-letter abbreviationsare used to identify adenine (A), guanine (G), cytosine(C), thymine (T), and uracil (U), whether free or pre-sent in nucleosides or nucleotides The prefix “d”

NH2

OH HO

OH

N

N N

OH

O

OH HO

OH O

OH HO

OH O

Figure 33–3. Ribonucleosides, drawn as the syn conformers.

OH HO O

O–

O P

OH O

Figure 33–5. The syn and anti conformers of sine differ with respect to orientation about the N-gly- cosidic bond.

adeno-288 / CHAPTER 33

Table 33–1 Bases, nucleosides, and nucleotides.

Nucleoside

Formula X = H Deoxyribose X = Ribose Phosphate

Adenine Adenosine Adenosine monophosphate

OH O

O O O P

O – – O O

O

N

HN O O

OH

O O P

O – – O

O – – O

N

O

N X

O N H

N X O

dX

O N H

N O

CH3

mammalian messenger RNAs (Figure 33–7) These

atypical bases function in oligonucleotide recognition

and in regulating the half-lives of RNAs Free

nu-cleotides include hypoxanthine, xanthine, and uric acid

(see Figure 34–8), intermediates in the catabolism of

adenine and guanine Methylated heterocyclic bases of

plants include the xanthine derivatives caffeine of

cof-fee, theophylline of tea, and theobromine of cocoa

(Fig-ure 33–8)

Posttranslational modification of preformed cleotides can generate additional bases such as

polynu-pseudouridine, in which D-ribose is linked to C-5 of

uracil by a carbon-to-carbon bond rather than by a

tRNA Similarly, methylation by S-adenosylmethionine

of a UMP of preformed tRNA forms TMP (thymidine

monophosphate), which contains ribose rather than

physio-Nucleoside Triphosphates Have High Group Transfer Potential

Acid anhydrides, unlike phosphate esters, have high

of the terminal phosphates of nucleoside triphosphates

transfer potential of purine and pyrimidine nucleosidetriphosphates permits them to function as group trans-fer reagents Cleavage of an acid anhydride bond typi-cally is coupled with a highly endergonic process such

as covalent bond synthesis—eg, polymerization of cleoside triphosphates to form a nucleic acid

nu-In addition to their roles as precursors of nucleicacids, ATP, GTP, UTP, CTP, and their derivativeseach serve unique physiologic functions discussed inother chapters Selected examples include the role ofATP as the principal biologic transducer of free energy;the second messenger cAMP (Figure 33–9); adenosine

sulfate donor for sulfated proteoglycans (Chapter 48)and for sulfate conjugates of drugs; and the methyl

group donor S-adenosylmethionine (Figure 33–11).

N

N H N N

CH3

O N

N N

HN

H2N

CH3N

N

O

NH2

N H

N

O

CH2OH

5-Methylcytosine 5-Hydroxymethylcytosine

Figure 33–7. Four uncommon naturally occurring

pyrimidines and purines.

OH O

OH O

N

CH2N

O

Figure 33–9. cAMP, 3 ′ ,5 ′ -cyclic AMP, and cGMP.

O N

N N N

CH3

O

CH3

H3C

Figure 33–8. Caffeine, a trimethylxanthine The

di-methylxanthines theobromine and theophylline are

similar but lack the methyl group at N-1 and at N-7,

re-spectively.

Adenine Ribose O SO3–

P P

Figure 33–10. Adenosine 3′-phosphate-5′ phosulfate.

-phos-290 / CHAPTER 33

GTP serves as an allosteric regulator and as an energy

source for protein synthesis, and cGMP (Figure 33–9)

serves as a second messenger in response to nitric oxide

(NO) during relaxation of smooth muscle (Chapter

48) UDP-sugar derivatives participate in sugar

epimer-izations and in biosynthesis of glycogen, glucosyl

disac-charides, and the oligosaccharides of glycoproteins and

proteoglycans (Chapters 47 and 48) UDP-glucuronic

acid forms the urinary glucuronide conjugates of

biliru-bin (Chapter 32) and of drugs such as aspirin CTP

participates in biosynthesis of phosphoglycerides,

sphingomyelin, and other substituted sphingosines

(Chapter 24) Finally, many coenzymes incorporate

nu-cleotides as well as structures similar to purine and

pyrimidine nucleotides (see Table 33–2)

Nucleotides Are Polyfunctional Acids

Nucleosides or free purine or pyrimidine bases are

un-charged at physiologic pH By contrast, the primary

phosphoryl groups (pK about 1.0) and secondary

phos-phoryl groups (pK about 6.2) of nucleotides ensure that

they bear a negative charge at physiologic pH

Nu-cleotides can, however, act as proton donors or

accep-tors at pH values two or more units above or below

neutrality

Nucleotides Absorb Ultraviolet Light

The conjugated double bonds of purine and pyrimidine

derivatives absorb ultraviolet light The mutagenic

ef-fect of ultraviolet light results from its absorption by

nucleotides in DNA with accompanying chemical

changes While spectra are pH-dependent, at pH 7.0 all

the common nucleotides absorb light at a wavelength

close to 260 nm The concentration of nucleotides and

nucleic acids thus often is expressed in terms of sorbance at 260 nm.”

“ab-SYNTHETIC NUCLEOTIDE ANALOGS ARE USED IN CHEMOTHERAPY

Synthetic analogs of purines, pyrimidines, nucleosides,and nucleotides altered in either the heterocyclic ring orthe sugar moiety have numerous applications in clinicalmedicine Their toxic effects reflect either inhibition ofenzymes essential for nucleic acid synthesis or their in-corporation into nucleic acids with resulting disruption

of base-pairing Oncologists employ fluoro- or iodouracil, 3-deoxyuridine, 6-thioguanine and 6-mer-captopurine, 5- or 6-azauridine, 5- or 6-azacytidine,and 8-azaguanine (Figure 33–12), which are incorpo-rated into DNA prior to cell division The purine ana-log allopurinol, used in treatment of hyperuricemia andgout, inhibits purine biosynthesis and xanthine oxidaseactivity Cytarabine is used in chemotherapy of cancer.Finally, azathioprine, which is catabolized to 6-mercap-topurine, is employed during organ transplantation tosuppress immunologic rejection

5-OH HO O

CH3

Methionine

CH2

CH2CH COO–

NH3+

Figure 33–11. S-Adenosylmethionine.

Table 33–2 Many coenzymes and related

compounds are derivatives of adenosinemonophosphate

NUCLEOTIDES / 291

(absent from DNA) functions as a nucleophile during

Polynucleotides Are Directional Macromolecules

ad-jacent monomers Each end of a nucleotide polymer

Polynucleotides Have Primary Structure The base sequence or primary structure of a polynu-

cleotide can be represented as shown below The phodiester bond is represented by P or p, bases by a sin-gle letter, and pentoses by a vertical line

N H N

N

N OH

4 5 6

3 2 1

O

OH HO

6

N

O HN

N

N

N H

H 2 N

8 N O

N H N

N H O

Synthetic nonhydrolyzable analogs of nucleoside

triphosphates (Figure 33–13) allow investigators to

dis-tinguish the effects of nucleotides due to phosphoryl

transfer from effects mediated by occupancy of

al-losteric nucleotide-binding sites on regulated enzymes

POLYNUCLEOTIDES

phosphodi-ester Most commonly, this second OH group is the

forms a dinucleotide in which the pentose moieties are

“backbone” of RNA and DNA

While formation of a dinucleotide may be sented as the elimination of water between two

repre-monomers, the reaction in fact strongly favors

phos-phodiester hydrolysis Phosphos-phodiesterases rapidly

cat-alyze the hydrolysis of phosphodiester bonds whose

spontaneous hydrolysis is an extremely slow process

Consequently, DNA persists for considerable periods

and has been detected even in fossils RNAs are far less

292 / CHAPTER 33

more compact notation is possible:

The most compact representation shows only the

-end on the right The phosphoryl groups are assumed

but not shown:

GGATCA pGpGpApTpCpA

O

O– O–P O O P

O–

O

O–P

Parent nucleoside triphosphate

O

O– O–P

O H N P

O–

O

O–P

β,γ-Imino derivative

O

O– O–P O

CH2P

O–

O

O–P

β,γ-Methylene derivative

Figure 33–13. Synthetic derivatives of nucleoside

triphosphates incapable of undergoing hydrolytic

re-lease of the terminal phosphoryl group (Pu/Py, a

purine or pyrimidine base; R, ribose or deoxyribose.)

Shown are the parent (hydrolyzable) nucleoside

triphosphate (top) and the unhydrolyzable β

-methyl-ene (center) and γ-imino derivatives (bottom).

SUMMARY

• Under physiologic conditions, the amino and oxotautomers of purines, pyrimidines, and their deriva-tives predominate

• Nucleic acids contain, in addition to A, G, C, T, and

U, traces of 5-methylcytosine,

• Most nucleosides contain D-ribose or ribose linked to N-1 of a pyrimidine or to N-9 of a

predominate

• A primed numeral locates the position of the

linked to the first by acid anhydride bonds form cleoside diphosphates and triphosphates

nu-• Nucleoside triphosphates have high group transferpotential and participate in covalent bond syntheses.The cyclic phosphodiesters cAMP and cGMP func-tion as intracellular second messengers

bonds form polynucleotides, directional

• Synthetic analogs of purine and pyrimidine bases andtheir derivatives serve as anticancer drugs either byinhibiting an enzyme of nucleotide biosynthesis or

by being incorporated into DNA or RNA

REFERENCES

Adams RLP, Knowler JT, Leader DP: The Biochemistry of the

Nu-cleic Acids, 11th ed Chapman & Hall, 1992.

Blackburn GM, Gait MJ: Nucleic Acids in Chemistry & Biology IRL

Press, 1990.

Bugg CE, Carson WM, Montgomery JA: Drugs by design Sci Am 1992;269(6):92.

Metabolism of Purine &

293

Victor W Rodwell, PhD

BIOMEDICAL IMPORTANCE

The biosynthesis of purines and pyrimidines is

strin-gently regulated and coordinated by feedback

mecha-nisms that ensure their production in quantities and at

times appropriate to varying physiologic demand

Ge-netic diseases of purine metabolism include gout,

Lesch-Nyhan syndrome, adenosine deaminase

defi-ciency, and purine nucleoside phosphorylase deficiency

By contrast, apart from the orotic acidurias, there are

few clinically significant disorders of pyrimidine

catab-olism

PURINES & PYRIMIDINES ARE

DIETARILY NONESSENTIAL

Human tissues can synthesize purines and pyrimidines

from amphibolic intermediates Ingested nucleic acids

and nucleotides, which therefore are dietarily

nonessen-tial, are degraded in the intestinal tract to

mononu-cleotides, which may be absorbed or converted to

purine and pyrimidine bases The purine bases are then

oxidized to uric acid, which may be absorbed and

ex-creted in the urine While little or no dietary purine or

pyrimidine is incorporated into tissue nucleic acids,

in-jected compounds are incorporated The incorporation

of injected [3H]thymidine into newly synthesized DNA

thus is used to measure the rate of DNA synthesis

BIOSYNTHESIS OF PURINE NUCLEOTIDES

Purine and pyrimidine nucleotides are synthesized in

vivo at rates consistent with physiologic need

Intracel-lular mechanisms sense and regulate the pool sizes of

nucleotide triphosphates (NTPs), which rise during

growth or tissue regeneration when cells are rapidly

di-viding Early investigations of nucleotide biosynthesis

employed birds, and later ones used Escherichia coli.

Isotopic precursors fed to pigeons established the source

of each atom of a purine base (Figure 34–1) and

initi-ated study of the intermediates of purine biosynthesis

Three processes contribute to purine nucleotidebiosynthesis These are, in order of decreasing impor-

tance: (1) synthesis from amphibolic intermediates

(synthesis de novo), (2) phosphoribosylation of purines,and (3) phosphorylation of purine nucleosides

INOSINE MONOPHOSPHATE (IMP)

IS SYNTHESIZED FROM AMPHIBOLIC INTERMEDIATES

Figure 34–2 illustrates the intermediates and reactionsfor conversion of α-D-ribose 5-phosphate to inosinemonophosphate (IMP) Separate branches then lead toAMP and GMP (Figure 34–3) Subsequent phosphoryltransfer from ATP converts AMP and GMP to ADPand GDP Conversion of GDP to GTP involves a sec-ond phosphoryl transfer from ATP, whereas conversion

of ADP to ATP is achieved primarily by oxidativephosphorylation (see Chapter 12)

Multifunctional Catalysts Participate in Purine Nucleotide Biosynthesis

In prokaryotes, each reaction of Figure 34–2 is alyzed by a different polypeptide By contrast, in eu-karyotes, the enzymes are polypeptides with multiplecatalytic activities whose adjacent catalytic sites facili-tate channeling of intermediates between sites Threedistinct multifunctional enzymes catalyze reactions 3,

cat-4, and 6, reactions 7 and 8, and reactions 10 and 11 ofFigure 34–2

Antifolate Drugs or Glutamine Analogs Block Purine Nucleotide Biosynthesis

The carbons added in reactions 4 and 5 of Figure 34–2are contributed by derivatives of tetrahydrofolate.Purine deficiency states, which are rare in humans, gen-erally reflect a deficiency of folic acid Compounds thatinhibit formation of tetrahydrofolates and thereforeblock purine synthesis have been used in cancerchemotherapy Inhibitory compounds and the reactionsthey inhibit include azaserine (reaction 5, Figure 34–2),diazanorleucine (reaction 2), 6-mercaptopurine (reac-tions 13 and 14), and mycophenolic acid (reaction 14)

294 / CHAPTER 34

N N Respiratory CO2

1

C2 3

C 6

C 8 9

Glycine Aspartate

Amide nitrogen of glutamine

tetrahydrofolate

-Methenyl-N10

-Formyl-

tetrahydro-folate

N H

N

5 C C 7

Figure 34–1. Sources of the nitrogen and carbon

atoms of the purine ring Atoms 4, 5, and 7 (shaded)

de-rive from glycine.

“SALVAGE REACTIONS” CONVERT

PURINES & THEIR NUCLEOSIDES TO

MONONUCLEOTIDES

Conversion of purines, their ribonucleosides, and their

deoxyribonucleosides to mononucleotides involves

so-called “salvage reactions” that require far less energy

than de novo synthesis The more important

mecha-nism involves phosphoribosylation by PRPP (structure

II, Figure 34–2) of a free purine (Pu) to form a purine

5′-mononucleotide (Pu-RP)

Two phosphoribosyl transferases then convert adenine

to AMP and hypoxanthine and guanine to IMP or

GMP (Figure 34–4) A second salvage mechanism

in-volves phosphoryl transfer from ATP to a purine

ri-bonucleoside (PuR):

Adenosine kinase catalyzes phosphorylation of

adeno-sine and deoxyadenoadeno-sine to AMP and dAMP, and

de-oxycytidine kinase phosphorylates dede-oxycytidine and

2′-deoxyguanosine to dCMP and dGMP

Liver, the major site of purine nucleotide

biosynthe-sis, provides purines and purine nucleosides for salvage

and utilization by tissues incapable of their

biosynthe-sis For example, human brain has a low level of PRPP

amidotransferase (reaction 2, Figure 34–2) and hence

depends in part on exogenous purines Erythrocytes

and polymorphonuclear leukocytes cannot synthesize

5-phosphoribosylamine (structure III, Figure 34–2)

PuR ATP + → PuR P ADP − +

on the activity of PRPP synthase, an enzyme sensitive

to feedback inhibition by AMP, ADP, GMP, andGDP

AMP & GMP Feedback-Regulate Their Formation From IMP

Two mechanisms regulate conversion of IMP to GMPand AMP AMP and GMP feedback-inhibit adenylo-succinate synthase and IMP dehydrogenase (reactions

12 and 14, Figure 34–3), respectively Furthermore,conversion of IMP to adenylosuccinate en route toAMP requires GTP, and conversion of xanthinylate(XMP) to GMP requires ATP This cross-regulationbetween the pathways of IMP metabolism thus serves

to decrease synthesis of one purine nucleotide whenthere is a deficiency of the other nucleotide AMP andGMP also inhibit hypoxanthine-guanine phosphoribo-syltransferase, which converts hypoxanthine and gua-nine to IMP and GMP (Figure 34–4), and GMP feed-back-inhibits PRPP glutamyl amidotransferase (reaction

2, Figure 34–2)

REDUCTION OF RIBONUCLEOSIDE DIPHOSPHATES FORMS

DEOXYRIBONUCLEOSIDE DIPHOSPHATES

Reduction of the 2′-hydroxyl of purine and pyrimidine

ribonucleotides, catalyzed by the ribonucleotide ductase complex (Figure 34–5), forms deoxyribonu-

re-cleoside diphosphates (dNDPs) The enzyme complex

is active only when cells are actively synthesizing DNA.Reduction requires thioredoxin, thioredoxin reductase,and NADPH The immediate reductant, reducedthioredoxin, is produced by NADPH:thioredoxin re-ductase (Figure 34–5) Reduction of ribonucleosidediphosphates (NDPs) to deoxyribonucleoside diphos-phates (dNDPs) is subject to complex regulatory con-trols that achieve balanced production of deoxyribonu-cleotides for synthesis of DNA (Figure 34–6)

2

Formylglycinamidine ribosyl-5-phosphate

R-5- P

(VI)

3 6

N 2 H 8

OOC

CO2C

CH

R-5- P 2

N

Aminoimidazole carboxylate ribosyl-5-phosphate

(VIII)

9

O

6 54 3

–

CH2

OOC –

Aspartate

CH

R-5- P 2

N

Aminoimidazole succinyl carboxamide ribosyl-5-phosphate

(IX)

O

6 5

3 41

OOC –

C 2 HC OOC –

C

C N H H

10

H4folate C

CH N N

H4folate

N 10

-Formyl-11 CH

N N

Formimidoimidazole carboxamide ribosyl-5-phosphate (XI)

O

C R-5- P

Ring closure

CH N N

Inosine monophosphate (IMP)

N H CH

N H C

N OOC

CH2H O

4

3 2 1

OH

O H H H OH

CH2O P

Mg 2

O 3

C 2 H

NH7 3+

OH

O H H H OH

H O P

Glycinamide ribosyl-5-phosphate

4

(IV)

H

C O

C 2 H

NH7 3+ 4 5

4 5

NH –

Methenyl-

-H4folate

H4folate

Formylglycinamide ribosyl-5-phosphate (V)

Glycine

5 Gln

H N CH

NH O

2 C

–

+

PRPP SYNTHASE

PRPP GLUTAMYL AMIDOTRANSFERASE

N

H2O NH3+

H

C COO–C

OOC – H2

GTP, Mg 2 +

N N N

Adenylosuccinate (AMPS)

NH

H

C OOC –

H2

–

N

N N N

Adenosine monophosphate

(AMP)

NH2 H

H C OOC

N H

Figure 34–7 summarizes the roles of the intermediates

and enzymes of pyrimidine nucleotide biosynthesis

The catalyst for the initial reaction is cytosolic carbamoyl

phosphate synthase II, a different enzyme from the

mi-tochondrial carbamoyl phosphate synthase I of urea

syn-thesis (Figure 29–9) Compartmentation thus provides

two independent pools of carbamoyl phosphate PRPP,

an early participant in purine nucleotide synthesis

(Fig-ure 34–2), is a much later participant in pyrimidine

biosynthesis

Multifunctional Proteins

Catalyze the Early Reactions

of Pyrimidine Biosynthesis

Five of the first six enzyme activities of pyrimidine

biosynthesis reside on multifunctional polypeptides

One such polypeptide catalyzes the first three reactions

of Figure 34–2 and ensures efficient channeling of

car-bamoyl phosphate to pyrimidine biosynthesis A second

bifunctional enzyme catalyzes reactions 5 and 6

THE DEOXYRIBONUCLEOSIDES OF URACIL & CYTOSINE ARE SALVAGED

While mammalian cells reutilize few free pyrimidines,

“salvage reactions” convert the ribonucleosides uridineand cytidine and the deoxyribonucleosides thymidineand deoxycytidine to their respective nucleotides ATP-dependent phosphoryltransferases (kinases) catalyze thephosphorylation of the nucleoside diphosphates 2′-de-oxycytidine, 2′-deoxyguanosine, and 2′-deoxyadenosine

to their corresponding nucleoside triphosphates In dition, orotate phosphoribosyltransferase (reaction 5,Figure 34–7), an enzyme of pyrimidine nucleotide syn-thesis, salvages orotic acid by converting it to orotidinemonophosphate (OMP)

ad-Methotrexate Blocks Reduction

dihydro-METABOLISM OF PURINE & PYRIMIDINE NUCLEOTIDES / 297

N N

HN O

N H

N

PPi

IMP Hypoxanthine

PRPP

N

HN

N H N

N N

O

HN

N N O

H2N

HYPOXANTHINE-GUANINE PHOSPHORIBOSYLTRANSFERASE

OH

O H H H OH

H O P

H

2 C

OH

O H H H OH

H O P

H 2C

N N

NH2

N N

N N

NH2

N H

N

PPi

AMP

ADENINE PHOSPHORIBOSYL TRANSFERASE

Adenine

PRPP

OH

O H H H OH

H O P

H 2C

Figure 34–4. Phosphoribosylation of adenine,

hy-poxanthine, and guanine to form AMP, IMP, and GMP,

respectively.

RIBONUCLEOTIDE REDUCTASE

THIOREDOXIN REDUCTASE

Reduced thioredoxin

Oxidized thioredoxin

Ribonucleoside diphosphate

2′-Deoxyribonucleoside diphosphate

Figure 34–5. Reduction of ribonucleoside phates to 2 ′-deoxyribonucleoside diphosphates.

diphos-folate For further pyrimidine synthesis to occur,

dihydro-folate must be reduced back to tetrahydrodihydro-folate, a

reac-tion catalyzed by dihydrofolate reductase Dividing cells,

which must generate TMP and dihydrofolate, thus are

es-pecially sensitive to inhibitors of dihydrofolate reductase

such as the anticancer drug methotrexate.

Certain Pyrimidine Analogs Are

Substrates for Enzymes of Pyrimidine

Nucleotide Biosynthesis

Orotate phosphoribosyltransferase (reaction 5, Figure

34–7) converts the drug allopurinol (Figure 33–12) to

a nucleotide in which the ribosyl phosphate is attached

to N-1 of the pyrimidine ring The anticancer drug

5-fluorouracil (Figure 33–12) is also

phosphoribosy-lated by orotate phosphoribosyl transferase

REGULATION OF PYRIMIDINE NUCLEOTIDE BIOSYNTHESISGene Expression & Enzyme Activity Both Are Regulated

The activities of the first and second enzymes of idine nucleotide biosynthesis are controlled by allosteric

2′dUDP

– – –

+ +

2 ′dCDP CDP

ATP

2′dCTP – – –

+ +

Figure 34–6. Regulation of the reduction of purine and pyrimidine ribonucleotides to their respective

2 ′-deoxyribonucleotides Solid lines represent chemical flow Broken lines show negative (– ) or positive (+ ) feedback regulation.

298 / CHAPTER 34

Dihydroorotic acid (DHOA)

HN O

N H O

C 4

1 62 5 2 O

H2O –

COO–CH

COO–

3 C N H

Carbamoyl aspartic acid (CAA)

2 Pi

3 H2O

HN

COO–

O C

2 CH CH O

N H C

NAD+

NADH + H+ 4

PPi PRPP 5 HN

O

N

CO26

Orotic acid (OA) OMP

HN O

N O

R-5-UMP

4

1 5 6 3 2

N O

HN O

N O dR

TMP

2

CH3

5 ,N10-Methylene H4folate 10

O

C4

1 2 5 O – CH

COO–

Carbamoyl phosphate (CAP)

6 C H

H3N +

2 O

H3N3 C +

H2folate +

ATP

ADP

CARBAMOYL PHOSPHATE SYNTHASE II

P

ASPARTATE TRANSCAR- BAMOYLASE

OROTASE

DIHYDRO-DIHYDROOROTATE DEHYDROGENASE

OROTATE PHOSPHORIBOSYL- TRANSFERASE

OROTIDYLIC ACID DECARBOXYLASE P

CTP

SYNTHASE

RIBONUCLEOTIDE REDUCTASE

THYMIDYLATE SYNTHASE

P

Figure 34–7. The biosynthetic pathway for pyrimidine nucleotides.

METABOLISM OF PURINE & PYRIMIDINE NUCLEOTIDES / 299

N N

NH2

N N

N N

Inosine

Ribose 1-phosphate

N HN O

NH N

NH HN

O N

Hypoxanthine

NH O

Xanthine

N HN O

NH N

N N

H HO

H

2 C

OH

O H H H OH

H HO

H HO

H

2 C

Figure 34–8. Formation of uric acid from purine nucleosides

by way of the purine bases hypoxanthine, xanthine, and

gua-nine Purine deoxyribonucleosides are degraded by the same

catabolic pathway and enzymes, all of which exist in the mucosa

of the mammalian gastrointestinal tract.

regulation Carbamoyl phosphate synthase II (reaction

1, Figure 34–7) is inhibited by UTP and purine

nu-cleotides but activated by PRPP Aspartate

transcar-bamoylase (reaction 2, Figure 34–7) is inhibited by

CTP but activated by ATP In addition, the first three

and the last two enzymes of the pathway are regulated

by coordinate repression and derepression

Purine & Pyrimidine Nucleotide

Biosynthesis Are Coordinately Regulated

Purine and pyrimidine biosynthesis parallel one

an-other mole for mole, suggesting coordinated control of

their biosynthesis Several sites of cross-regulation

char-acterize purine and pyrimidine nucleotide biosynthesis

The PRPP synthase reaction (reaction 1, Figure 34–2),

which forms a precursor essential for both processes, is

feedback-inhibited by both purine and pyrimidine

nu-cleotides

HUMANS CATABOLIZE PURINES

TO URIC ACID

Humans convert adenosine and guanosine to uric acid

(Figure 34–8) Adenosine is first converted to inosine

by adenosine deaminase In mammals other than

higher primates, uricase converts uric acid to the

water-soluble product allantoin However, since humans lack

uricase, the end product of purine catabolism in

hu-mans is uric acid

GOUT IS A METABOLIC DISORDER

OF PURINE CATABOLISM

Various genetic defects in PRPP synthetase (reaction 1,

Figure 34–2) present clinically as gout Each defect—

eg, an elevated Vmax, increased affinity for ribose

5-phosphate, or resistance to feedback inhibition—results

in overproduction and overexcretion of purine

catabo-lites When serum urate levels exceed the solubility

limit, sodium urate crystalizes in soft tissues and joints

and causes an inflammatory reaction, gouty arthritis.

However, most cases of gout reflect abnormalities in

renal handling of uric acid

300 / CHAPTER 34

OTHER DISORDERS OF

PURINE CATABOLISM

While purine deficiency states are rare in human

sub-jects, there are numerous genetic disorders of purine

ca-tabolism Hyperuricemias may be differentiated based

on whether patients excrete normal or excessive

quanti-ties of total urates Some hyperuricemias reflect specific

enzyme defects Others are secondary to diseases such

as cancer or psoriasis that enhance tissue turnover

Lesch-Nyhan Syndrome

Lesch-Nyhan syndrome, an overproduction

hyper-uricemia characterized by frequent episodes of uric acid

lithiasis and a bizarre syndrome of self-mutilation,

re-flects a defect in hypoxanthine-guanine

phosphoribo-syl transferase, an enzyme of purine salvage (Figure

34–4) The accompanying rise in intracellular PRPP

re-sults in purine overproduction Mutations that decrease

or abolish hypoxanthine-guanine

phosphoribosyltrans-ferase activity include deletions, frameshift mutations,

base substitutions, and aberrant mRNA splicing

Von Gierke’s Disease

Purine overproduction and hyperuricemia in von

Gierke’s disease (glucose-6-phosphatase deficiency)

occurs secondary to enhanced generation of the PRPP

precursor ribose 5-phosphate An associated lactic

aci-dosis elevates the renal threshold for urate, elevating

total body urates

Hypouricemia

Hypouricemia and increased excretion of hypoxanthine

and xanthine are associated with xanthine oxidase

de-ficiency due to a genetic defect or to severe liver

dam-age Patients with a severe enzyme deficiency may

ex-hibit xanthinuria and xanthine lithiasis

Adenosine Deaminase & Purine

Nucleoside Phosphorylase Deficiency

Adenosine deaminase deficiency is associated with an

immunodeficiency disease in which both

thymus-derived lymphocytes (T cells) and bone

marrow-de-rived lymphocytes (B cells) are sparse and

dysfunc-tional Purine nucleoside phosphorylase deficiency is

associated with a severe deficiency of T cells but

appar-ently normal B cell function Immune dysfunctions

ap-pear to result from accumulation of dGTP and dATP,

which inhibit ribonucleotide reductase and thereby

de-plete cells of DNA precursors

CATABOLISM OF PYRIMIDINES PRODUCES WATER-SOLUBLE METABOLITES

Unlike the end products of purine catabolism, those

of pyrimidine catabolism are highly water-soluble:

CO2, NH3, β-alanine, and β-aminoisobutyrate (Figure34–9) Excretion of β-aminoisobutyrate increases inleukemia and severe x-ray radiation exposure due to in-creased destruction of DNA However, many persons

of Chinese or Japanese ancestry routinely excrete β-aminoisobutyrate Humans probably transaminate β-aminoisobutyrate to methylmalonate semialdehyde,which then forms succinyl-CoA (Figure 19–2)

Pseudouridine Is Excreted Unchanged

Since no human enzyme catalyzes hydrolysis or phorolysis of pseudouridine, this unusual nucleoside isexcreted unchanged in the urine of normal subjects

phos-OVERPRODUCTION OF PYRIMIDINE CATABOLITES IS ONLY RARELY ASSOCIATED WITH CLINICALLY SIGNIFICANT ABNORMALITIES

Since the end products of pyrimidine catabolism arehighly water-soluble, pyrimidine overproduction results

in few clinical signs or symptoms In hyperuricemia sociated with severe overproduction of PRPP, there isoverproduction of pyrimidine nucleotides and in-creased excretion of β-alanine Since N5,N10-methyl-ene-tetrahydrofolate is required for thymidylate synthe-sis, disorders of folate and vitamin B12 metabolismresult in deficiencies of TMP

as-Orotic Acidurias

The orotic aciduria that accompanies Reye’s syndrome

probably is a consequence of the inability of severelydamaged mitochondria to utilize carbamoyl phosphate,which then becomes available for cytosolic overproduc-

tion of orotic acid Type I orotic aciduria reflects a

de-ficiency of both orotate phosphoribosyltransferase andorotidylate decarboxylase (reactions 5 and 6, Figure

34–7); the rarer type II orotic aciduria is due to a

defi-ciency only of orotidylate decarboxylase (reaction 6,Figure 34–7)

Deficiency of a Urea Cycle Enzyme Results

in Excretion of Pyrimidine Precursors

Increased excretion of orotic acid, uracil, and uridineaccompanies a deficiency in liver mitochondrial or-nithine transcarbamoylase (reaction 2, Figure 29–9)

METABOLISM OF PURINE & PYRIMIDINE NUCLEOTIDES / 301

CH2

C H

CH3COO −

H H

CH3O

O HN

Thymine

C

O NH

H H H O

O HN

NADP +

NADPH + H +

Uracil

N H

O

O HN

NH2

CH3

H3N + CH

2 CH COO − COO −

Figure 34–9. Catabolism of pyrimidines.

Excess carbamoyl phosphate exits to the cytosol, where

it stimulates pyrimidine nucleotide biosynthesis The

resulting mild orotic aciduria is increased by

high-nitrogen foods

Drugs May Precipitate Orotic Aciduria

Allopurinol (Figure 33–12), an alternative substrate fororotate phosphoribosyltransferase (reaction 5, Figure34–7), competes with orotic acid The resulting nu-cleotide product also inhibits orotidylate decarboxylase

(reaction 6, Figure 34–7), resulting in orotic aciduria and orotidinuria 6-Azauridine, following conversion

to 6-azauridylate, also competitively inhibits orotidylatedecarboxylase (reaction 6, Figure 34–7), enhancing ex-cretion of orotic acid and orotidine

SUMMARY

• Ingested nucleic acids are degraded to purines andpyrimidines New purines and pyrimidines areformed from amphibolic intermediates and thus aredietarily nonessential

• Several reactions of IMP biosynthesis require folatederivatives and glutamine Consequently, antifolatedrugs and glutamine analogs inhibit purine biosyn-thesis

• Oxidation and amination of IMP forms AMP andGMP, and subsequent phosphoryl transfer fromATP forms ADP and GDP Further phosphoryltransfer from ATP to GDP forms GTP ADP is con-verted to ATP by oxidative phosphorylation Reduc-tion of NDPs forms dNDPs

• Hepatic purine nucleotide biosynthesis is stringentlyregulated by the pool size of PRPP and by feedbackinhibition of PRPP-glutamyl amidotransferase byAMP and GMP

• Coordinated regulation of purine and pyrimidinenucleotide biosynthesis ensures their presence in pro-portions appropriate for nucleic acid biosynthesisand other metabolic needs

• Humans catabolize purines to uric acid (pKa 5.8),present as the relatively insoluble acid at acidic pH or

as its more soluble sodium urate salt at a pH nearneutrality Urate crystals are diagnostic of gout.Other disorders of purine catabolism include Lesch-Nyhan syndrome, von Gierke’s disease, and hypo-uricemias

• Since pyrimidine catabolites are water-soluble, theiroverproduction does not result in clinical abnormali-ties Excretion of pyrimidine precursors can, how-ever, result from a deficiency of ornithine transcar-bamoylase because excess carbamoyl phosphate isavailable for pyrimidine biosynthesis

302 / CHAPTER 34

REFERENCES

Benkovic SJ: The transformylase enzymes in de novo purine

biosynthesis Trends Biochem Sci 1994;9:320.

Brooks EM et al: Molecular description of three macro-deletions

and an Alu-Alu recombination-mediated duplication in the

HPRT gene in four patients with Lesch-Nyhan disease.

Mutat Res 2001;476:43.

Curto R, Voit EO, Cascante M: Analysis of abnormalities in purine

metabolism leading to gout and to neurological dysfunctions

in man Biochem J 1998;329:477.

Harris MD, Siegel LB, Alloway JA: Gout and hyperuricemia Am

Family Physician 1999;59:925.

Lipkowitz MS et al: Functional reconstitution, membrane

target-ing, genomic structure, and chromosomal localization of a

human urate transporter J Clin Invest 2001;107:1103.

Martinez J et al: Human genetic disorders, a phylogenetic tive J Mol Biol 2001;308:587.

perspec-Puig JG et al: Gout: new questions for an ancient disease Adv Exp Med Biol 1998;431:1.

Scriver CR et al (editors): The Metabolic and Molecular Bases of

In-herited Disease, 8th ed McGraw-Hill, 2001.

Tvrdik T et al: Molecular characterization of two deletion events involving Alu-sequences, one novel base substitution and two tentative hotspot mutations in the hypoxanthine phosphori- bosyltransferase gene in five patients with Lesch-Nyhan- syndrome Hum Genet 1998;103:311.

Zalkin H, Dixon JE: De novo purine nucleotide synthesis Prog Nucleic Acid Res Mol Biol 1992;42:259.

Nucleic Acid Structure & Function 35

303

Daryl K Granner, MD

BIOMEDICAL IMPORTANCE

The discovery that genetic information is coded along

the length of a polymeric molecule composed of only

four types of monomeric units was one of the major

sci-entific achievements of the twentieth century This

polymeric molecule, DNA, is the chemical basis of

heredity and is organized into genes, the fundamental

units of genetic information The basic information

pathway—ie, DNA directs the synthesis of RNA,

which in turn directs protein synthesis—has been

eluci-dated Genes do not function autonomously; their

replication and function are controlled by various gene

products, often in collaboration with components of

various signal transduction pathways Knowledge of the

structure and function of nucleic acids is essential in

understanding genetics and many aspects of

pathophys-iology as well as the genetic basis of disease

DNA CONTAINS THE

GENETIC INFORMATION

The demonstration that DNA contained the genetic

in-formation was first made in 1944 in a series of

experi-ments by Avery, MacLeod, and McCarty They showed

that the genetic determination of the character (type) of

the capsule of a specific pneumococcus could be

trans-mitted to another of a different capsular type by

intro-ducing purified DNA from the former coccus into the

latter These authors referred to the agent (later shown

to be DNA) accomplishing the change as “transforming

factor.” Subsequently, this type of genetic manipulation

has become commonplace Similar experiments have

recently been performed utilizing yeast, cultured

mam-malian cells, and insect and mammam-malian embryos as

re-cipients and cloned DNA as the donor of genetic

infor-mation

DNA Contains Four Deoxynucleotides

The chemical nature of the monomeric

deoxynucleo-tide units of DNA—deoxyadenylate, deoxyguanylate,

deoxycytidylate, and thymidylate—is described in

Chapter 33 These monomeric units of DNA are held

in polymeric form by 3′,5′-phosphodiester bridges

con-stituting a single strand, as depicted in Figure 35–1

The informational content of DNA (the genetic code)resides in the sequence in which these monomers—purine and pyrimidine deoxyribonucleotides—are or-dered The polymer as depicted possesses a polarity;one end has a 5′-hydroxyl or phosphate terminal whilethe other has a 3′-phosphate or hydroxyl terminal Theimportance of this polarity will become evident Sincethe genetic information resides in the order of themonomeric units within the polymers, there must exist

a mechanism of reproducing or replicating this specificinformation with a high degree of fidelity That re-quirement, together with x-ray diffraction data fromthe DNA molecule and the observation of Chargaffthat in DNA molecules the concentration of de-oxyadenosine (A) nucleotides equals that of thymidine(T) nucleotides (A = T), while the concentration of de-oxyguanosine (G) nucleotides equals that of deoxycyti-dine (C) nucleotides (G = C), led Watson, Crick, andWilkins to propose in the early 1950s a model of a dou-ble-stranded DNA molecule The model they proposed

is depicted in Figure 35–2 The two strands of this

double-stranded helix are held in register by hydrogen bonds between the purine and pyrimidine bases of the

respective linear molecules The pairings between thepurine and pyrimidine nucleotides on the oppositestrands are very specific and are dependent upon hydro-

gen bonding of A with T and G with C (Figure 35–3).

This common form of DNA is said to be handed because as one looks down the double helix thebase residues form a spiral in a clockwise direction Inthe double-stranded molecule, restrictions imposed bythe rotation about the phosphodiester bond, the fa-vored anti configuration of the glycosidic bond (Figure33–8), and the predominant tautomers (see Figure33–3) of the four bases (A, G, T, and C) allow A to paironly with T and G only with C, as depicted in Figure35–3 This base-pairing restriction explains the earlierobservation that in a double-stranded DNA moleculethe content of A equals that of T and the content of Gequals that of C The two strands of the double-helical

right-molecule, each of which possesses a polarity, are tiparallel; ie, one strand runs in the 5′ to 3′ directionand the other in the 3′ to 5′ direction This is analogous

an-to two parallel streets, each running one way but ing traffic in opposite directions In the double-stranded DNA molecules, the genetic information re-

G

NH2O

H

CH2

O

H H

CH2

H

O

H H

H

NH2 N

O P

O P

O

O P

O

O P

Figure 35–1. A segment of one strand of a DNA molecule in which the purine and pyrimidine bases guanine (G), cytosine (C), thymine (T), and adenine (A) are held together by a phosphodiester backbone between 2 ′-de-

oxyribosyl moieties attached to the nucleobases by an N-glycosidic bond Note that the backbone has a polarity

(ie, a direction) Convention dictates that a single-stranded DNA sequence is written in the 5 ′ to 3′ direction (ie, pGpCpTpA, where G, C, T, and A represent the four bases and p represents the interconnecting phosphates).

sides in the sequence of nucleotides on one strand, the

template strand This is the strand of DNA that is

copied during nucleic acid synthesis It is sometimes

re-ferred to as the noncoding strand The opposite strand

is considered the coding strand because it matches the

RNA transcript that encodes the protein

The two strands, in which opposing bases are held

together by hydrogen bonds, wind around a central axis

in the form of a double helix Double-stranded DNA

exists in at least six forms (A–E and Z) The B form is

usually found under physiologic conditions (low salt,

high degree of hydration) A single turn of B-DNA

about the axis of the molecule contains ten base pairs

The distance spanned by one turn of B-DNA is 3.4

nm The width (helical diameter) of the double helix in

B-DNA is 2 nm

As depicted in Figure 35–3, three hydrogen bonds

hold the deoxyguanosine nucleotide to the

deoxycyti-dine nucleotide, whereas the other pair, the A–T pair, isheld together by two hydrogen bonds Thus, the G–Cbonds are much more resistant to denaturation, or

“melting,” than A–T-rich regions

The Denaturation (Melting) of DNA

Is Used to Analyze Its Structure

The double-stranded structure of DNA can be rated into two component strands (melted) in solution

sepa-by increasing the temperature or decreasing the saltconcentration Not only do the two stacks of bases pullapart but the bases themselves unstack while still con-nected in the polymer by the phosphodiester backbone.Concomitant with this denaturation of the DNA mole-cule is an increase in the optical absorbance of thepurine and pyrimidine bases—a phenomenon referred

to as hyperchromicity of denaturation Because of the

NUCLEIC ACID STRUCTURE & FUNCTION / 305

P P

P P

S

G C

Figure 35–2. A diagrammatic representation of the

Watson and Crick model of the double-helical structure

of the B form of DNA The horizontal arrow indicates

the width of the double helix (20 Å), and the vertical

arrow indicates the distance spanned by one complete

turn of the double helix (34 Å) One turn of B-DNA

in-cludes ten base pairs (bp), so the rise is 3.4 Å per bp.

The central axis of the double helix is indicated by the

vertical rod The short arrows designate the polarity of

the antiparallel strands The major and minor grooves

are depicted (A, adenine; C, cytosine; G, guanine;

T, thymine; P, phosphate; S, sugar [deoxyribose].)

H

H

H N

N H

stacking of the bases and the hydrogen bonding

be-tween the stacks, the double-stranded DNA molecule

exhibits properties of a rigid rod and in solution is a

vis-cous material that loses its viscosity upon denaturation

The strands of a given molecule of DNA separateover a temperature range The midpoint is called the

melting temperature, or T m The Tmis influenced by

the base composition of the DNA and by the salt

con-centration of the solution DNA rich in G–C pairs,

which have three hydrogen bonds, melts at a higher

tem-perature than that rich in A–T pairs, which have two

hy-drogen bonds A tenfold increase of monovalent cation

concentration increases the Tmby 16.6 °C Formamide,

which is commonly used in recombinant DNA

experi-ments, destabilizes hydrogen bonding between bases,

thereby lowering the Tm This allows the strands of DNA

or DNA-RNA hybrids to be separated at much lowertemperatures and minimizes the phosphodiester bondbreakage that occurs at high temperatures

Renaturation of DNA Requires Base Pair Matching

Separated strands of DNA will renature or reassociatewhen appropriate physiologic temperature and salt con-ditions are achieved The rate of reassociation dependsupon the concentration of the complementary strands.Reassociation of the two complementary DNA strands

of a chromosome after DNA replication is a physiologicexample of renaturation (see below) At a given temper-ature and salt concentration, a particular nucleic acidstrand will associate tightly only with a complementarystrand Hybrid molecules will also form under appro-priate conditions For example, DNA will form a hy-brid with a complementary DNA (cDNA) or with acognate messenger RNA (mRNA; see below) Whencombined with gel electrophoresis techniques that sepa-rate hybrid molecules by size and radioactive labeling toprovide a detectable signal, the resulting analytic tech-

niques are called Southern (DNA/cDNA) and ern blotting (DNA/RNA), respectively These proce-