Voet D., Voet Ju.G. Biochemistry

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Section 26-4. Amino Acids as Biosynthetic Precursors 1051

Figure 26-36 X-ray structure of human porphobilinogen

synthase (PBGS). (a) PBGS in covalent complex with its

porphobiligen (PBG) product.A monomer of this

homooctameric protein is viewed perpendicular to the axis of its

␣/␤ barrel and is drawn in gray with its ␤ strands cyan and the

loop forming its flexible lid (residues 201–222) magenta.The

PBG, product, Lys 252 to which it is covalently linked, and the

three Cys side chains that ligand the active site Zn

2⫹

ion (blue

sphere) are shown in stick form with PBG C pink, side chain C

green, N blue, O red, S yellow, and the N¬C bond linking Lys

252 to PBG gold.The active site Zn

2⫹

ion is liganded (black

lines) by the S atoms of Cys 122, Cys 124, Cys 132, and the PBG

amino group. Lys 199, which lies directly behind Lys 252 in this

view, appears to be properly positioned to act as an acid–base

catalyst. [Based on an X-ray structure by Jonathan Cooper,

University of Southampton, U.K. PDBid 1E51.] (b) Quaternary

structural changes between the low activity hexameric state of

the F12L mutant of human PBGS (blue) and the high activity

octameric state of the wild-type enzyme (red). In the upper

panel, the proteins are viewed along their 3-fold and 4-fold axes

with the subunits closest to the viewer more darkly colored. In

the lower panel, the outer drawings are viewed along their 2-fold

axes with one dimer drawn in ribbon form and the others in

space-filling form.The inner drawings show only the dimers into

which the oligomers are assumed to dissociate before

reassembling to an alternate quaternary state. [Courtesy of Sarah

Lawrence and Eileen Jaffe,The Fox Chase Cancer Center,

Philadelphia, Pennsylvania. The X-ray structure of the F12L

mutant was determined by Eileen Jaffe. PDBid 1PV8.]

(a)

(b)

JWCL281_c26_1019-1087.qxd 4/20/10 9:26 AM Page 1051

1052 Chapter 26. Amino Acid Metabolism

Figure 26-37 The synthesis of uroporphyrinogen III from

PBG as catalyzed by porphobilinogen deaminase and

uroporphyrinogen III synthase. (1a) General base-catalyzed

elimination of NH

to form a methylene pyrrolinene

intermediate. (1b) Addition to the methylene pyrrolinene

intermediate of the enzyme’s covalently linked dipyrromethane

cofactor to form a covalent adduct. (2–4) Sequential addition of

a second, third, and fourth PBG through successive NH

eliminations from PBG to form methylene pyrrolinene, as in

Reaction 1a, followed by addition of a pyrrole ring carbon atom

from the growing chain, as in Reaction 1b. (5) Hydrolysis of the

methylbilane–enzyme to yield hydroxymethylbilane and

regenerate the free enzyme–dipyrromethane complex.

(6) Synthesis of uroporphyrinogen III via a spiro intermediate by

porphobilinogen deaminase and uroporphyrinogen III synthase.

(7) Spontaneous cyclization of hydroxymethylbilane in the

absence of uroporphyrinogen III synthase. A and P represent

acetyl and propionyl groups.

A A

Methylene

pyrrolinene

Enz

NH HN

Enz

Enzyme–

dipyrromethane

PBG

COO

–

A =

COO

–

P = CH

PBG

NH HN

uroporphy-

rinogen III

synthase

Hydroxymethylbilane

Methylbilane–enzyme

Spiro intermediate

Uroporphyrinogen III

Uroporphyrinogen I

uroporphyrinogen III

synthase

JWCL281_c26_1019-1087.qxd 4/20/10 9:26 AM Page 1052

d. The Porphyrin Ring Is Formed

from Four PBG Molecules

The next phase of heme biosynthesis is the condensation

of four PBG molecules to form uroporphyrinogen III, the

porphyrin nucleus, in a series of reactions catalyzed by por-

phobilinogen deaminase (alternatively, hydroxymethylbilane

synthase or uroporphyrinogen synthase) and uroporphyrino-

gen III synthase.The reaction (Fig. 26-37) begins with the en-

zyme’s displacement of the amino group in PBG to form a

covalent adduct. A second, third, and fourth PBG then se-

quentially add through the displacement of the primary

amino group on one PBG by a carbon atom on the pyrrole

ring of the succeeding PBG to yield a linear tetrapyrrole that

is hydrolyzed and released from the enzyme as hydrox-

ymethylbilane (also called preuroporphyrinogen).

e. Porphobilinogen Deaminase Has a

Dipyrromethane Cofactor

Peter Shoolingin-Jordan and Alan Battersby independ-

ently showed that porphobilinogen deaminase contains a

unique dipyrromethane cofactor (two pyrroles linked by a

methylene bridge; rings C

and C

in Fig. 26-37), which is co-

valently linked to the enzyme via a C¬S bond to an enzyme

Cys residue.Thus, the methylbilane–enzyme complex really

contains a linear hexapyrrole.The subsequent reaction step,

also catalyzed by porphobilinogen deaminase (Step 5 in Fig.

26-37), is the hydrolysis of the bond linking the second and

third pyrrole units of the hexapyrrole to yield hydroxy-

methylbilane and the dipyrromethane cofactor. This cofac-

tor is still linked to the enzyme, which is therefore ready to

catalyze a new round of hydroxymethylbilane synthesis.

How is the dipyrromethane cofactor assembled?

Shoolingin-Jordan has shown that porphobilinogen deami-

nase synthesizes its own cofactor from two PBG units us-

ing, it appears, the same catalytic machinery with which it

synthesizes methylbilane. However, the enzyme Cys reacts

much more rapidly with presynthesized hydroxymethylbi-

lane to form a reaction intermediate (the product of Step 2

in Fig. 26-37) that continues to add two more PBG units.

When hydroxymethylbilane is released,the enzyme retains

its dipyrromethane cofactor.

The X-ray structure of human porphobilinogen deaminase

(whose sequence is ⬎45% identical to those of the E. coli en-

zyme), in covalent complex with its dipyrromethane cofactor,

indicates that this monomeric, 364-residue protein folds into

three nearly equal sized domains (Fig 26-38). The

dipyrromethane cofactor lies deep in a cleft between domains

1 and 2 such that there is still considerable unoccupied space

in the cleft. Although the enzyme sequentially appends four

PBG residues to the cofactor, it has only one catalytic site.

If the enzyme has only one catalytic site, how does it

reposition the polypyrrole chain after each catalytic cycle

so that it can further extend this chain? One possibility is

that the polypyrrole chain fills the cavity next to the cofac-

tor. This model provides a simple steric rationale for why

the length of the polypyrrole chain is limited to six residues

(the final four of which are hydrolytically cleaved away by

the enzyme to yield the hydroxymethylbilane product and

regenerate the dipyrromethane cofactor).

f. Protoporphyrin IX Biosynthesis Requires

Four More Reactions

Cyclization of the hydroxymethylbilane product re-

quires uroporphyrinogen III synthase (Fig. 26-37). In the

absence of this enzyme, hydroxymethylbilane is released

from the synthase and rapidly cyclizes nonenzymatically to

the symmetric uroporphyrinogen I. Heme, however, is an

asymmetric molecule; the methyl substituent of pyrrole

ring D has an inverted placement compared to those of

rings A, B, and C (Fig. 26-32). This ring reversal to yield

uroporphyrinogen III has been shown by Battersby to pro-

ceed through attachment of the methylenes from rings A

and C to the same carbon of ring D so as to form a spiro

compound (a bicyclic compound with a carbon atom com-

mon to both rings; Fig. 26-37).

Heme biosynthesis takes place partly in the mitochon-

drion and partly in the cytosol (Fig. 26-39). ALA is mito-

chondrially synthesized and is transported to the cytosol for

conversion to PBG and then to uroporphyrinogen III. Pro-

toporphyrin IX, to which Fe is added to form heme, is pro-

duced from uroporphyrinogen III in a series of reactions

catalyzed by (1) uroporphyrinogen decarboxylase, which

decarboxylates all four acetate side chains (A) to form

methyl groups (M); (2) coproporphyrinogen oxidase, which

oxidatively decarboxylates two of the propionate side

chains (P) to vinyl groups (V); and (3) protoporphyrinogen

Section 26-4. Amino Acids as Biosynthetic Precursors 1053

Figure 26-38 X-ray structure of human porphobilinogen

deaminase in covalent complex with its dipyrromethane cofactor.

The protein is shown in ribbon form with domain 1 (residues

1–116 and 216–239) magenta, domain 2 (residues 117–215) cyan,

and domain 3 (residues 240–364) orange. The dipyrromethane

cofactor and the Cys side chain to which it is covalently linked are

drawn in stick form with C green, N blue, O red, and C yellow.

[Based on an X-ray structure by Zhi-Jie Liu, Institute of

Biophysics, Beijing, China. PDBid 3ECR.]

JWCL281_c26_1019-1087.qxd 8/9/10 9:40 AM Page 1053

1054 Chapter 26. Amino Acid Metabolism

M V

P M

HC CH

Heme

NH HN

M V

P M

Protoporphyrinogen IX

M V

P M

HC CH

Protoporphyrin IX

CoA

–

Succinyl-CoA

COO

–

Glycine

–

-Aminolevulinic acid (ALA)

δ-aminolevulinic

acid synthase

ferrochelatase

protoporphyrinogen

oxidase

2 CO

copropor-

phyrinogen

oxidase

NH HN

M P

P M

Coproporphyrinogen III

NH HN

A P

P A

Uroporphyrinogen III

uroporphyrinogen

decarboxylase

porphobilinogen

deaminase

uropor-

phyrinogen III

synthase

COO

–

ALA

porphobilinogen

synthase

Porphobilinogen (PBG)

4 CO

4 NH

Citric

acid

cycle

Mitochondrion

Cytosol

Figure 26-39 The overall pathway of heme biosynthesis.

␦-Aminolevulinic acid (ALA) is synthesized in the mitochondrion

by ALA synthase. ALA (left) leaves the mitochondrion and is

converted to PBG, four molecules of which condense to form a

porphyrin ring.The next two reactions involve oxidation of the

pyrrole ring substituents yielding protoporphyrinogen IX whose

formation is accompanied by its transport back into the

mitochondrion.After oxidation of the methylene groups linking

the pyrroles to yield protoporphyrin IX, ferrochelatase catalyzes

the insertion of Fe

2⫹

to yield heme. A, P, M, and V, respectively,

represent acetyl, propionyl, methyl,and vinyl (¬CH

“CH

) groups.

C atoms originating as the carboxyl group of acetate are red.

JWCL281_c26_1019-1087.qxd 6/8/10 9:38 AM Page 1054

oxidase, which oxidizes the methylene groups linking the

pyrrole rings to methenyl groups. Altogether, six carboxyl

groups originally from carboxyl-labeled acetate are lost as

.The only remaining C atoms from carboxyl-labeled ac-

etate are the carboxyl groups of heme’s two propionate side

chains (P). During the coproporphyrinogen oxidase reac-

tion, the macrocycle is transported back into the mito-

chondrion for the pathway’s final reactions.

g. Ferrochelatase Catalyzes the Insertion of Fe(II)

Into Protoporphyrin IX to Form Heme

Protoporphyrin IX is converted to heme by the inser-

tion of Fe(II) into the tetrapyrrole nucleus by fer-

rochelatase, a protein that is associated with the inner mi-

tochondrial membrane on the matrix side. The X-ray

structure of human ferrochelatase in complex with its

heme product (Fig. 26-40), determined by Harry Dailey

and William Lanzilotta, reveals that the 369-residue sub-

units of this homodimeric protein consist of two struc-

turally similar domains and a C-terminal extension that oc-

curs only in animal ferrochelatases (Fig. 26-40). This

C-terminal extension participates in hydrogen bonding be-

tween the monomers; bacterial ferrochelatases, which lack

this extension, are monomeric although they are otherwise

structurally similar to the human enzyme despite their only

⬃10% sequence identity. In addition,the C-terminal exten-

sion is bound to the N-terminal domain by an unusual

[2Fe–2S] cluster that is coordinated by C196 of the

N-terminal domain and C403,C406, and C411 of the C-termi-

nal extension.The function of this [2Fe–2S] cluster, which is

distant from the active site, is unclear although it appears

likely that it only has a structural role. Nevertheless, three

mutations, C406Y, C406S, and C411G, that inactivate the

enzyme and thereby cause the rare inherited disease ery-

thropoietic protoporphyria (see below) demonstrate the

importance of the [2Fe–2S] cluster for activity.

The ferrochelatase active site (Fig. 26-40) consists of two

hydrophobic lips that, it has been proposed, participate in

the enzyme’s association with the inner mitochondrial

membrane. The ferrochelatase reaction follows an ordered

mechanism in which the Fe(II) binds to the enzyme before

the porphyrin. The reaction requires that the two pyrrole

NH protons be removed from the porphyrin prior to the

binding of the Fe(II) (Fig. 26-39). The invariant H263 ap-

pears properly positioned to abstract these protons from

the porphyrin (Fig. 26-40), a hypothesis that is supported

by mutagenesis studies. Structural and spectroscopic stud-

ies indicate that the metalation reaction is accompanied by

the folding of the porphyrin by ⬃12° to a nonplanar con-

formation. Product release is then facilitated by the partial

unwinding of a structurally conserved ␲ helix (Fig. 26-40;

␲ helices are discussed in Section 8-1Bb).

h. Heme Biosynthesis Is Regulated Differently

in Erythroid and Liver Cells

The two major sites of heme biosynthesis are erythroid

cells, which synthesize ⬃85% of the body’s heme groups,

and the liver, which synthesizes ⬃80% of the remainder.

An important function of heme in liver is as the prosthetic

groups of the cytochromes P450, a family of oxidative en-

zymes involved in detoxification (Section 15-4Bc), whose

members are required throughout a liver cell’s lifetime in

amounts that vary with conditions. In contrast, erythroid

cells, in which heme is, of course,a hemoglobin component,

engage in heme synthesis only on differentiation, when

they synthesize hemoglobin in vast quantities.This is a one-

time synthesis; the heme must last the erythrocyte’s life-

time (normally 120 days) since heme and hemoglobin syn-

thesis stop on red cell maturation (protein synthesis stops

on the loss of nuclei and ribosomes). The different ways in

which heme biosynthesis is regulated in liver and in ery-

throid cells reflect these different demands: In liver, heme

biosynthesis must really be “controlled,” whereas in ery-

throid cells, the process is more like breaking a dam.

In liver, the main control target in heme biosynthesis is

ALA synthase, the enzyme catalyzing the pathway’s first

committed step. Heme, or its Fe(III) oxidation product

hemin, controls this enzyme’s activity through three mech-

anisms: (1) feedback inhibition, (2) inhibition of the trans-

port of ALA synthase (ALAS) from its site of synthesis in

the cytosol to its reaction site in the mitochondrion

(Fig. 26-39), and (3) repression of ALAS synthesis.

Section 26-4. Amino Acids as Biosynthetic Precursors 1055

Figure 26-40 X-ray structure of human ferrochelatase in

complex with its heme product. A subunit of this homodimeric

protein is drawn in ribbon form colored in rainbow order from

its N-terminus (blue) to its C-terminus (red), but with its

conserved ␲ helix (residues 340–360) magenta.The bound heme

and [2Fe–2S] cluster are shown in space-filling form with C

green, N blue, O red, S yellow, and Fe orange.The side chain of

the catalytically essential His 263 is drawn in stick form with C

cyan and N blue. [Based on an X-ray structure by Harry Dailey

and William Lanzilotta, University of Georgia. PDBid 2QD3.]

JWCL281_c26_1019-1087.qxd 6/8/10 9:38 AM Page 1055

In erythroid cells, heme exerts quite a different effect on

its biosynthesis. Heme induces, rather than represses, pro-

tein synthesis in reticulocytes (immature erythrocytes).Al-

though the vast majority of the protein synthesized by

reticulocytes is globin, heme may also induce these cells to

synthesize the enzymes of the heme biosynthesis pathway.

Moreover, the rate-determining step of heme biosynthesis

in erythroid cells may not be the ALA synthase reaction,

which in mammalian reticulocytes is catalyzed by a differ-

ent isozyme (ALAS-2) than the ALA synthase that is ex-

pressed in other cells (ALAS-1). Experiments on various

systems of differentiating erythroid cells implicate fer-

rochelatase and porphobilinogen deaminase in the control

of heme biosynthesis in these cells. There are also indica-

tions that cellular uptake of iron may be rate limiting. Iron

is transported in the plasma complexed with the iron

transport protein transferrin. The rate at which the

iron–transferrin complex enters most cells, including those

of liver, is controlled by receptor-mediated endocytosis

(Section 12-5Bc). However, lipid-soluble iron complexes

that diffuse directly into reticulocytes stimulate in vitro

heme biosynthesis. The existence of several control points

supports the supposition that when erythroid heme biosyn-

thesis is “switched on,” all of its steps function at their maxi-

mal rates rather than any one step limiting the flow through

the pathway. Heme-stimulated synthesis of globin also en-

sures that heme and globin are synthesized in the correct ra-

tio for assembly into hemoglobin (Section 32-4Aa).

i. Porphyrias Have Bizarre Symptoms

Seven sets of genetic defects in heme biosynthesis, in

liver or erythroid cells, are recognized.All involve the accu-

mulation of porphyrin and/or its precursors and are there-

fore known as porphyrias (Greek: porphyra, purple). Two

such defects are known to affect erythroid cells: uropor-

phyrinogen III synthase deficiency (congenital erythropoi-

etic porphyria) and ferrochelatase deficiency (erythropoi-

etic protoporphyria). The former results in accumulation

of uroporphyrinogen I and its decarboxylation product

coproporphyrinogen I. Excretion of these compounds col-

ors the urine red, their deposition in the teeth turns them a

fluorescent reddish brown, and their accumulation in the

skin renders it extremely photosensitive such that it ulcer-

ates and forms disfiguring scars. Increased hair growth is

also observed in afflicted individuals such that fine hair

may cover much of the face and extremities. These symp-

toms have prompted speculation that the werewolf legend

has a biochemical basis.

The most common porphyria that primarily affects liver

is porphobilinogen deaminase deficiency (acute intermittent

porphyria). This disease is marked by intermittent attacks of

abdominal pain and neurological dysfunction, often brought

about by infection, fasting, certain drugs, alcohol, steroids,

and other chemicals, all of which induce the expression of

ALAS-1. Excessive amounts of ALA and PBG are excreted

in the urine during and after such attacks.The urine may be-

come red resulting from the excretion of excess porphyrins

synthesized from PBG in nonhepatic cells although the skin

does not become unusually photosensitive. King George III,

who ruled England during the American Revolution, and

who has been widely portrayed as being mad, in fact had at-

tacks characteristic of acute intermittent porphyria, was re-

ported to have urine the color of port wine, and had several

descendants who were diagnosed as having this disease.

American history might have been quite different had

George III not inherited this metabolic defect.

j. Heme Is Degraded to Bile Pigments

At the end of their lifetime, red cells are removed from the

circulation and their components degraded. Heme catabo-

lism (Fig.26-41) begins with oxidative cleavage, by heme oxy-

genase, of the porphyrin between rings A and B to form

biliverdin, a green linear tetrapyrrole. Biliverdin’s central

methenyl bridge (between rings C and D) is then reduced to

form the red-orange bilirubin. The changing colors of a heal-

ing bruise are a visible manifestation of heme degradation.

The highly lipophilic bilirubin is insoluble in aqueous

solutions. Like other lipophilic metabolites, such as free

fatty acids, it is transported in the blood in complex with

serum albumin. In the liver, its aqueous solubility is in-

creased by esterification of its two propionate side groups

with glucuronic acid, yielding bilirubin diglucuronide,

which is secreted into the bile. Bacterial enzymes in the

large intestine hydrolyze the glucuronic acid groups and, in

a multistep process, convert bilirubin to several products,

most notably urobilinogen. Some urobilinogen is reab-

sorbed and transported via the bloodstream to the kidney,

where it is converted to the yellow urobilin and excreted,

thus giving urine its characteristic color. Most of the uro-

bilinogen, however, is microbially converted to the deeply

red-brown stercobilin, the major pigment of feces.

When the blood contains excessive amounts of bilirubin,

the deposition of this highly insoluble substance colors the

skin and the whites of the eyes yellow.This condition, called

jaundice (French: jaune, yellow), signals either an abnor-

mally high rate of red cell destruction, liver dysfunction, or

bile duct obstruction. Newborn infants, particularly when

premature, often become jaundiced because their livers do

not yet make sufficient bilirubin UDP-glucuronosyltrans-

ferase to glucuronidate the incoming bilirubin.Jaundiced in-

fants are treated by bathing them with light from a fluores-

cent lamp; this photochemically converts bilirubin to more

soluble isomers that the infant can degrade and excrete.

k. Hemoglobin’s Reduced Affinity

for CO Prevents Asphyxiation

In the reaction forming biliverdin, the methenyl bridge

carbon between porphyrin rings A and B is released as CO

(Fig. 26-41, top), which, we have seen, is a tenacious heme

ligand (with 200-fold greater affinity for hemoglobin and

myoglobin than O

; Section 10-1A). Consequently, ⬃1% of

hemoglobin’s O

-binding sites are blocked by CO, even in

the absence of air pollution. However, free heme in solu-

tion binds CO with 20,000-fold greater affinity than it binds

. Thus, the globin (protein) portion of hemoglobin (and

likewise myoglobin) somehow lowers the affinity of its

bound heme for CO, thereby making O

transport possible.

How does the globin do so?

1056 Chapter 26. Amino Acid Metabolism

JWCL281_c26_1019-1087.qxd 4/20/10 9:26 AM Page 1056

Early X-ray structures of carboxymyoglobin (myoglo-

bin with a CO ligand) indicated that the bound CO was in-

clined from the normal to the heme plane by 40° to 60° (the

Fe¬C¬O bond angle appeared to be 120° to 140°), ap-

proximately the same angle with which O

binds to heme

(Fig. 10-12).Yet, in complexes of CO with porphyrins in the

absence of protein, the CO is normal to the heme plane.

This suggested that the globin (in both myoglobin and

hemoglobin) sterically bends the bound CO away from its

preferred linear geometry, thereby reducing its affinity for

CO and hence permitting the CO to be slowly exhaled.

However,a variety of spectroscopic investigations together

with highly accurate X-ray structures of carboxymyoglobin

revealed that the bound CO is, in fact, inclined from the

normal to the heme plane by ⬃7°, a distortion that is too

small to explain the reduced affinity of myoglobin for CO.

Section 26-4. Amino Acids as Biosynthetic Precursors 1057

Figure 26-41 The heme degradation pathway. M,V, P, and E, respectively, represent methyl,

vinyl, propionyl, and ethyl groups.

M V

Heme

+ CO +

M V

M P

M V

Biliverdin

Bilirubin

NADP

NADPH + H

Bilirubin diglucuronide

COO

–

C O

bilirubin

UDP–glucuronosyl

transferase

(liver)

microbial

enzymes

(large intestine)

UDP UDP–glucuronate

Glucuronate

Urobilinogen

8H•

microbial enzymes

2H•

(kidney)

UrobilinStercobilin

2H•

microbial enzymes

(large intestine)

P M

M V

M P

M V

P M

M V

H H

M V

H H

M E

M P

P M

H H

M E

M P

M E

P M

M E

M P

M E

P M

HHH H

COO

–

JWCL281_c26_1019-1087.qxd 8/9/10 9:40 AM Page 1057

Of course, this reduced affinity might instead be explained

by the distortions that the upright CO ligand imposes on

the globin, presumably via the distal His (E7, the His

residue that hydrogen bonds to the bound O

; Section

10-2A). However, studies of the energetics of binding of

CO and O

to myoglobins in which His E7 has been mutated

to nonpolar residues of comparable bulk (e.g., Leu) indicate

that this is not the main determinant of the ligand affinity

changes. Rather, the reduction in affinity of myoglobin for

CO relative to that for O

has been shown to arise from the

greater hydrogen bonding affinity that His E7 has for O

rel-

ative to CO together with electrostatic effects due to the dif-

fering charge distributions in the O

and CO ligands.

l. Chloroquine Prevents Malaria by Inhibiting

Plasmodial Heme Sequestration

Malaria is caused by the mosquito-borne parasite Plas-

modium falciparum (Section 7-3Ab), which multiplies

within and destroys red blood cells in a 2-day cycle. During

the intraerythrocytic stages of its life cycle, the parasite par-

tially meets its nutritional needs by proteolyzing up to

⬃80% of the host cell’s hemoglobin in its so-called acid food

vacuole, whose pH is 4.7. This process releases heme, which

in its soluble form,is toxic to the parasite because it damages

cell membranes and inhibits a variety of enzymes. Since, un-

like their human hosts, plasmodia cannot degrade heme,

they sequester it within their food vacuoles in the form of

harmless dark brown granules known as hemozoin, which

consist of crystals of dimerized hemes linked together by re-

ciprocal iron–carboxylate bonds between the ferric ions

and the propionate side chains of adjacent molecules. He-

mozoin has been found to be identical to ␤-hematin,

Fe(III)

␤-Hematin (hemozoin)

≥

whose X-ray structure has been determined. Dimers inter-

act in the crystals through hydrogen bonds between the re-

maining carboxyl groups.

Chloroquine,

a member of the quinoline ring–containing family of anti-

malarials, which includes quinine, is one of the most suc-

cessful antimicrobial agents that has been produced. It is

effective against plasmodia only during their intraerythro-

cytic stages. This drug, being a weak base that can readily

pass through membranes in its uncharged form, accumu-

lates in the plasmodial acid food vacuole in its acidic

(charged) form in millimolar concentrations. Chloroquine

and several other quinoline-containing antimalarials in-

hibit the crystallization of hemes to form hemozoin. This

inhibition in vivo is almost certainly responsible for the an-

timalarial properties of these drugs. The mechanism of in-

hibition is as yet unclear although a plausible hypothesis is

that the drug adsorbs onto crystallized hemozoin, inhibit-

ing further crystallization.

The massive use of chloroquine has, unfortunately, led

to the appearance of chloroquine-resistant plasmodia in

nearly every malarial region of the world. Resistant plas-

modia do not concentrate chloroquine in their food vac-

uoles to the high levels found in sensitive parasites.

Rather, they export this drug out of their food vacuoles at

an ⬃50-fold higher rate than do sensitive organisms. Since

chloroquine activity and chloroquine resistance have dif-

ferent mechanisms, it has been possible to modify existing

quinoline-containing structures and to develop new he-

mozoin crystallization inhibitors that are effective anti-

malarial agents but to which plasmodia are not (yet)

resistant.

B. Biosynthesis of Physiologically Active Amines

Epinephrine, norepinephrine, dopamine, serotonin (5-

hydroxytryptamine), ␥-aminobutyric acid (GABA), and

NH CH

N(C

)

Chloroquine

Quinine

1058 Chapter 26. Amino Acid Metabolism

JWCL281_c26_1019-1087.qxd 7/21/10 6:26 PM Page 1058

histamine

are hormones and/or neurotransmitters derived from amino

acids. For instance, epinephrine, as we have seen, activates

muscle adenylate cyclase, thereby stimulating glycogen

breakdown (Section 18-3E); deficiency in dopamine pro-

duction is associated with Parkinson’s disease, a degenera-

tive condition causing “shaking palsy”; serotonin causes

smooth muscle contraction; GABA is one of the brain’s

major inhibitory neurotransmitters (Section 20-5Cf), being

released at 30% of its synapses; and histamine is involved

in allergic responses (as allergy sufferers who take antihis-

tamines will realize), as well as in the control of acid secre-

tion by the stomach (Section 20-3C).

The biosynthesis of each of these physiologically active

amines involves decarboxylation of the corresponding pre-

cursor amino acid. Amino acid decarboxylases are PLP-

dependent enzymes that form a PLP–Schiff base with the

CCH

NH R

= OH, R = CH

Epinephrine (Adrenalin)

= OH, R = H Norepinephrine

= H, R = H Dopamine

Serotonin

(5-hydroxytryptamine)

–

OOC CH

γ-Aminobutyric acid (GABA)

Histamine

substrate so as to stabilize the C

␣

carbanion formed on

␣

¬COO

⫺

bond cleavage (Section 26-1Aa):

Formation of histamine and GABA are one-step

processes (Fig. 26-42). In the synthesis of serotonin from

tryptophan, the decarboxylation is preceded by a hydroxy-

lation (Fig. 26-43) by tryptophan hydroxylase, one of three

mammalian enzymes that has a 5,6,7,8-tetrahydrobiopterin

cofactor (Section 26-3Ha). This hydroxylation involves an

NIH shift similar to that occurring in phenylalanine hy-

droxylase (Fig. 26-30), although no epoxide intermediate

has been observed in this case. Dopamine, norepinephrine,

and epinephrine are all termed catecholamines because

they are amine derivatives of catechol:

The conversion of tyrosine to these various catecholamines

occurs as follows (Fig. 26-44):

1. Tyrosine is hydroxylated to 3,4-dihydroxyphenylala-

nine (

L-DOPA) by tyrosine hydroxylase, another 5,6,7,8-

tetrahydrobiopterin-requiring enzyme.

L-DOPA is decarboxylated to dopamine.

3. A second hydroxylation yields norepinephrine.

Catechol

...

–

2–

Section 26-4. Amino Acids as Biosynthetic Precursors 1059

Figure 26-42 The formation of ␥-aminobutyric acid (GABA) and histamine. The reactions

involve the decarboxylations of glutamate to form GABA and of histidine to form histamine.

Glutamate

COO

–

glutamate

decarboxylase

(PLP dependent)

–

OOC

␥-Aminobutyric acid

(GABA)

–

OOC

Histidine

COO

–

histidine

decarboxylase

(PLP dependent)

Histamine

JWCL281_c26_1019-1087.qxd 4/20/10 9:26 AM Page 1059

4. Methylation of norepinephrine’s amino group by

S-adenosylmethionine (SAM; Section 26-3Ea) produces

epinephrine.

The specific catecholamine that a cell produces depends

on which enzymes of the pathway are present. In adrenal

medulla, which functions to produce hormones (Section

19-1F), epinephrine is the predominant product. In some

areas of the brain, norepinephrine is more common. In

other areas, most prominently the substantia nigra, the

pathway stops at dopamine synthesis. Indeed, Parkinson’s

disease, which is caused by degeneration of the substantia

nigra, has been treated with some success by the admin-

istration of

L-DOPA, dopamine’s immediate precursor.

Dopamine itself is ineffective because it cannot cross the

blood–brain barrier.

L-DOPA, however, is able to get to its

sites of action where it is decarboxylated to dopamine. The

enzyme catalyzing this reaction, aromatic amino acid decar-

boxylase, decarboxylates all aromatic amino acids and is

therefore also responsible for serotonin formation.

L-DOPA

is also a precursor of the black skin pigment melanin.

C. Glutathione

Glutathione (GSH; ␥-glutamylcysteinylglycine),

a tripeptide that contains an unusual ␥-amide bond, partici-

pates in a variety of detoxification, transport, and metabolic

processes (Fig. 26-45). For instance, it is a substrate for

Glutathione

(GSH; ␥-glutamylcysteinylglycine)

C NHCH

COO

–

COO

–

1060 Chapter 26. Amino Acid Metabolism

Tryptophan

COO

–

5-Hydroxytryptophan

COO

–

Serotonin

aromatic amino

acid decarboxylase

(PLP dependent)

tryptophan

hydroxylase

Tetrahydro-

biopterin

Pterin-4a-

carbinolamine

Figure 26-43 The

formation of serotonin.

The biosynthesis involves

the hydroxylation and

subsequent

decarboxylation of

tryptophan.

Figure 26-44 The sequential synthesis of

L-DOPA, dopamine, norepinephrine,

and epinephrine from tyrosine.

L-DOPA is also the precursor of the black skin

pigment melanin, an oxidized polymeric material.

CHO

Tyrosine

COO

–

Tetrahydrobiopterin + O

tyrosine

hydroxylase

Pterin-4a-carbinolamine

CHO

Dihydroxyphenylalanine

(

L-DOPA)

COO

–

Melanin

aromatic amino

acid decarboxylase

Dopamine

+ Ascorbate

dopamine

␤-hydroxylase

O + Dehydroascorbate

Norepinephrine

S-Adenosylmethionine

phenylethanolamine

-methyltransferase

S-Adenosylhomocysteine

Epinephrine

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