Voet D., Voet Ju.G. Biochemistry

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COO

–

Tryptophan

COO

–

-Formylkynurenine

COO

–

Kynurenine

2-Amino-3-carboxymuconate-

6-semialdehyde

C COO

–

3-Hydroxyanthranilate

COO

–

3-Hydroxykynurenine

COO

–

Alanine

COO

–

OOC

OCH

Quinolinate

COO

–

COO

–

2-Aminomuconate-

6-semialdehyde

–

OOC

OCH

2-Aminomuconate

–

OOC

–

OOC

-Ketoadipate

–

OOC

–

OOC

COO

–

Acetoacetate

HCOO

–

O + NADP

+ NADPH

spontaneous

To NAD

and NADP

NADH

NAD

NAD(P)

NAD(P)H

101112131415

and ␣-ketoglutarate dehydrogenase (Sections 21-2A and

21-3D). Reactions 6, 8, and 9 are standard reactions of fatty

acyl-CoA oxidation: dehydrogenation by FAD, hydration,

and dehydrogenation by NAD

⫹

. Reactions 10 and 11 are

standard reactions in ketone body formation. Two mole-

cules of CO

are produced at Reactions 5 and 7 of the

pathway.

The saccharopine pathway is thought to predominate in

mammals because a genetic defect in the enzyme that cat-

alyzes Reaction 1 in the sequence results in hyperlysinemia

and hyperlysinuria (elevated levels of lysine in the blood and

urine, respectively) along with mental and physical retarda-

tion.This is yet another example of how the study of rare in-

herited disorders has helped to trace metabolic pathways.

Leucine’s carbon skeleton,as we have seen, is converted

to one molecule each of acetoacetate and acetyl-CoA,

whereas that of lysine is converted to one molecule of ace-

toacetate and two of CO

. Since neither acetoacetate nor

acetyl-CoA can be converted to glucose in animals, leucine

and lysine are purely ketogenic amino acids.

G. Tryptophan Is Degraded to Alanine

and Acetoacetate

The complexity of the major tryptophan degradation path-

way (Fig. 26-24) precludes detailed discussion of all of its

reactions. However,one reaction in the pathway is of partic-

ular interest. Reaction 4, cleavage of 3-hydroxykynurenine

Section 26-3. Metabolic Breakdown of Individual Amino Acids 1041

Figure 26-24 The pathway of tryptophan degradation. The

enzymes involved are (1) tryptophan-2,3-dioxygenase,

(2) formamidase, (3) kynurenine-3-monooxygenase,

(4) kynureninase (PLP dependent), (5) 3-hydroxyanthranilate-

3,4-dioxygenase, (6) amino carboxymuconate semialdehyde

decarboxylase, (7) aminomuconate semialdehyde dehydrogenase,

(8) hydratase, (9) dehydrogenase, and (10–16) enzymes of

Reactions 5 through 11 in lysine degradation (Fig. 26-23).

2-Amino-3-carboxymuconate-6-semialdehyde, in addition to

undergoing Reaction 6, spontaneously forms quinolinate, an

NAD

⫹

and NADP

⫹

precursor (Section 28-5A).

JWCL281_c26_1019-1087.qxd 4/20/10 9:26 AM Page 1041

COO

–

3-Hydroxykynurenine

COO

–

Enzyme–PLP Schiff base

•

COO

–

•

COO

–

COO

–

)NH

COO

–

COO

–

Acyl–enzyme

3-Hydroxy-

anthranilate

•

COO

–

Alanine

Enzyme–PLP

Schiff base

␥

␤

␣

to alanine and 3-hydroxyanthranilate, is catalyzed by

kynureninase, a PLP-dependent enzyme. The reaction fur-

ther demonstrates the enormous versatility of PLP. We

have seen how PLP can labilize an ␣-amino acid’s C

␣

¬H

and C

␣

¬C

␤

bonds (Fig. 26-16). Here we see the facilitation

of C

␤

¬C

␥

bond cleavage. The reaction follows the same

steps as transamination reactions but does not hydrolyze

the tautomerized Schiff base (Fig. 26-25).The proposed re-

action mechanism involves an attack of an enzyme nucle-

ophile on the carbonyl carbon (C

␥

) of the tautomerized

3-hydroxykynurenine–PLP Schiff base (Fig. 26-25, Step 3).

This is followed by C

␤

¬C

␥

bond cleavage to generate an

acyl–enzyme intermediate together with a tautomerized

alanine–PLP adduct (Fig. 26-25, Step 4). Hydrolysis of the

acyl–enzyme then yields 3-hydroxyanthranilate, whose fur-

ther degradation yields ␣-ketoadipate (Fig. 26-24, Reac-

tions 5–9). ␣-Ketoadipate is also an intermediate in lysine

breakdown (Fig. 26-23, Reaction 4) so that the last seven

reactions in the degradation of both these amino acids are

identical, forming acetoacetate and two molecules of CO

1042 Chapter 26. Amino Acid Metabolism

Figure 26-25 Proposed mechanism for the PLP-dependent

kynureninase-catalyzed C

␤

¬C

␥

bond cleavage of

3-hydroxykynurenine. The reaction occurs in eight steps:

(1) transimination; (2) tautomerization; (3) attack of an enzyme

nucleophile, X; (4) C

␤

¬C

␥

bond cleavage with formation of an

acyl–enzyme intermediate; (5) acyl–enzyme hydrolysis; (6) and

(7) tautomerization; and (8) transimination.

JWCL281_c26_1019-1087.qxd 6/8/10 9:38 AM Page 1042

CHHO

Tyrosine

COO

–

p-Hydroxyphenylpyruvate

COO

–

α-Ketoglutarate

Glutamate

Tetrahydrobiopterin + O

Dihydrobiopterin + H

COO

–

COO

–

Homogentisate

COO

–

4-Maleylacetoacetate

–

OOC

COO

–

4-Fumarylacetoacetate

Fumarate

COO

–

OOC

Acetoacetate

CCH

Phenylalanine

COO

–

COO–

⌬

⌬x

Pteridine

C B A

Isoalloxazine

R⬘

Flavin

Pterin

(2-amino-4-oxopteridine)

Biopterin: R =

–

OOC

COO

–

Folate: R =

H. Phenylalanine and Tyrosine Are Degraded

to Fumarate and Acetoacetate

Since the first reaction in phenylalanine degradation is its

hydroxylation to tyrosine, a single pathway (Fig. 26-26) is

responsible for the breakdown of both of these amino

acids.The final products of the six-reaction degradation are

fumarate, a citric acid cycle intermediate, and acetoacetate,

a ketone body.

a. Pterins Are Redox Cofactors

The hydroxylation of phenylalanine by the nonheme

iron–containing homotetrameric enzyme phenylalanine

hydroxylase (PAH) requires O

and that the iron be in the

Fe(II) state. The enzyme also requires the participation of

biopterin, a pterin derivative. Pterins are compounds that

contain the pteridine ring (Fig. 26-27). Note the resem-

blance between the pteridine ring and the isoalloxazine

ring of the flavin coenzymes; the positions of the nitrogen

atoms in pteridine are identical with those of the B and C

rings of isoalloxazine. Folate derivatives also contain the

pterin ring (Section 26-4D). Pterins, like flavins, participate

in biological oxidations. The active form of biopterin is the

fully reduced form, 5,6,7,8-tetrahydrobiopterin (BH

). It is

produced from 7,8-dihydrobiopterin and NADPH, in what

Section 26-3. Metabolic Breakdown of Individual Amino Acids 1043

Figure 26-26 The pathway of phenylalanine degradation. The

enzymes involved are (1) phenylalanine hydroxylase,

(2) aminotransferase, (3) p-hydroxyphenylpyruvate dioxygenase,

(4) homogentisate dioxygenase, (5) maleylacetoacetate

isomerase, and (6) fumarylacetoacetase. The symbols labeling the

various carbon atoms serve to indicate the group migration that

occurs in Reaction 3 of the pathway (see Fig. 26-31).

Figure 26-27 The pteridine ring, the nucleus of biopterin

and folate. Note the similar structures of pteridine and the

isoalloxazine ring of flavin coenzymes.

JWCL281_c26_1019-1087.qxd 4/20/10 9:26 AM Page 1043

7,8-Dihydrobiopterin

5,6,7,8-Tetrahydrobiopterin (BH

)

7,8-Dihydrobiopterin

(quinoid form)

COO

–

Phenylalanine

COO

–

Tyrosine

phenylalanine

hydroxylase

pterin-4a-

carbinolamine

dehydratase

dihydropteridine

reductase

NAD(P)H

NAD(P)

NADP

NADPH + H

dihydrofolate reductase

Pterin-4a-carbinolamine

may be considered a priming reaction, by dihydrofolate

reductase (Fig. 26-28).

Each 452-residue subunit of the PAH homotetramer

contains three domains, an N-terminal regulatory do-

main, a catalytic domain, and a C-terminal tetrameriza-

tion domain. However, the 325-residue catalytic domain

alone forms catalytically competent dimers. The X-ray

structure of the catalytic domain of PAH in its Fe(II) state

in complex with BH

, determined by Edward Hough, re-

veals that the Fe(II) is octahedrally coordinated by His

285, His 290, Glu 330, and three water molecules, and that

atom O4 of BH

is hydrogen bonded to two of these

waters (Fig. 26-29).

In the phenylalanine hydroxylase reaction, 5,6,7,8-

tetrahydrobiopterin is hydroxylated to pterin-4a-carbinol-

amine (Fig. 26-28), which is converted to 7,8-dihydro-

biopterin (quinoid form) by pterin-4a-carbinolamine

dehydratase. The quinoid is subsequently reduced by the

NAD(P)H-requiring enzyme dihydropteridine reductase

to regenerate the active cofactor. Note that although

dihydrofolate reductase and dihydropteridine reductase

produce the same product, they utilize different tau-

tomers of the substrate.Although this suggests that these

enzymes may be evolutionarily related, the comparison

of their X-ray structures indicates that this is not the

case: Dihydropteridine reductase resembles nicotinamide

1044 Chapter 26. Amino Acid Metabolism

Figure 26-28 The formation, utilization, and regeneration of 5,6,7,8-tetrahydrobiopterin (BH

)

in the phenylalanine hydroxylase reaction.

JWCL281_c26_1019-1087.qxd 6/8/10 9:38 AM Page 1044

coenzyme–requiring flavin-dependent enzymes such as

glutathione reductase and dihydrolipoyl dehydrogenase

(Section 21-2B).

b. Phenylalanine Hydroxylase Is Controlled by

Phosphorylation and by Allosteric Interactions

PAH initiates the detoxification of high concentrations

of phenylalanine as well as the synthesis of the cate-

cholamine hormones and neurotransmitters (Section 26-4B).

It is allosterically activated by its substrate, phenylalanine,

and by phosphorylation at its Ser 16 by the cAMP-dependent

protein kinase A (PKA; Section 18-3Cb). Its second sub-

strate, BH

, allosterically inhibits the enzyme.

c. The NIH Shift

An unexpected aspect of the PAH reaction is that a

atom, which begins on C4 of phenylalanine’s phenyl ring,

ends up on C3 of this ring in tyrosine (Fig. 26-28, right)

rather than being lost to the solvent by replacement with

the OH group. The mechanism postulated to account for

this NIH shift (so called because it was first characterized

by chemists at the National Institutes of Health) involves

the activation of oxygen by the pterin and Fe cofactors to

form the pterin-4a-carbinolamine and a reactive oxyferryl

group [Fe(IV)“O

2⫺

; Fig. 26-30, Steps 1 and 2] that reacts

with the substrate to form an epoxide across the phenyl

ring’s 3,4 bond (Fig. 26-30, Step 3).This is followed by epox-

ide opening to form a carbocation at C3 (Fig.26-30, Step 4).

Migration of a hydride from C4 to C3 forms a more stable

carbocation (an oxonium ion; Fig. 26-30, Step 5). This mi-

gration is followed by ring aromatization to form tyrosine

(Fig. 26-30, Step 6). Tyrosine hydroxylase and tryptophan

hydroxylase (Section 26-4B) are both homologous to

phenylalanine hydroxylase and utilize this same NIH shift

reaction mechanism, although there may not be an epoxide

intermediate in these cases.

Reaction 3 in the phenylalanine degradation pathway

(Fig. 26-26) provides another example of an NIH shift.

This reaction, which is catalyzed by the Fe(II)-containing

p-hydroxyphenylpyruvate dioxygenase, involves the oxida-

tive decarboxylation of an ␣-keto acid as well as ring hy-

droxylation. In this case, the NIH shift involves migration

of an alkyl group rather than of a hydride ion to form a

more stable carbocation (Fig. 26-31). This shift, which has

been demonstrated through isotope-labeling studies (rep-

resented by the different symbols in Figs. 26-26 and 26-31),

accounts for the observation that C3 is bonded to C4 in

p-hydroxyphenylpyruvate but to C5 in homogentisate.

d. Alkaptonuria and Phenylketonuria Result from

Defects in Phenylalanine Degradation

Archibald Garrod realized in the early 1900s that human

genetic diseases result from specific enzyme deficiencies.

We have repeatedly seen how this realization has con-

tributed to the elucidation of metabolic pathways. The first

such disease to be recognized was alkaptonuria, which,Gar-

rod observed, resulted in the excretion of large quantities of

homogentisic acid.This condition results from deficiency of

homogentisate dioxygenase (Fig. 26-26, Reaction 4).Alkap-

tonurics suffer no ill effects other than arthritis later in life

(although their urine darkens alarmingly because of the

rapid air oxidation of the homogentisate they excrete).

Individuals suffering from phenylketonuria (PKU) are

not so fortunate. Severe mental retardation occurs within a

few months of birth if the disease is not detected and treated

immediately (see below). Indeed, ⬃1% of the patients in

mental institutions were, at one time (before routine screen-

ing), phenylketonurics. PKU is caused by the inability to hy-

droxylate phenylalanine (Fig. 26-26, Reaction 1) and there-

fore results in increased blood levels of phenylalanine

(hyperphenylalaninemia). The excess phenylalanine is

transaminated to phenylpyruvate

by an otherwise minor pathway. The “spillover” of phen-

ylpyruvate (a phenylketone) into the urine was the first ob-

servation connected with the disease and gave the disease

its name, although it has since been demonstrated that it is

the high concentration of phenylalanine itself that gives rise

to brain dysfunction. All babies born in the United States

are now screened for PKU immediately after birth by test-

ing for elevated levels of phenylalanine in the blood.

Classic PKU results from a deficiency in phenylalanine

hydroxylase (PAH). When this was established in 1947, it

was the first human inborn error of metabolism whose ba-

sic biochemical defect had been identified. Since then, over

Phenylpyruvate

COO

–

Section 26-3. Metabolic Breakdown of Individual Amino Acids 1045

Figure 26-29 The active site of the Fe(II) form of phenylalanine

hydroxylase (PAH) in complex with 5,6,7,8-tetrahydrobiopterin

(BH

). The Fe(II) (orange sphere) is octahedrally coordinated

(gray lines) by His 285, His 290, and Glu 330 (C green, N blue,

and O red) and three water molecules (red spheres). BH

atom

O4 is hydrogen bonded (black dashed lines) to two of these

water molecules. [Based on an X-ray structure by Edward

Hough, University of Tromsø, Norway. PDBid 1J8U.]

JWCL281_c26_1019-1087.qxd 6/8/10 9:38 AM Page 1045

400 mutations have been identified in PAH. Because all of

the tyrosine breakdown enzymes are normal, treatment

consists in providing the patient with a low-phenylalanine

diet and monitoring the blood level of phenylalanine to en-

sure that it remains within normal limits for the first 5 to 10

years of life (the adverse effects of hyperphenylalaninemia

seem to disappear after that age). PAH deficiency also ac-

counts for another common symptom of PKU: Its victims

have lighter hair and skin color than their siblings. This is

because tyrosine hydroxylation, the first reaction in the for-

mation of the black skin pigment melanin (Section 26-4B),

is inhibited by elevated phenylalanine levels.

Other causes of hyperphenylalaninemia have been discov-

ered since the introduction of infant screening techniques.

These result from deficiencies in the enzymes catalyzing the

formation or regeneration of 5,6,7,8-tetrahydrobiopterin

(BH

), the PAH cofactor (Fig. 26-28). In such cases, patients

must also be supplied with

L-3,4-dihydroxyphenylalanine

(

L-DOPA) and 5-hydroxytryptophan, metabolic precursors

of the neurotransmitters norepinephrine and serotonin, re-

spectively,since tyrosine hydroxylase and tryptophan hydrox-

ylase, the PAH homologs that produce these physiologically

active amines, also require 5,6,7,8-tetrahydrobiopterin (Sec-

tion 26-4B). Unfortunately, simply adding BH

to the diet of

1046 Chapter 26. Amino Acid Metabolism

hydrogen ion

migration

R ⫽

Fe(IV) O

2–

Fe(II)

Pterin-4a-carbinolamine

N H

Fe(II)

5,6,7,8-Tetrahydrobiopterin (BH

)

Fe(II)

Phenylalanine

–

Tyrosine

–

Resonance-stabilized oxonium ion

–

Figure 26-30 Proposed mechanism of the NIH shift in the phenylalanine hydroxylase

reaction. The mechanism involves (1 and 2) activation of oxygen by the enzyme’s BH

and Fe(II) cofactors to yield pterin-4a-carbinolamine and a reactive oxyferryl species

[Fe(IV)“O

2–

]; (3) reaction of the Fe(IV)“O

2–

with the phenylalanine substrate to form

an epoxide across its phenyl ring’s 3,4 bond; (4) epoxide opening to form a carbocation at

C3; (5) migration of a hydride from C4 to C3 to form a more stable carbocation (an

oxonium ion); and (6) ring aromatization to form tyrosine.

JWCL281_c26_1019-1087.qxd 4/20/10 9:26 AM Page 1046

an affected individual is not an effective treatment because

is unstable and cannot cross the blood–brain barrier.

4 AMINO ACIDS AS

BIOSYNTHETIC PRECURSORS

Certain amino acids, in addition to their major function as

protein building blocks, are essential precursors of a variety

of important biomolecules, including nucleotides and nu-

cleotide coenzymes, heme, various hormones and neuro-

transmitters, and glutathione. In this section, we consider

the pathways producing some of these substances. We be-

gin by discussing the biosynthesis of heme from glycine and

succinyl-CoA. We then examine the pathways by which ty-

rosine, tryptophan, glutamate, and histidine are converted

to various neurotransmitters and study certain aspects of

glutathione biosynthesis and the involvement of this tripep-

tide in amino acid transport and other processes. Finally, we

consider the role of folate derivatives in the biosynthetic

transfer of C

units. The biosynthesis of nucleotides and

nucleotide coenzymes is the subject of Chapter 28.

A. Heme Biosynthesis and Degradation

Heme (Fig. 26-32), as we have seen, is an Fe-containing

prosthetic group that is an essential component of many

proteins, notably hemoglobin, myoglobin, and the cy-

tochromes. The initial reactions of heme biosynthesis are

common to the formation of other tetrapyrroles including

Section 26-4. Amino Acids as Biosynthetic Precursors 1047

Figure 26-31 The NIH shift in the p-hydroxyphenylpyruvate

dioxygenase reaction. Carbon atoms are labeled as an aid to

following the group migration constituting the shift.

Figure 26-32 Structure of heme. Heme’s C and N atoms are

derived from those of glycine and acetate.

–

p-Hydroxyphenypyruvate

⌬

•

⌬

–

•

⌬

–

•

⌬

–

•

⌬

–

•

⌬

–

•

Resonance-stabilized

oxonium ion

Homogentisate

alkyl group

migration

COO

–

COO

–

COO

–

COO

–

Glycine

Acetate

␣

␥

␤

␦

Heme

JWCL281_c26_1019-1087.qxd 4/20/10 9:26 AM Page 1047

chlorophyll in plants and bacteria (Section 24-2A) and

coenzyme B

in bacteria (Section 25-2Eb).

a. Porphyrins Are Derived from Succinyl-CoA

and Glycine

Elucidation of the heme biosynthesis pathway involved

some interesting detective work. David Shemin and David

Rittenberg, who were among the first to use isotopic trac-

ers in the elucidation of metabolic pathways, demon-

strated, in 1945, that all of heme’s C and N atoms can be de-

rived from acetate and glycine. Only glycine, out of a variety

N-labeled metabolites they tested (including ammo-

nia, glutamate, leucine, and proline), yielded

N-labeled

heme in the hemoglobin of experimental subjects to whom

these metabolites were administered. Similar experiments,

using acetate labeled with

C in its methyl or carboxyl

groups, or [

␣

]glycine, demonstrated that 24 of heme’s 34

carbon atoms are derived from acetate’s methyl carbon, 2

from acetate’s carboxyl carbon, and 8 from glycine’s C

␣

atom (Fig. 26-32). None of the heme atoms is derived from

glycine’s carboxyl carbon atom.

Figure 26-32 indicates that heme C atoms derived from

acetate methyl groups occur in groups of three linked

atoms. Evidently, acetate is first converted to some other

metabolite that has this labeling pattern. Shemin and Rit-

tenberg postulated that this metabolite is succinyl-CoA

based on the following reasoning (Fig. 26-33):

1. Acetate is metabolized via the citric acid cycle (Sec-

tion 21-1B).

2. Labeling studies indicate that atom C3 of the citric acid

cycle intermediate succinyl-CoA is derived from acetate’s

methyl C atom, whereas atom C4 comes from acetate’s

carboxyl C atom.

3. After many turns of the citric acid cycle, C1 and C2 of

succinyl-CoA likewise become fully derived from acetate’s

methyl C atom.

We shall see that this labeling pattern indeed leads to that

of heme.

In the mitochondria of yeast and animals as well as in

some bacteria, the first phase of heme biosynthesis is a con-

densation of succinyl-CoA with glycine followed by decar-

boxylation to form ␦-aminolevulinic acid (ALA) as catalyzed

by the PLP-dependent enzyme ␦-aminolevulinate synthase

(ALA synthase or ALAS; Fig. 26-34). The carboxyl group

lost in the decarboxylation (Fig. 26-34, Reaction 5) originates

in glycine, which is why heme contains no label from this

group.

b. The Pyrrole Ring Is the Product

of Two ALA Molecules

The pyrrole ring is formed in the next phase of the path-

way through linkage of two molecules of ALA to yield por-

phobilinogen (PBG). The reaction is catalyzed by porpho-

bilinogen synthase [PBGS; alternatively, ␦-aminolevulinic

acid dehydratase (ALAD)], which in yeast and mammals,

is Zn

2⫹

-dependent and involves Schiff base formation of

one of the substrate molecules with an enzyme amine

group (in some bacteria and all plants, Mg

2⫹

substitutes for

2⫹

). One possible mechanism of this condensation–

elimination reaction involves formation of a second Schiff

base between the ALA–enzyme Schiff base and the second

ALA molecule (Fig. 26-35). At this point, if we continue

tracing the acetate and glycine labels through the PBG syn-

thase reaction (Fig. 26-35), we can begin to see how heme’s

labeling pattern arises.

The X-ray structure of human PBGS in covalent com-

plex with its product PBG, determined by Jonathan

Cooper, indicates that this enzyme is a homooctamer with

symmetry. Each of its 330-residue subunits consists of

an ␣/␤ barrel and a 39-residue N-terminal tail that wraps

around a neighboring monomer (related to it by 2-fold

symmetry) so that the protein is better described as a rela-

tively loosely organized tetramer of compact dimers. As is

the case with nearly all ␣/␤ barrel enzymes, PBGS’s active

site (Fig. 26-36a) lies at the mouth of the barrel at the

1048 Chapter 26. Amino Acid Metabolism

Figure 26-33 The origin of the C atoms of succinyl-CoA as

derived from acetate via the citric acid cycle. C atoms labeled

with triangles and squares are derived, respectively, from

acetate’s methyl and carboxyl C atoms. Filled symbols label

atoms derived from acetate in the present round of the citric acid

cycle, whereas open symbols label atoms derived from acetate in

previous rounds of the citric acid cycle. Note that the C1 and C4

atoms of succinyl-CoA are scrambled on forming the 2-fold

symmetric succinate.

CoASH + ATP

AMP

+ PP

COO

–

SCoA

CoASH

Acetate

COO

–

COO

–

CHO

COO

–

SCoA

COO

–

Acetyl-CoA

Citric

acid

cycle

COO

–

COO

–

Citrate

Succinyl-CoA

Succinate

Oxaloacetate

COO

–

COO

–

JWCL281_c26_1019-1087.qxd 4/20/10 9:26 AM Page 1048

C-terminal ends of its ␤ strands. The active site is covered

by a loop that comparison with other PBGS structures indi-

cates forms a flexible lid over the substrate, an arrange-

ment that is reminiscent of the glycolytic enzyme triose

phosphate isomerase (TIM; Fig. 17-11). PBG is covalently

bound to Lys 252 and its free amino group is coordinated to

the active site Zn

2⫹

ion. Lys 199 appears to be properly

positioned to act as a general acid–base catalyst.

Inhibition of PBG synthase by Pb

2⫹

(a competitor of its

active site Zn

2⫹

ion) is one of the major manifestations of

lead poisoning, which is among the most common acquired

environmental diseases. Indeed, it has been suggested that

the accumulation, in the blood, of ALA, which resembles

the neurotransmitter ␥-aminobutyric acid (Section 26-4B),

is responsible for the psychosis that often accompanies

lead poisoning.

Section 26-4. Amino Acids as Biosynthetic Precursors 1049

Figure 26-34 The mechanism of action of the PLP-dependent

enzyme ␦-aminolevulinate synthase (ALAS). The reaction steps

are (1) transimination, (2) PLP-stabilized carbanion formation,

Enz

␦-Aminolevulinate (ALA)

Enz

COO

–

COO

–

COO

–

CoASH

SCoA

COO

–

COO

–

COO

–

SCoA

COO

–

COO

–

COO

–

COO

–

Succinyl-CoA

Enz

PLP

Enz

Glycine

PLP

Enz

(3) C¬C bond formation, (4) CoA elimination, (5) decarboxylation

facilitated by the PLP–Schiff base, and (6) transimination

yielding ALA and regenerating the PLP–enzyme.

JWCL281_c26_1019-1087.qxd 4/20/10 9:26 AM Page 1049

c. PBGS Has Two Quaternary States

with Different Enzymatic Activities

Although the X-ray structure of wild-type human PBGS

indicates that it is a D

-symmetric homooctamer, the X-ray

structure of its rare F12L mutant form, determined by

Eileen Jaffe, is a D

-symmetric homohexamer (Fig. 26-36b).

Moreover, the F12L mutant has far less activity than does

wild-type PBGS, even though residue 12 is far from the en-

zyme’s active site in both proteins.This is apparently due to

a conformational change in PBGS’s N-terminal arm: In the

octamer, the N-terminal arms of two adjacent monomers

wrap around each other’s barrel to form a so-called hug-

ging dimer, whereas in the hexamer, the N-terminal arms

extend away from the core of the protein to form a so-

called detached dimer (Fig. 26-36b). Nevertheless, the ␣/␤

barrels of these two oligomeric forms are closely superim-

posable. The difference in the activities of these two qua-

ternary forms is caused by the binding of an allosteric Mg

2⫹

ion in the octamer’s “hugging” interface that is absent in

the detached dimer.

The alternate oligomeric forms have been observed in

solutions of wild-type PBGS and the equilibrium between

these forms can be varied by changing the pH, the enzyme

concentration, and the substrate concentration, as well as

through mutation. Jaffe has therefore proposed that this

quaternary structural change is an allosteric mechanism for

controlling the activity of the enzyme. In contrast, in the

symmetry and sequential models of allosterism (Sections

10-4B and 10-4C), the quaternary state of the enzyme does

not change during an allosteric transition.

Jaffe has termed enzymes that control their activities by

changing their oligomeric states morpheeins [pronounced

morph-ee¿-in; derived from the verb “to morph” and the

classic pronunciation of the word protein (pro-tee¿-in)].Al-

though PBGS is the first known morpheein, several others

have since been identified (e.g., ribonucleotide reductase;

Section 28-3Ad). In fact, it may be that morpheeins are rel-

atively common because when an enzyme is purified, inac-

tive fractions are usually assumed to be denatured and are

therefore discarded rather than being characterized.

1050 Chapter 26. Amino Acid Metabolism

COO

–

COO

–

ALA

Enz

COO

–

COO

–

Enz

COO

–

ALA

Porphobilinogen

(PBG)

COO

–

Enz

COO

–

COO

–

Enz

COO

–

COO

–

COO

–

COO

–

COO

–

Figure 26-35 A possible mechanism for porphobilinogen synthase. The reaction

involves (1) Schiff base formation, (2) second Schiff base formation, (3) formation

of a carbanion ␣ to a Schiff base, (4) cyclization by an aldol-type condensation,

(5) elimination of the enzyme¬NH

group, and (6) tautomerization.

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