Voet D., Voet Ju.G. Biochemistry

complex deadenylylates glutamine synthetase when P

uridylylated (also at a Tyr residue) and adenylylates gluta-

mine synthetase when P

lacks UMP residues.The level of P

uridylylation, in turn, depends on the relative activities of

two enzymatic activities located on the same protein: a

uridylyltransferase that uridylylates P

and a uridylyl-

removing enzyme that hydrolytically excises the attached

UMP groups of P

(Fig. 26-56).The uridylyltransferase is ac-

tivated by ␣-ketoglutarate and ATP and inhibited by gluta-

mine and P

, whereas uridylyl-removing enzyme is insensi-

tive to these metabolites. This complex metabolic cascade

therefore renders the activity of E. coli glutamine synthetase

extremely responsive to the cell’s nitrogen requirements.

Glutamate Is the Precursor of

Proline, Ornithine, and Arginine

Conversion of glutamate to proline (Fig. 26-57, Reac-

tions 1– 4) involves the reduction of the ␥-carboxyl group to

an aldehyde followed by the formation of an internal Schiff

base whose further reduction yields proline. Reduction of

the glutamate ␥-carboxyl group to an aldehyde is an ender-

gonic process that is facilitated by the carboxyl group’s

prior phosphorylation by ␥-glutamyl kinase. The unstable

product,glutamate-5-phosphate, has not been isolated from

reaction mixtures but is presumed to be the substrate for

the reduction that follows. The resulting glutamate-5-

semialdehyde cyclizes spontaneously to form the internal

Schiff base ⌬

-pyrroline-5-carboxylate. The final reduction

to proline is catalyzed by pyrroline-5-carboxylate reductase.

Whether the enzyme requires NADH or NADPH is unclear.

The E. coli pathway from glutamate to ornithine and

hence to arginine likewise involves the ATP-driven reduc-

tion of the glutamate ␥-carboxyl group to an aldehyde (Fig.

26-57, Reactions 6 and 7). Spontaneous cyclization of this in-

termediate, N-acetylglutamate-5-semialdehyde, is prevented

by prior acetylation of its amino group by N-acetylglutamate

synthase to form N-acetylglutamate (Fig. 26-57, Reaction 5).

N-Acetylglutamate-5-semialdehyde, in turn, is converted to

the corresponding amine by transamination (Fig. 26-57, Re-

action 8). Hydrolysis of the acetyl protecting group finally

yields ornithine, which, as we have seen (Section 26-2),is con-

verted to arginine via the urea cycle. In humans, however, the

pathway to ornithine is more direct.The N-acetylation of glu-

tamate that protects it from cyclization does not occur.

Rather, glutamate-5-semialdehyde, which is in equilibrium

with ⌬

-pyrroline-5-carboxylate, is directly transaminated

to yield ornithine in a reaction catalyzed by ornithine-␦-

aminotransferase (Fig. 26-57, Reaction 10).

e. Serine, Cysteine, and Glycine Are Derived from

3-Phosphoglycerate

Serine is formed from the glycolytic intermediate

3-phosphoglycerate in a three-reaction pathway (Fig. 26-58):

1. Conversion of 3-phosphoglycerate’s 2-OH group to a

ketone yielding 3-phosphohydroxypyruvate, serine’s phos-

phorylated keto acid analog.

2. Transamination of 3-phosphohydroxypyruvate to

phosphoserine.

3. Hydrolysis of phosphoserine to yield serine.

Serine participates in glycine synthesis in two ways (Sec-

tion 26-3B):

1. Direct conversion of serine to glycine by serine hy-

droxymethyl transferase in a reaction that also yields

-methylene-THF (Fig. 26-12, Reaction 4 in reverse).

2. Condensation of the N

-methylene-THF with

and by the glycine cleavage system (Fig. 26-12,

Reaction 3 in reverse).

We have already discussed the synthesis, in animals, of cys-

teine from serine and homocysteine, a breakdown product

of methionine (Section 26-3Ea). Homocysteine combines

with serine to yield cystathionine, which subsequently forms

cysteine and ␣-ketobutyrate (Fig. 26-18, Reactions 5 and 6).

Since cysteine’s sulfhydryl group is derived from the essen-

tial amino acid methionine, cysteine is really an essential

amino acid. In plants and microorganisms, however, cys-

teine is synthesized from serine in a two-step reaction in-

volving the activation of the serine ¬OH by converting it to

O-acetylserine followed by the displacement of acetate by

sulfide (Fig. 26-59a). The sulfide required is produced from

sulfate in an 8-electron reduction that occurs in E. coli as

shown in Fig. 26-59b. Sulfate is first activated by the

⫹

Section 26-5. Amino Acid Biosynthesis 1071

Figure 26-58 The conversion of 3-phosphoglycerate to serine.

The pathway enzymes are (1) 3-phosphoglycerate

dehydrogenase, (2) a PLP-dependent aminotransferase,

and (3) phosphoserine phosphatase.

COO

–

OHCH

OPO

–

3-Phosphoglycerate

NADH

NAD

COO

–

OPO

–

3-Phosphohydroxypyruvate

Glutamate

COO

–

OPO

–

3-Phosphoserine

COO

–

Serine

α-Ketoglutarate

JWCL281_c26_1019-1087.qxd 7/21/10 6:26 PM Page 1071

enzymes ATP sulfurylase (which is used in the pyrosequenc-

ing of DNA; Section 7-2Ca) and adenosine-5ⴕ-phosphosulfate

(APS) kinase. The activated sulfate is then reduced to sulfite

by 3ⴕ-phosphoadenosine-5ⴕ-phosphosulfate (PAPS) reduc-

tase and to sulfide by sulfite reductase.

B. Biosynthesis of the Essential Amino Acids

Essential amino acids, like nonessential amino acids, are syn-

thesized from familiar metabolic precursors. Their synthetic

pathways are present only in microorganisms and plants,

however, and usually involve more steps than those of the

nonessential amino acids. For example, lysine, methionine,

and threonine are all synthesized from aspartate in pathways

whose common first reaction is catalyzed by aspartokinase,

an enzyme that is present only in plants and microorganisms.

Similarly, valine and leucine are formed from pyruvate;

isoleucine is formed from pyruvate and ␣-ketobutyrate; and

tryptophan, phenylalanine, and tyrosine are formed from

phosphoenolpyruvate and erythrose-4-phosphate. The en-

zymes that synthesize essential amino acids were apparently

lost early in animal evolution, possibly because of the ready

availability of these amino acids in the diet.

Time and space prevent a detailed discussion of the many

interesting reactions that occur in these pathways.The biosyn-

thetic pathways of the aspartate family of amino acids, the

pyruvate family, the aromatic family, and histidine are pre-

sented in Figs. 26-60 through 26-63 and 26-65 together with

lists of the enzymes involved. Several agriculturally useful

1072 Chapter 26. Amino Acid Metabolism

Figure 26-59 Cysteine biosynthesis. (a) The synthesis of

cysteine from serine in plants and microorganisms. (b) The

8-electron reduction of sulfate to sulfide in E. coli.

Serine

serine acetyltransferase

COO

–

COO

–

SCoA

CoASH

Cysteine

COO

–

O-Acetylserine

O-acetylserine (thiol) lyase

COO

–

OPO

(a) (b)

ATP sulfurylase

+ATP SO

–

ATP

APS kinase

ADP

NADPH

3⬘-Phospho-AMP

+ NADP

3NADP

PAPS reductase

sulfite reductase

Sulfite

3ⴕ-Phosphoadenosine-5ⴕ-phosphosulfate (PAPS)

Adenosine-5ⴕ-phosphosulfate (APS)

3NADPH

–

OPO

–

Figure 26-60 (Opposite) The biosynthesis of the “aspartate

family” of amino acids: lysine, methionine, and threonine. The

pathway enzymes are (1) aspartokinase, (2) ␤-aspartate

semialdehyde dehydrogenase, (3) homoserine dehydrogenase,

(4) homoserine kinase, (5) threonine synthase (a PLP enzyme),

(6) homoserine acyltransferase, (7) cystathionine ␥-synthase,

(8) cystathionine ␤-lyase, (9) methionine synthase (alternatively

homocysteine methyltransferase, which also occurs in mammals;

Section 26-3Ec), (10) dihydrodipicolinate synthase,

(11) dihydrodipicolinate reductase, (12) N-succinyl-2-amino-6-

ketopimelate synthase, (13) succinyl-diaminopimelate

aminotransferase (a PLP enzyme), (14) succinyl-diaminopimelate

desuccinylase, (15) diaminopimelate epimerase, and

(16) diaminopimelate decarboxylase.

JWCL281_c26_1019-1087.qxd 6/8/10 9:38 AM Page 1072

Section 26-5. Amino Acid Biosynthesis 1073

COO

–

ADP

ATP

CoASH

COO

–

–2

NADP

+ P

Glutamate

α-Ketoglutarate

Aspartate

Aspartyl-

-phosphate

COO

–

-Aspartate-semialdehyde

Pyruvate

spontaneous

NADP

NADPH

COO

–

Homoserine

ADP

ATP

COO

–

Phosphohomoserine

–

COO

–

Threonine

Succinyl-CoA

COO

–

Succ

O-Succinylhomoserine

Succinate

Cysteine

COO

–

COO

–

Cystathionine

Pyruvate

+ NH

COO

–

Homocysteine

THF

COO

–

Methionine

COO

––

OOC

Dihydrodipicolinate

4-Hydroxy-tetrahydrodipicolinate

NADPH

NADP

COO

––

OOC

Tetrahydrodipicolinate

COO

–

OOC

COO

––

OOC

N-Succinyl-2-amino-

6-keto-

L-pimelate

CoASH

Succinyl

-CoA + H

Succ

COO

––

OOC

N-Succinyl-L,L-

-diamino-

pimelate

Succ

Succinate

)

C H

COO

–

(

COO

–

L,L

-α

-Diamino-

pimelate

)

COO

–

(

COO

–

(CH

)

COO

–

meso-

-Diamino-

pimelate

Lysine

-Methyl-THF

JWCL281_c26_1019-1087.qxd 4/20/10 9:26 AM Page 1073

1074 Chapter 26. Amino Acid Metabolism

NAD(P)H

NAD(P)

Glutamate

α-Ketoglutarate

COO

–

Threonine

α-Ketobutyrate

COO

–

COO

–

Pyruvate

COO

–

Pyruvate

TPP

–

TPP

COO

–

COO

–

α-Acetolactate

COO

–

α,β-Dihydroxy-

isovalerate

COO

–

α-Ketoisovalerate

COO

–

Valine

COO

–

α-Isopropylmalate

COO

–

COO

–

β-Isopropylmalate

COO

–

α-Ketoisocaproate

COO

–

Leucine

COO

–

COO

–

α-Aceto-α-

hydroxybutyrate

COO

–

COO

–

α,β-Dihydroxy-

β-methylvalerate

α-Keto-β-

methylvalerate

COO

–

Isoleucine

COO

–

Acetyl-CoA

CoA

NAD

NADH + CO

NAD(P)H

NAD(P)

α-Ketoglutarate

Glutamate

α-Ketoglutarate

JWCL281_c26_1019-1087.qxd 4/20/10 9:26 AM Page 1074

herbicides are specific inhibitors of some of these enzymes.

Such herbicides have little toxicity toward animals and hence

pose minimal risk to human health and the environment.

a. The Aspartate Family: Lysine, Methionine,

and Threonine

In bacteria, aspartate is the common precursor of lysine,

methionine, and threonine (Fig. 26-60).The biosyntheses of

these essential amino acids all begin with the aspartoki-

nase-catalyzed phosphorylation of aspartate to yield

aspartyl-␤-phosphate. We have seen that the control of

metabolic pathways commonly occurs at the first commit-

ted step of the pathway. One might therefore expect lysine,

methionine, and threonine biosynthesis to be controlled as

a group. Each of these pathways is, in fact, independently

controlled. E. coli does so via three isozymes of aspartoki-

nase that respond differently to the three amino acids in

terms both of feedback inhibition of enzyme activity and

repression of enzyme synthesis. Table 26-3 summarizes this

differential control. In addition, the pathway direction is

controlled by feedback inhibition at the branch points by

the individual amino acids. Thus methionine inhibits the

O-acylation of homoserine (Fig. 26-60, Reaction 6), and

lysine inhibits dihydrodipicolinate synthase (Fig. 26-60,

Reaction 10).

The Pyruvate Family: Leucine, Isoleucine,

and Valine

Valine and isoleucine are both synthesized via the same

five-step pathway (Fig. 26-61), the only difference being in

the first step of the series. In this TPP-dependent reaction,

which resembles those catalyzed by pyruvate decarboxy-

lase (Section 17-3Ba) and transketolase (Section 23-4Ca),

pyruvate forms an adduct with TPP, which is decarboxy-

lated to hydroxyethyl-TPP. This resonance-stabilized car-

banion adds either to the keto group of a second pyruvate

to form acetolactate on the way to valine, or to the keto

group of threonine-derived ␣-ketobutyrate to form

␣-aceto-␣-hydroxybutyrate on the way to isoleucine. The

leucine biosynthetic pathway branches off from the valine

pathway at ␣-ketoisovalerate (Fig. 26-61, Reaction 6). Re-

actions 6 to 8 in Fig. 26-61 are reminiscent of the first three

reactions of the citric acid cycle (Sections 21-3A–C). Here,

acetyl-CoA condenses with ␣-ketoisovalerate to form

␣-isopropylmalate, which then undergoes a dehydration/

hydration reaction, followed by oxidative decarboxylation

and transamination, to yield leucine.

c. The Aromatic Amino Acids: Phenylalanine,

Tyrosine, and Tryptophan

The precursors to the aromatic amino acids are the gly-

colytic intermediate phosphoenolpyruvate (PEP) and

erythrose-4-phosphate (an intermediate in the pentose

phosphate pathway; Section 23-4Cb). Their condensation

forms 2-keto-3-deoxy-

D-arabinoheptulosonate-7-phosphate,

a C

compound that cyclizes and is ultimately converted to

chorismate (Fig. 26-62), the branch point for tryptophan

biosynthesis. Chorismate is converted either to anthrani-

late and then on to tryptophan, or to prephenate and on to

either tyrosine or phenylalanine (Fig. 26-63). Although

mammals synthesize tyrosine by the hydroxylation of

phenylalanine (Section 26-3Ha), many microorganisms

synthesize it directly from prephenate.

Since the synthesis of aromatic amino acids only occurs

in plants and microorganisms, this pathway is a natural tar-

get for herbicides that will not be toxic to animals. For ex-

ample, glyphosate,

the active ingredient in one of the most widely used weed

killers, Roundup, is a competitive inhibitor with respect to

PEP in the 5-enolpyruvylshikimate-3-phosphate (EPSP)

synthase reaction (Reaction 6 of Fig. 26-62).

d. A Protein Tunnel Channels the Intermediate

Product of Tryptophan Synthase between

Two Active Sites

The final two reactions in tryptophan biosynthesis, Re-

actions 5 and 6 in Fig. 26-63, are both catalyzed by trypto-

phan synthase:

1. The ␣ subunit (268 residues) of this ␣

␤

bifunctional

enzyme cleaves indole-3-glycerol phosphate, yielding in-

dole and glyceraldehyde-3-phosphate (Reaction 5).

2. The ␤ subunit (396 residues) joins indole with

L-serine in a PLP-dependent reaction to form L-tryptophan

(Reaction 6).

Either subunit alone is enzymatically active, but when they

are joined in the ␣

␤

tetramer, the rates of both reactions

Glyphosate

⫺2

P ¬ CH

¬ NH ¬ CH

¬ COO

⫺

Section 26-5. Amino Acid Biosynthesis 1075

Table 26-3 Differential Control of Aspartokinase

Isoenzymes in E. Coli

Feedback

Enzyme Inhibitor Corepressor(s)

Aspartokinase I Threonine Threonine and isoleucine

Aspartokinase II None Methionine

Aspartokinase III Lysine Lysine

Compounds whose presence results in the transcriptional repression of

enzyme synthesis (Section 31-3G).

Figure 26-61 (Opposite) The biosynthesis of the “pyruvate

family” of amino acids: isoleucine, leucine, and valine. The

pathway enzymes are (1) acetolactate synthase (a TPP enzyme),

(2) acetolactate mutase, (3) reductase, (4) dihydroxy acid

dehydratase, (5) valine aminotransferase (a PLP enzyme),

(6) ␣-isopropylmalate synthase, (7) ␣-isopropylmalate

dehydratase, (8) isopropylmalate dehydrogenase, (9) leucine

aminotransferase (a PLP enzyme), and (10) threonine deaminase

(serine dehydratase, a PLP enzyme).

JWCL281_c26_1019-1087.qxd 6/8/10 9:38 AM Page 1075

and their substrate affinities are increased by 1 to 2 orders of

magnitude. Indole, the intermediate product, does not ap-

pear free in solution; the enzyme apparently sequesters it.

The X-ray structure of tryptophan synthase from Sal-

monella typhimurium, determined by Craig Hyde, Edith

Miles, and David Davies, explains the latter observation.

The protein forms a 150-Å-long, 2-fold symmetric

␣–␤–␤–␣ complex (Fig. 26-64) in which the active sites of

neighboring ␣ and ␤ subunits are separated by ⬃25 Å.

These active sites are joined by a solvent-filled tunnel that is

wide enough to permit the passage of the intermediate sub-

strate, indole. This structure, the first in which the presence

of a tunnel between active sites was observed, suggests

the following series of events. The indole-3-glycerol phos-

phate substrate binds to the ␣ subunit through an opening

into its active site, its “front door,” and the glyceraldehyde-

3-phosphate product leaves via the same route. Similarly,

the ␤ subunit active site has a “front door” opening to the

solvent through which serine enters and tryptophan leaves.

Both active sites also have “back doors” that are connected

1076 Chapter 26. Amino Acid Metabolism

OPO

–

COO

–

COO

–

Phosphoenol-

pyruvate (PEP)

Erythrose-4-

phosphate

2-Keto-3-deoxyarabino-

heptulosonate-7-

phosphate (DAHP)

+ NAD

NAD

COO

–

5-Dehydroquinate

COO

–

5-Dehydroshikimate

NADHNAD

COO

–

COO

–

COO

–

COO

–

Shikimate

ATPADP

Shikimate-5-

phosphate

2–

COO

–

PEP

5-Enolpyruvylshikimate-

3-phosphate

2–

COO

–

Chorismate

COO

–

Figure 26-62 The biosynthesis of chorismate, the aromatic

amino acid precursor. The pathway enzymes are (1) 2-keto-3-

deoxy-

D-arabinoheptulosonate-7-phosphate synthase,

(2) dehydroquinate synthase (an NAD

⫹

-requiring reaction that

yields an unchanged NAD

⫹

product and is thereby indicative of

an oxidized intermediate as similarly occurs in the

UDP–galactose-4-epimerase reaction; Section 17-5B),

(3) 5-dehydroquinate dehydratase, (4) shikimate dehydrogenase,

(5) shikimate kinase, (6) 5-enolpyruvylshikimate-3-phosphate

synthase, and (7) chorismate synthase.

JWCL281_c26_1019-1087.qxd 4/20/10 9:26 AM Page 1076

by the tunnel.The indole intermediate presumably diffuses

between the two active sites via the tunnel and hence does

not escape to the solvent.

Allosteric interactions between the subunits to control

the activity of the ␣ subunit also serve to ensure that indole

is only released when the ␤ subunit is ready to accept it.

Section 26-5. Amino Acid Biosynthesis 1077

Serine

COO

–

Chorismate

COO

–

COO

–

Anthranilate

HO OH

–

OOC

2–

-(5ⴕ-Phosphoribosyl)-

anthranilate

COO

–

OPO

–

Enol-1- -carboxyphenylamino-

1-deoxyribulose phosphate

C C

OPO

–

Indole-3-glycerol

phosphate

Indole

Tryptophan

COO

–

OOC

Prephenate

COO

–

+ CO

NAD

4-Hydroxyphenyl-

pyruvate

COO

–

Phenylpyruvate

COO

–

Tyrosine

COO

–

Phenylalanine

COO

–

Glutamate

α-Ketoglutarate

Glutamate

α-Ketoglutarate

+ NADH

Glutamine

Pyruvate + Glutamate

5-Phosphoribosyl-

α-pyrophosphate (PRPP)

Glyceraldehyde-

3-phosphate

810

911

Figure 26-63 The biosynthesis of phenylalanine, tryptophan,

and tyrosine from chorismate. The pathway enzymes are

(1) anthranilate synthase, (2) anthranilate

phosphoribosyltransferase, (3) N-(5¿-phosphoribosyl)

anthranilate isomerase, (4) indole-3-glycerol phosphate synthase,

(5) tryptophan synthase, ␣ subunit, (6) tryptophan synthase,

␤ subunit, (7) chorismate mutase, (8) prephenate dehydrogenase,

(9) aminotransferase, (10) prephenate dehydratase, and

(11) aminotransferase.

JWCL281_c26_1019-1087.qxd 6/8/10 9:38 AM Page 1077

Michael Dunn has shown that the elimination of water

from the serine–PLP Schiff base on the ␤ subunit to form

an aminoacrylate–PLP Schiff base intermediate

triggers a conformational change that activates the ␣ sub-

unit to produce indole. The diffusion of the indole to the ␤

2–

minoacrylate–PLP Schiff base

–

COO

–

subunit to react with this intermediate then results in the

formation of tryptophan.

Channeling may be particularly important for indole

since this nonpolar molecule otherwise can escape the bac-

terial cell by diffusing through its plasma and outer mem-

branes. We have seen similar phenomena in reactions

involving glutamine amidotransferases (Sections 26-2Aa

and 26-5Aa), as well as in the series of reactions catalyzed

by fatty acid synthase, in which the growing product is kept

in the vicinity of the multifunctional enzyme’s active site by

covalent attachment to the enzyme’s flexible phosphopan-

tetheine arm (Section 25-4Ca). Channeling is also implicated

in the multistep biosyntheses of purines and pyrimidines

(Sections 28-1A and 28-2A).

e. Histidine Biosynthesis

Five of histidine’s six C atoms are derived from 5-phos-

phoribosyl-␣-pyrophosphate (PRPP; Fig. 26-65), an inter-

mediate also involved in the biosynthesis of tryptophan

(Fig. 26-63, Reaction 2), purine nucleotides (Section 28-1A),

and pyrimidine nucleotides (Section 28-2A). The histi-

dine’s sixth carbon originates from ATP. The ATP atoms

that are not incorporated into histidine are eliminated as

5-aminoimidazole-4-carboxamide ribonucleotide (AICAR;

Fig. 26-65, Reaction 5), which is also an intermediate in

purine biosynthesis (Section 28-1A).

The unusual biosynthesis of histidine from a purine has

been cited as evidence supporting the hypothesis that life

was originally RNA based (Section 1-5Ca). His residues, as

we have seen, are often components of enzyme active sites,

where they act as nucleophiles and/or general acid–base

catalysts. The discovery that RNA can have catalytic prop-

erties (Section 31-4Ae) therefore suggests that the imida-

zole moiety of purines plays a similar role in these RNA

enzymes (ribozymes). This further suggests that the histi-

dine biosynthesis pathway is a “fossil” of the transition to

more efficient protein-based life-forms.

6 NITROGEN FIXATION

The most prominent chemical elements in living systems

are O, H, C, N, and P. The elements O, H, and P occur widely

in metabolically available forms (e.g., H

O, O

, and P

However, the major available forms of C and N, CO

and

, are extremely stable (unreactive); for example, the

N‚N triple bond has a bond energy of 945 kJ ⴢ mol

⫺1

1078 Chapter 26. Amino Acid Metabolism

Figure 26-64 A ribbon diagram of the bifunctional enzyme

tryptophan synthase from S. typhimurium. Only one ␣␤

protomer of this 2-fold symmetric ␣␤␤␣ heterotetramer is

shown.The ␣ subunit is blue, the ␤ subunit’s N-terminal domain

is orange, its C-terminal domain is red-orange, and all ␤ sheets

are tan.The active site of the ␣ subunit is located by its bound

competitive inhibitor, indolepropanol phosphate (IPP; red

ball-and-stick model), whereas that of the ␤ subunit is marked by

its PLP coenzyme (yellow ball-and-stick model). The

solvent-accessible surface of the ⬃25-Å-long “tunnel” connecting

the active sites of the ␣ and ␤ subunits is outlined by a yellow dot

surface. Several indole molecules (green ball-and-stick models)

have been modeled into the tunnel in head to tail fashion,

thereby demonstrating that the tunnel has sufficient width to

permit the indole product of the ␣ subunit to pass through the

tunnel to the ␤ subunit’s active site. [Courtesy of Craig Hyde,

Edith Miles, and David Davies, National Institutes of Health.]

See Interactive Exercise 25

Figure 26-65 (Opposite) The biosynthesis of histidine. The

pathway enzymes are (1) ATP phosphoribosyltransferase,

(2) pyrophosphohydrolase, (3) phosphoribosyl–AMP

cyclohydrolase, (4) phosphoribosylformimino-5-aminoimidazole

carboxamide ribonucleotide isomerase, (5) imidazole glycerol

phosphate synthase (a glutamine amidotransferase),

(6) imidazole glycerol phosphate dehydratase, (7)

L-histidinol

phosphate aminotransferase, (8) histidinol phosphate

phosphatase, and (9) histidinol dehydrogenase.

JWCL281_c26_1019-1087.qxd 10/19/10 9:46 AM Page 1078

Section 26-6. Nitrogen Fixation 1079

H O

HO OH

2–

5-Phosphoribosyl-

α-pyrophosphate (PRPP)

Ribose

P P P

ATP

Ribose

P P P

H H

OH OH

-5ⴕ-Phosphoribosyl-ATP

Ribose

H H

OH OH

-5ⴕ-Phosphoribosyl-AMP

Ribose

H H

HO OH

-5ⴕ-Phosphoribosylformimino-

5-aminoimidazole-4-

carboxamide ribonucleotide

Ribose

-5ⴕ-Phosphoribulosylformimino-

5-aminoimidazole-4-

carboxamide ribonucleotide

OPO

–

OPO

–

OPO

–

OPO

–

Ribose

Imidazole glycerol

phosphate

5-Aminoimidazole-

4-carboxamide

ribonucleotide (AICAR)

Glutamine

Glutamate

To purine biosynthesis

Imidazole acetol

phosphate

L-Histidinol

phosphate

Glutamate α-Ketoglutarate

L-Histidinol Histidine

2NADH2NAD

789

COO

–

C C

JWCL281_c26_1019-1087.qxd 4/20/10 9:26 AM Page 1079

2. The MoFe-protein, an ␣

␤

heterotetramer that con-

tains Fe and Mo.

The X-ray structure of Azotobacter vinelandii nitrogenase

in complex with the inhibitor ADP ⴢ AIF

⫺

(which mimics

the transition state in ATP hydrolysis), determined by

Douglas Rees, reveals that each MoFe-protein associates

with two molecules of Fe-protein (Fig. 26-67).

Each Fe-protein dimer’s single [4Fe–4S] cluster is lo-

cated in a solvent-exposed cleft between the two subunits

1080 Chapter 26. Amino Acid Metabolism

Figure 26-66 Photograph showing the root nodules of the

legume bird’s-foot trefoil. [Vu/Cabisco/Visuals Unlimited.]

Figure 26-67 X-ray structure of the A. vinelandii nitrogenase

in complex with ADP ⴢ AIF

ⴚ

. The enzyme, which is viewed along

its molecular 2-fold axis, is an (␣␤␥

)

heterooctamer in which

the ␤–␣–␣–␤ assembly, the MoFe-protein, is flanked by two ␥

Fe-proteins whose 289-residue subunits are related by local

2-fold symmetry. The homologous ␣ subunits (cyan and magenta;

491 residues) and ␤ subunits (light red and light blue; 522

residues) are related by pseudo-2-fold symmetry. The two ␥

subunits forming each Fe-protein (pink and green with their

Switch I and Switch II segments red and blue) bind to the

MoFe-protein with the 2-fold axis relating them coincident with

the pseudo-2-fold axis relating the MoFe-protein’s ␣ and ␤

subunits.The ADP ⴢ AIF

⫺

, [4Fe–4S] cluster, FeMo-cofactor, and

P-cluster are drawn in space-filling form with C green, N blue, O

red, S yellow, Fe orange, Mo pink, and the AlF

⫺

ion purple.

[Based on an X-ray structure by Douglas Rees, California

Institute of Technology. PDBid 1N2C.]

See Interactive

Exercise 26

(versus 351 kJ ⴢ mol

⫺1

for a C¬O single bond). CO

, with

only minor exceptions, is metabolized (fixed) only by pho-

tosynthetic organisms (Chapter 24). N

fixation is even less

common; this element is converted to metabolically useful

forms by only a few strains of bacteria, named diazatrophs.

Diazatrophs of the genus Rhizobium live in symbiotic

relationship with root nodule cells of legumes (plants be-

longing to the pea family, including beans, clover, and al-

falfa; Fig. 26-66) where they convert N

to NH

The NH

thus formed can be incorporated either into glu-

tamate by glutamate dehydrogenase (Section 26-1B) or

into glutamine by glutamine synthetase (Section 26-5Ab).

This nitrogen-fixing system produces more metabolically

useful nitrogen than the legume needs; the excess is ex-

creted into the soil, enriching it. It is therefore common

agricultural practice to plant a field with alfalfa every few

years to build up the supply of usable nitrogen in the soil

for later use in growing other crops.

a. Nitrogenase Contains Novel Redox Centers

Nitrogenase, which catalyzes the reduction of N

, is a complex of two proteins:

1. The Fe-protein, a homodimer that contains one

[4Fe–4S] cluster and two ATP binding sites.

2NH

⫹ H

⫹ 16ADP ⫹ 16P

⫹ 8H

⫹

⫹ 16ATP ⫹ 16H

O ⫹ 8e

⫺

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Voet D., Voet Ju.G. Biochemistry

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