Voet D., Voet Ju.G. Biochemistry

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peroxidase reactions, helping to destroy peroxides generated

by oxidases; it is involved in leukotriene biosynthesis (Sec-

tion 25-7Cb); and the balance between its reduced (GSH)

and oxidized (GSSG) forms maintains the sulfhydryl groups

of intracellular proteins in their correct oxidation states.

The ␥-glutamyl cycle, which was elucidated by Alton

Meister, provides a vehicle for the energy-driven transport

of amino acids into cells through the synthesis and break-

down of GSH (Fig. 26-46). GSH is synthesized from gluta-

mate, cysteine, and glycine by the consecutive action of ␥-

glutamylcysteine synthetase and GSH synthetase (Fig.

26-46, Reactions 1 and 2).ATP hydrolysis provides the free

energy for each reaction. The carboxyl group is activated

for peptide bond synthesis by formation of an acyl phos-

phate intermediate:

The breakdown of GSH is catalyzed by ␥-glutamyl

transpeptidase, ␥-glutamyl cyclotransferase, 5-oxoprolinase,

and an intracellullar protease (Fig. 26-46, Reactions 3–6).

Amino acid transport occurs because, whereas GSH is

synthesized intracellularly and is located largely within the

cell,␥-glutamyl transpeptidase, which catalyzes GSH break-

down (Fig. 26-46, Reaction 3), is situated on the cell mem-

brane’s external surface and accepts amino acids, notably

cysteine and methionine. GSH is first transported to the

ATP

–

R⬘

ADP

OPO

2–

Section 26-4. Amino Acids as Biosynthetic Precursors 1061

Figure 26-45 Some reactions involving glutathione. The

reactions and enzymes are (1) peroxide detoxification by

glutathione peroxidase, (2) regeneration of GSH from GSSG by

glutathione reductase (Section 21-2Ba), (3) thiol transferase

modulation of protein thiol–disulfide balance, and

(4) leukotriene biosynthesis by a glutathione-S-transferase.

Figure 26-46 Glutathione synthesis as part of the ␥-glutamyl

cycle of glutathione metabolism. The cycle’s reactions are

catalyzed by (1) ␥-glutamylcysteine synthetase, (2) glutathione

Leukotrienes

2GSH

NADP

NADPH

ROOR

Protein

2ROH

GSSG

Protein

NCH

COO

–

COO

–

COO

–

C N

Glutathione

Cys-Gly

Glycine

NCH

Amino acid

(internal)

COO

–

AAC

C COO

–

OOC CH

Cysteine

5-Oxoproline

NCH

COO

–

COO

–

␥-Glu-Cys

NCH

COO

–

COO

–

ADP

+ P

ATP

ADP

+ P

ADP + P

ATP

Glutamate

Amino acid

(external)

synthetase, (3) ␥-glutamyl transpeptidase, (4) ␥-glutamyl

cyclotransferase, (5) 5-oxoprolinase, and (6) an intracellular

protease.

JWCL281_c26_1019-1087.qxd 4/20/10 9:26 AM Page 1061

external surface of the cell membrane, where the transfer of

the ␥-glutamyl group from GSH to an external amino acid

occurs. The ␥-glutamyl amino acid is then transported back

into the cell and converted to glutamate by a two-step

process in which the transported amino acid is released and

5-oxoproline is formed as an intermediate. The last step in

the cycle, the hydrolysis of 5-oxoproline, requires ATP hy-

drolysis.This surprising observation (amide bond hydrolysis

is almost always an exergonic process) is a consequence of

5-oxoproline’s unusually stable internal amide bond.

D. Tetrahydrofolate Cofactors: The Metabolism

of C

Units

Many biosynthetic processes involve the addition of a C

unit to a metabolic precursor. A familiar example is car-

boxylation. For instance, gluconeogenesis from pyruvate

begins with the addition of a carboxyl group to form ox-

aloacetate (Section 23-1Aa). The coenzyme involved in

this and most other carboxylation reactions is biotin (Sec-

tion 23-1Ab). In contrast, S-adenosylmethionine functions

as a methylating agent (Section 26-3Ea).

Tetrahydrofolate (THF) is more versatile than the above

cofactors in that it functions to transfer C

units in several

oxidation states. THF is a 6-methylpterin derivative linked

in sequence to p-aminobenzoic acid and Glu residues

(Fig. 26-47). Up to five additional Glu residues may be

linked to the first glutamate via isopeptide bonds to form a

polyglutamyl tail.

THF is derived from folic acid (Latin:folium, leaf), a dou-

bly oxidized form of THF that must be enzymatically re-

duced before it becomes an active coenzyme (Fig. 26-48).

Both reductions are catalyzed by dihydrofolate reductase

(DHFR). Mammals cannot synthesize folic acid and so must

obtain it from their diets or from intestinal microorganisms.

units are covalently attached to THF at its positions

N5, N10, or both N5 and N10.These C

units, which may be

at the oxidation levels of formate, formaldehyde, or

methanol (Table 26-1), are all interconvertible by enzy-

matic redox reactions (Fig. 26-49).

The main entry of C

units into the THF pool is as

-methylene-THF through the conversion of serine

to glycine by serine hydroxymethyltransferase (Sections

26-3Bb and 26-5Ae) and the cleavage of glycine by glycine

synthase (the glycine cleavage system; Section 26-3Ba, Fig.

26-14). Histidine also contributes C

units through its

degradation with the formation of N

-formimino-THF

(Fig. 26-17, Reaction 11).

A C

unit in the THF pool can have several fates

(Fig. 26-50):

1. It may be used directly as N

-methylene-THF in

the conversion of the deoxynucleotide dUMP to dTMP by

thymidylate synthase (Section 28-3Bb).

2. It may be reduced to N

-methyl-THF for the synthe-

sis of methionine from homocysteine (Section 26-3Ea).

3. It may be oxidized through N

-methenyl-THF to

-formyl-THF for use in the synthesis of purines (Section

1062 Chapter 26. Amino Acid Metabolism

Figure 26-47 Tetrahydrofolate (THF).

Figure 26-48 The two-stage reduction of folate to THF. Both reactions are catalyzed by

dihydrofolate reductase (DHFR).

CCH

C OH

冣

冢

COO

–

2-Amino-4-oxo-

6-methylpterin

p-Aminobenzoic

acid

Glutamates

(n ⴝ 1ⴚ6)

Tetrahydropteroic acid

Tetrahydropteroylglutamic acid (tetrahydrofolate; THF)

NADPH

NADP

NADPH

NADP

Folate

7,8-Dihydrofolate

(DHF)

Tetrahydrofolate

(THF)

JWCL281_c26_1019-1087.qxd 6/8/10 9:38 AM Page 1062

28-1A). Since the purine ring of ATP is involved in histidine

biosynthesis in microorganisms and plants (Section 26-5Be),

-formyl-THF is indirectly involved in this pathway as

well.Prokaryotes use N

-formyl-THF in a formylation reac-

tion yielding formylmethionyl-tRNA, which they require for

the initiation of protein synthesis (Section 32-3Ca).

Section 26-4. Amino Acids as Biosynthetic Precursors 1063

Table 26-1 Oxidation Levels of C

Groups Carried by THF

Oxidation Level Group Carried THF Derivative(s)

Methanol Methyl (¬CH

) N

-Methyl-THF

Formaldehyde Methylene (¬CH

¬) N

-Methylene-THF

Formate Formyl (¬CH“O) N

-Formyl-THF, N

-formyl-THF

Formimino (¬CH“NH) N

-Formimino-THF

Methenyl (¬CH“) N

-Methenyl-THF

Figure 26-49 Interconversion of the C

units carried by THF.

-Methyl-THF

NADP

NADPH

-Methylene-THF

NADP

NADPH

, N

-Methenyl-THF

-Formyl-THF

-Formimino-THF

HHN

ADP +

ATP

ADP

-formimino-THF

cyclodeaminase

Histidine

Glutamate

THF

, N

-methenyl-THF

reductase

ADP +

ATP+

HCO

–

Formate

THF

-formyl-THF

synthetase

Homocysteine Methionine

methionine synthase

, N

-methenyl-THF

cyclohydrolase

-methylene-THF

reductase

serine

hydroxymethyl-

transferase

glycine

cleavage

system

+CO

+ NADH

COOCH

NAD

Glycine

–

Glycine

THF

COOC

–

Serine

O O

-formyl-THF

isomerase

JWCL281_c26_1019-1087.qxd 6/8/10 9:38 AM Page 1063

A deficiency in folic acid results in megloblastic anemia, a

condition in which erythrocytes in the blood are replaced by

fewer abnormally large erythrocytes known as macrocytes.

This occurs as a consequence of the lack of N

-methyl-

ene-THF,which is required for the synthesis of the DNA pre-

cursor deoxythymidine monophosphate from deoxyuridine

monophosphate (Section 28-3Bb). Pernicious anemia (vita-

min B

deficiency; Section 25-2Ee) has similar anemic

symptoms. This is because the irreversibility of the reaction

forming N

-methyl-THF and the now inactive coenzyme

–dependent methionine synthase (top of Fig. 26-49), the

only mammalian enzyme that utilizes N

-methyl-THF, result

in folates becoming trapped in their N

-methyl-THF form.

Consequently, the anemic symptoms of pernicious anemia

may be alleviated by the administration of folic acid but this

does not relieve its neurological symptoms.

Sulfonamides (sulfa drugs) such as sulfanilamide are an-

tibiotics that are structural analogs of the p-aminobenzoic

acid constituent of THF:

They competitively inhibit bacterial synthesis of THF at

the p-aminobenzoic acid incorporation step, thereby block-

ing the above THF-requiring reactions. The inability of

mammals to synthesize folic acid leaves them unaffected

by sulfonamides, which accounts for the medical utility of

these widely used antibacterial agents.

5 AMINO ACID BIOSYNTHESIS

Many amino acids are synthesized by pathways that are

present only in plants and microorganisms. Since mammals

must obtain these amino acids in their diets, these sub-

stances are known as essential amino acids. The other

amino acids, which can be synthesized by mammals from

common intermediates, are termed nonessential amino

Sulfonamides

(R = H, sulfanilamide)

-Aminobenzoic acid

acids. Their ␣-keto acid carbon skeletons are converted to

amino acids by transamination reactions (Section 26-1A)

utilizing the preformed ␣-amino nitrogen of another amino

acid, usually glutamate.Yet, although it was originally pre-

sumed that glutamate can be synthesized from ammonia

and ␣-ketoglutarate by glutamate dehydrogenase acting in

reverse, it now appears that the predominant physiological

direction of this enzyme is glutamate breakdown (Section

26-1B). Consequently, preformed ␣-amino nitrogen should

also be considered to be an essential nutrient. In this con-

text, it is interesting to note that, in addition to the four

well-known taste receptors, those for sweet, sour, salty, and

bitter tastes, a fifth taste receptor has been characterized,

that for the meaty taste of monosodium glutamate (MSG),

which is known as umami (Japanese: flavor).

The essential and nonessential amino acids for humans

are listed in Table 26-2. Arginine is classified as essential,

even though it is synthesized by the urea cycle (Section

26-2D), because it is required in greater amounts than can

be produced by this route during the normal growth and

development of children (but not adults). The essential

amino acids occur in animal and vegetable proteins. Dif-

ferent proteins, however,contain different proportions of the

essential amino acids. Milk proteins, for example, contain

them all in the proportions required for proper human nutri-

tion. Bean protein, on the other hand, contains an abundance

of lysine but is deficient in methionine, whereas wheat is

deficient in lysine but contains ample methionine.A balanced

protein diet therefore must contain a variety of different

protein sources that complement each other to supply the

proper proportions of all the essential amino acids.

In this section we study the pathways involved in the

formation of the nonessential amino acids. We also briefly

consider such pathways for the essential amino acids as

they occur in plants and microorganisms. You should note,

however, that although we discuss some of the most com-

mon pathways for amino acid biosynthesis, there is consid-

erable variation in these pathways among different species.

In contrast, as we have seen, the basic pathways of carbo-

hydrate and lipid metabolism are all but universal.

A. Biosynthesis of the Nonessential Amino Acids

All the nonessential amino acids except tyrosine are synthe-

sized by simple pathways leading from one of four common

metabolic intermediates: pyruvate, oxaloacetate, ␣-ketoglu-

tarate, and 3-phosphoglycerate. Tyrosine, which is really

misclassified as nonessential, is synthesized by the one-step

hydroxylation of the essential amino acid phenylalanine

(Section 26-3H). Indeed, the dietary requirement for

phenylalanine reflects the need for tyrosine as well. The

presence of dietary tyrosine therefore decreases the need

for phenylalanine. Since preformed ␣-amino nitrogen in the

form of glutamate is an essential nutrient for nonessential

1064 Chapter 26. Amino Acid Metabolism

Figure 26-50 The biosynthetic fates of the C

units in the THF

pool.

-Methyl-THF methionine SAM

epinephrine

phosphatidylcholine

histidinepurines

formylmethionyl-tRNA

-Methylene-THF

-Methenyl-THF

thymidylate (dTMP)

-Formyl-THF

JWCL281_c26_1019-1087.qxd 4/20/10 9:26 AM Page 1064

amino acid biosynthesis, we first discuss its production by

plants and microorganisms.

a. Glutamate Is Synthesized by Glutamate Synthase

Glutamate synthase, an enzyme that occurs only in

microorganisms, plants, and lower animals, converts

␣-ketoglutarate and ammonia originating in glutamine to

glutamate.The electrons required for this reductive amina-

tion come from NADPH or ferredoxin, depending on the

organism. The NADPH-dependent glutamate synthase

from the nitrogen-fixing bacterium Azospirillum brasilense,

the best characterized such enzyme, is an ␣

␤

hetero-

tetramer that binds an FAD and two [4Fe–4S] clusters on

each ␤ subunit,and an FMN and a [3Fe–4S] cluster on each

␣ subunit.The overall reaction is

and involves five steps that occur at three distinct active

sites (Fig. 26-51):

1. Electrons are transferred from NADPH to FAD at

active site 1 on the ␤ subunit to yield FADH

2. The electrons are transferred from the FADH

FMN at site 2 on a specific ␣ subunit through the iron–sul-

fur clusters to yield FMNH

3. Glutamine is hydrolyzed to ␣-glutamate and ammo-

nia at site 3 on the ␣ subunit.

4. The ammonia produced is transferred to site 2 where

it reacts with ␣-ketoglutarate to form ␣-iminoglutarate.

2 glutamate ⫹ NADP

⫹

NADPH ⫹ H

⫹

⫹ glutamine ⫹␣-ketoglutarate

Section 26-5. Amino Acid Biosynthesis 1065

Table 26-2 Essential and Nonessential Amino

Acids in Humans

Essential Nonessential

Arginine

Alanine

Histidine Asparagine

Isoleucine Aspartate

Leucine Cysteine

Lysine Glutamate

Methionine Glutamine

Phenylalanine Glycine

Threonine Proline

Tryptophan Serine

Valine Tyrosine

Although mammals synthesize arginine, they cleave most of it to form

urea (Sections 26-2D and 26-2E).

Figure 26-51 The sequence of reactions catalyzed by glutamate synthase.

Glutamine

COO

–

+ H

Glutamate

␣-Ketoglutarate

COO

–

+ H

␣ Subunit—site 3

␣ Subunit—site 2

Overall: NADPH

+ H

+ glutamine + ␣-ketoglutarate

␤ Subunit—site 1

COO

––

OOC

channeling

COO

–

Glutamate

COO

––

OOC

FMNH

FMN

–

␣-Iminoglutarate

–

OOC

FAD

FADH

NADPH + H

2 glutamate

+ NADP

NADP

JWCL281_c26_1019-1087.qxd 4/20/10 9:26 AM Page 1065

5. The ␣-iminoglutarate is reduced by FMNH

to form

glutamate.

In the absence of the ␤ subunit, the ␣ subunit can synthe-

size glutamate from glutamine and ␣-ketoglutarate using

an artificial electron donor; moreover,it is homologous and

functionally similar to ferredoxin-dependent glutamine

synthases. The A. brasilense ␣ subunit is therefore consid-

ered to be the enzyme’s catalytic core.

The X-ray structure of the 1479-residue ␣ subunit of A.

brasilense glutamate synthase in complex with a [3Fe–4S]

cluster, an FMN, an ␣-ketoglutarate substrate,and a methio-

nine sulfone inhibitor (a tetrahedral transition state analog)

was determined by Andrea Mattevi (Fig. 26-52). The subunit

has four domains, an N-terminal glutamine amidotransferase

domain, a central domain, an FMN-binding domain, and a C-

terminal ␤-helix domain (see below). The methionine sul-

fone binds in the N-terminal amidotransferase domain (site

3), where glutamine is normally hydrolyzed via the nucle-

ophilic attack of the Cys 1 sulfydryl group on the glutamine

␥

atom to transiently form a tetrahedral intermediate that

the tetrahedral sulfone group mimics. The FMN-binding do-

main consists in large part of an ␣/␤ barrel, at the mouth of

which the ␣-ketoglutarate and the [3Fe–4S] cluster bind.The

distance between the methyl group on methionine sulfone

(the analog of the glutamine amido group) and the ␣-keto

group of the ␣-ketoglutarate is 31 Å.These two sites are con-

nected by a tunnel through which ammonia must diffuse in

order to react with ␣-ketoglutarate (Reaction 4, Fig. 26-51).

However, the tunnel is blocked by the main chain atoms of

four residues that protrude into the tunnel. Consequently,

there must be at least a 2- to 3-Å shift in the structure during

the reaction to permit ammonia to diffuse between these

sites. Such gating of the tunnel may well be crucial for the

control of enzyme function so as to avoid the wasteful hy-

drolysis of glutamine. In fact, glutamine is hydrolyzed only

when the enzyme has bound ␣-ketoglutarate and reducing

equivalents are available for iminoglutarate reduction.

The C-terminal domain of glutamate synthase consists

largely of a 7-turn, right-handed ␤ helix (Fig. 26-53). In this

unusual motif, the polypeptide chain is wrapped in a wide

helix such that neighboring turns of chain interact as do the

strands of a parallel ␤ sheet.The 43-Å-long glutamate syn-

thase ␤ helix has an elliptical cross section of 16 by 23 Å.

␤ Helices have been observed in only a handful of mainly

bacterial enzymes. The ␤ helix of glutamate synthase does

–

Glutamine

–

Methionine sulfone

not contain a residue that is involved in catalysis or elec-

tron transfer. However, it does appear to have an impor-

tant structural role because some of its residues line the

tunnel through which ammonia passes.

Glutamine amidotransferase domains or subunits are

part of several protein structures in which glutamine is the

ammonia donor for a further reaction. In glutamate syn-

thase, it is a domain within the ␣ subunit (Fig. 26-52). In

E. coli carbamoyl phosphate synthetase (CPS; Section

26-2Aa), it is a complete subunit within a heterodimer.

These domains or subunits occur in one of two families

that are differentiated by their active site structures. In

CPS, it belongs to the triad family, so called because it con-

tains an active site Cys in a catalytic triad reminiscent of

the catalytic triad in serine proteases (Section 15-3). The

glutamine amidotransferase domain of glutamate syn-

thase belongs to the N-terminal nucleophile (Ntn) family,

which, as we have seen, has an N-terminal Cys that acts as

the active site nucleophile. Other enzymes involved in

1066 Chapter 26. Amino Acid Metabolism

Iminoglutarate

reduction

L-Gln hydrolysis

3Fe–4S

FMN

Figure 26-52 X-ray structure of the ␣ subunit of A. brasilense

glutamate synthase as represented by its C

␣

backbone. The

subunit has four domains, an N-terminal glutamine

amidotransferase domain (blue) to which methionine sulfone (a

glutamine tetrahedral transition state analog) is bound; a central

domain (red); an FMN-binding domain (green) to which an

FMN, a [3Fe–4S] cluster, and an ␣-ketoglutarate are bound; and a

␤ helix domain (purple).The foregoing ligands are drawn in

black in ball-and-stick form.The 31-Å-long tunnel from the

methyl group on the methionine sulfone (analogous to the amido

group of glutamine) to the ␣-keto group of the ␣-ketoglutarate is

outlined by a gray surface. The tunnel is blocked in this structure

(it is divided into two cavities) by the main chain atoms of four

residues that protrude into the tunnel. [Courtesy of Andrea

Mattevi, Universitá degli Studi di Pavia, Italy. PDBid 1EA0.]

JWCL281_c26_1019-1087.qxd 4/20/10 9:26 AM Page 1066

amino acid biosynthesis and having a glutamine amidotrans-

ferase domain include asparagine synthetase (Fig. 26-54,

Reaction 4; see below), a member of the Ntn family, and

imidazole glycerol phosphate synthase (Fig. 26-65, Re-

action 5), which belongs to the triad family. All of these

enzymes have an ammonia-channeling tunnel that connects

the amidotransferase site with the ammonia-utilizing site.

b. Alanine, Asparagine, Aspartate, Glutamate, and

Glutamine Are Synthesized from Pyruvate,

Oxaloacetate, and ␣-Ketoglutarate

Pyruvate, oxaloacetate, and ␣-ketoglutarate are the

keto acids that correspond to alanine, aspartate, and gluta-

mate, respectively. Indeed, as we have seen (Section 26-1),

the synthesis of each of these amino acids is a one-step

Section 26-5. Amino Acid Biosynthesis 1067

Figure 26-53 The ␤ helix of A. brasilense glutamate synthase. The polypeptide

backbone (residues 1225–1416) is colored in rainbow order from its N-terminus

(blue) to its C-terminus (red). Neighboring turns of polypeptide chain within the

␤ helix interact as do the strands of parallel ␤ sheets. Nevertheless, the

conformations of many of these segments lie outside the normal range for ␤

strands, and hence they are drawn in coil form. [Based on an X-ray structure by

Andrea Mattevi, Universitá degli Studi di Pavia, Italy. PDBid 1EA0.]

Glutamate

COO

–

OPO

–

-Ketoglutarate

COO

–

ADP

ATP

-Glutamylphosphate

intermediate

COO

–

glutamine

synthetase

Glutamine

COO

–

Aspartate

COO

–

Oxaloacetate

COO

–

AMP

ATP

Asparagine

COO

–

asparagine

synthetase

Glutamate

Glutamine

Pyruvate

COO

–

Amino

acid

α-Keto

acid

aminotransferase

Alanine

COO

–

aminotransferase

Amino

acid

α-Keto

acid

Amino

acid

α-Keto

acid

Figure 26-54 The syntheses of alanine, aspartate, glutamate, asparagine, and

glutamine. These reactions involve the transaminations of (1) pyruvate, (2) oxaloacetate,

and (3) ␣-ketoglutarate, and the amidations of (4) aspartate and (5) glutamate.

JWCL281_c26_1019-1087.qxd 6/8/10 9:38 AM Page 1067

(b)

transamination reaction (Fig. 26-54, Reactions 1–3). As-

paragine and glutamine are, respectively, synthesized from

aspartate and glutamate by amidation (Fig. 26-54, Reactions

4 and 5). Glutamine synthetase catalyzes the formation of

glutamine in a reaction in which ATP is hydrolyzed to ADP

and P

via the intermediacy of ␥-glutamylphosphate and

is the amino group donor (Fig. 26-54, Reaction 5). Cu-

riously, aspartate amidation by asparagine synthetase to form

asparagine follows a different route; it utilizes glutamine as

its amino group donor and hydrolyzes ATP to AMP⫹PP

(Fig. 26-54, Reaction 4). This enzyme is composed of a gluta-

mine amidotransferase domain of the Ntn family (see above)

and a second domain in which ␤-aspartyl-AMP

is synthesized from Asp and ATP and then reacts with am-

monia to form Asn. As with other glutamine amidotrans-

ferase–containing enzymes, the two domains are connected

by a tunnel that channels ammonia between their two

active sites.

c. Glutamine Synthetase Is a Central Control Point

in Nitrogen Metabolism

Glutamine, as we have seen, is the amino group donor in

the formation of many biosynthetic products, as well as be-

ing a storage form of ammonia. Glutamine synthetase’s

consequent pivotal position in nitrogen metabolism makes

its control a vital aspect of this process. In fact, mammalian

glutamine synthetases are activated by ␣-ketoglutarate, the

product of glutamate’s oxidative deamination.This control

presumably prevents the accumulation of the ammonia

produced by that reaction.

Bacterial glutamine synthetase, as Earl Stadtman showed,

has a much more elaborate control system. This enzyme,

which consists of 12 identical 469-residue subunits arranged

with D

symmetry (Fig. 26-55), is regulated by several effec-

tors as well as by covalent modification.Although a complete

description of this complex enzyme is not given here, several

aspects of its catalytic and control systems bear note.

The X-ray structure of Salmonella typhimurium gluta-

mine synthetase in complex with the glutamate structural

analog phosphinothricin,

determined by David Eisenberg, reveals that its catalytic

sites occur at the interface between the C-terminal domain

COO

–

Phosphinothricin

–

COO

–

Glutamate

O P

COO

–

␤-Aspartyl-AMP

–

OH OH

⫹

1068 Chapter 26. Amino Acid Metabolism

Figure 26-55 X-ray structure of S. typhimurium glutamine

synthetase. The enzyme consists of 12 identical subunits, here

represented by their C

␣

backbones, arranged with D

symmetry

(the symmetry of a hexagonal prism). (a) View down the 6-fold

axis of symmetry showing only the six subunits of the upper ring

in alternating blue and green.The subunits of the lower ring are

roughly directly below those of the upper ring.The protein,

including its side chains (not shown), has a diameter of 143 Å.

The six active sites shown are marked by the pairs of Mn

2⫹

ions

(magenta spheres; divalent metal ions, physiologically Mg

2⫹

, are

required for enzymatic activity). Each adenylylation site,Tyr 397

(yellow), lies between two subunits at a higher radius than the

corresponding active site. Also drawn in one active site are ADP

(cyan) and phosphinothricin (orange), a competitive inhibitor of

glutamate. (b) Side view along one of the enzyme’s 2-fold axes

showing only the eight nearest subunits.The molecule extends

103 Å along the 6-fold axis, which is vertical in this view. [Based

on an X-ray structure by David Eisenberg, UCLA. PDBid 1FPY.]

(a)

JWCL281_c26_1019-1087.qxd 4/20/10 9:26 AM Page 1068

of one subunit and the N-terminal domain of an adjacent

subunit. These catalytic sites have a shape described as a

“bifunnel” that opens at both the exposed top (ATP bind-

ing) and bottom (Glu and binding) of the molecule

(between the two hexameric rings) and is narrow in the

plane of its essential metal ions (two per subunit). Nu-

cleotide binding induces conformational changes that in-

crease the enzyme’s affinity for glutamate and ammonium

ion, leading to an ordered sequential mechanism.

Nine feedback inhibitors cumulatively control the activity

of bacterial glutamine synthetase: histidine, tryptophan, car-

bamoyl phosphate (as synthesized by carbamoyl phosphate

synthetase II), glucosamine-6-phosphate, AMP, and CTP are

all end products of pathways leading from glutamine,

whereas alanine, serine, and glycine reflect the cell’s nitrogen

⫹

level. Several of these inhibitors act in a competitive manner,

binding either to the glutamate binding site (serine, glycine,

and alanine) or to the ATP binding site (AMP and CTP).

E. coli glutamine synthetase is covalently modified by

adenylylation of a specific Tyr residue (Fig. 26-56). The en-

zyme’s susceptibility to cumulative feedback inhibition in-

creases, and its activity therefore decreases, with its degree

of adenylylation. The level of adenylylation is controlled by

a complex metabolic cascade that is conceptually similar to

the one controlling glycogen phosphorylase (although the

type of covalent modification differs in that glycogen phos-

phorylase is phosphorylated at a specific Ser residue;Section

18-3Ca). Both adenylylation and deadenylylation of gluta-

mine synthetase are catalyzed by adenylyltransferase in

complex with a tetrameric regulatory protein, P

. This

Section 26-5. Amino Acid Biosynthesis 1069

α-Ketoglutarate

ATP

Glutamine

Glutamine synthetase (less active)

Adenylyltransferase

–

OH OH

Adenine

Glutamine synthetase (more active)

–

POO

OH OH

Uracil

•

Adenylyltransferase

Uridylyltransferase

Uridylyl-removing enzyme

•

ADP

UTP

OUMP

Figure 26-56 The regulation of bacterial

glutamine synthetase. The

adenylylation/deadenylylation of a

specific Tyr residue is controlled by the

level of uridylylation of a specific

adenylyltransferase ⭈ P

Tyr residue.

This uridylylation level, in turn, is

controlled by the relative activities of

uridylyltransferase, which is

sensitive to the levels of a

variety of nitrogen

metabolites, and

uridylyl-removing

enzyme, whose

activity is

independent

of these

metabolite

levels.

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1070 Chapter 26. Amino Acid Metabolism

COO

–

CoASH CoASH

Glutamate

–

COO

–

N-Acetylglutamate

N-Acetylglutamate-5-phosphate

–

COO

–

Glutamate-5-phosphate

–

COO

–

COO

–

nonenzymatic

Glutamate-5-semialdehyde

⌬

-Pyrroline-5-carboxylate

Proline

COO

–

Arginine

ATP

ADP

ATP

ADP

NAD(P)H

NAD(P)

Glutamate

α-Ketoglutarate

Glutamate

α-Ketoglutarate

NAD(P)H

NAD(P)

COO

–

COO

–

COO

–

N-Acetylglutamate-5-semialdehyde

N-Acetylornithine

Ornithine

NAD(P)H

NAD(P)

COO

–

COO

–

COO

–

urea cycle

Figure 26-57 The biosynthesis of the “glutamate family” of amino

acids: arginine, ornithine, and proline. The enzymes catalyzing proline

biosynthesis are (1) ␥-glutamyl kinase, (2) dehydrogenase, (3)

nonenzymatic, and (4) pyrroline-5-carboxylate reductase. The enzymes

catalyzing ornithine biosynthesis are (5) N-acetylglutamate synthase,

(6) acetylglutamate kinase, (7) N-acetyl-␥-glutamyl phosphate

reductase, (8) N-acetylornithine-␦-aminotransferase, and

(9) acetylornithine deacetylase. An alternate pathway to ornithine is

through Reaction 10, catalyzed by ornithine-␦-aminotransferase.

Ornithine is converted to arginine (11) via the urea cycle (Fig. 26-7,

Reactions 2–4).

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