Voet D., Voet Ju.G. Biochemistry

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condensation of its aldehyde group with the ε-amino group

of an enzymatic Lys residue (Fig. 26-1d). This Schiff base,

which is conjugated to the coenzyme’s pyridinium ring, is

the focus of the coenzyme’s activity.

Esmond Snell,Alexander Braunstein, and David Metzler

demonstrated that the aminotransferase reaction occurs

via a Ping Pong Bi Bi mechanism whose two stages consist

of three steps each (Fig. 26-2):

Section 26-1. Amino Acid Deamination 1021

...

Enzyme

()

–

R C

COO

–

...

Enzyme

()

–

R C

COO

–

...

Enzyme

()

–

R C

COO

–

Enzyme–PLP

Schiff base

-Amino acid

␣

Geminal diamine

intermediate

•

Amino acid–PLP Schiff

base (aldimine)

...

–

C COO

–

Resonance-stabilized intermediate

–

...

–

C COO

–

EnzymeEnzyme

...

–

C COO

–

Enzyme

–

Steps 2 & 2ⴕ: Tautomerization:

Ketimine

–

C COO

–

Carbinolamine

H C H

Steps 1 & 1ⴕ: Transimination:

Steps 3 & 3ⴕ: Hydrolysis:

Lys

Lys Lys

EnzymeLys

–

C COO

–

Pyridoxamine

phosphate (PMP)–

enzyme

H C H

-Keto acid

EnzymeLys

•••

•

2–

Figure 26-2 The mechanism of PLP-dependent enzyme-

catalyzed transamination. The first stage of the reaction, in which

the ␣-amino group of an amino acid is transferred to PLP

yielding an ␣-keto acid and PMP, consists of three steps:

(1) transimination; (2) tautomerization, in which the Lys released

during the transimination reaction acts as a general acid–base

catalyst; and (3) hydrolysis. The second stage of the reaction, in

which the amino group of PMP is transferred to a different

␣-keto acid to yield a new ␣-amino acid and PLP, is essentially

the reverse of the first stage: Steps 3¿,2¿, and 1¿ are, respectively,

the reverse of Steps 3, 2, and 1.

See the Animated Figures

JWCL281_c26_1019-1087.qxd 4/20/10 9:26 AM Page 1021

b. Stage I: Conversion of an Amino Acid

to an ␣-Keto Acid

Step 1. The amino acid’s nucleophilic amino group

attacks the enzyme–PLP Schiff base carbon atom in a tran-

simination (trans-Schiffization) reaction to form an amino

acid–PLP Schiff base (aldimine), with concomitant release

of the enzyme’s Lys amino group. This Lys is then free to

act as a general base at the active site.

Step 2. The amino acid–PLP Schiff base tautomerizes

to an ␣-keto acid–PMP Schiff base by the active site

Lys–catalyzed removal of the amino acid ␣ hydrogen and

protonation of PLP atom C4¿ via a resonance-stabilized

carbanion intermediate. This resonance stabilization facili-

tates the cleavage of the C

␣

¬H bond.

Step 3. The ␣-keto acid–PMP Schiff base is hydrolyzed

to PMP and an ␣-keto acid.

c. Stage II: Conversion of an ␣-Keto Acid

to an Amino Acid

To complete the aminotransferase’s catalytic cycle,

the coenzyme must be converted from PMP back to the

enzyme–PLP Schiff base. This involves the same three

steps as above, but in reverse order:

Step 3ⴕ. PMP reacts with an ␣-keto acid to form a

Schiff base.

Step 2ⴕ. The ␣-keto acid–PMP Schiff base tautomer-

izes to form an amino acid–PLP Schiff base.

Step 1ⴕ. The ε-amino group of the active site Lys

residue attacks the amino acid–PLP Schiff base in a trans-

imination reaction to regenerate the active enzyme–PLP

Schiff base, with release of the newly formed amino acid.

The reaction’s overall stoichiometry therefore is

Examination of the amino acid–PLP Schiff base’s struc-

ture (Fig. 26-2, Step 1) reveals why this system is called “an

electron-pusher’s delight.” Cleavage of any of the amino

acid C

␣

atom’s three bonds (labeled a, b, and c) produces a

resonance-stabilized C

␣

carbanion whose electrons are

␣-keto acid 1 ⫹ amino acid 2

Amino acid 1 ⫹␣-keto acid 2 Δ

delocalized all the way to the coenzyme’s protonated pyri-

dinium nitrogen atom; that is, PLP functions as an electron

sink. For transamination reactions, this electron-withdrawing

capacity facilitates removal of the ␣ proton (a bond cleav-

age) in the tautomerization of the Schiff base. PLP-

dependent reactions involving b bond cleavage (amino acid

decarboxylation) and c bond labilization are discussed in

Section 26-4B and in Sections 26-3Bb and 26-3G,respectively.

Aminotransferases differ in their specificity for amino

acid substrates in the first stage of the transamination reac-

tion, thereby producing the correspondingly different

␣-keto acid products. Most aminotransferases, however,

accept only ␣-ketoglutarate or (to a lesser extent) oxalo-

acetate as the ␣-keto acid substrate in the second stage of

the reaction, thereby yielding glutamate or aspartate as

their only amino acid products. The amino groups of most

amino acids are consequently funneled into the formation of

glutamate or aspartate, which are themselves interconverted

by glutamate–aspartate aminotransferase:

Oxidative deamination of glutamate (Section 26-1B) yields

ammonia and regenerates ␣-ketoglutarate for another

round of transamination reactions. Ammonia and aspartate

are the two amino group donors in the synthesis of urea.

d. The Glucose–Alanine Cycle Transports Nitrogen

to the Liver

An important exception to the foregoing is a group of

muscle aminotransferases that accept pyruvate as their

␣-keto acid substrate. The product amino acid, alanine, is

released into the bloodstream and transported to the liver,

where it undergoes transamination to yield pyruvate for

use in gluconeogenesis (Section 23-1A).The resulting glu-

cose is returned to the muscles, where it is glycolytically

degraded to pyruvate. This is the glucose–alanine cycle

(Fig. 26-3). The amino group ends up in either ammonium

ion or aspartate for urea biosynthesis. Evidently, the

glucose–alanine cycle functions to transport nitrogen from

muscle to liver.

During starvation the glucose formed in the liver by this

route is also used by the other peripheral tissues, breaking the

␣-ketoglutarate ⫹ aspartate

Glutamate ⫹ oxaloacetate Δ

1022 Chapter 26. Amino Acid Metabolism

Figure 26-3 The glucose–alanine cycle. See the Animated Figures

Blood MuscleLiver

Glucose

AlanineAlanine

Glycogen

Pyruvate

Gluconeogenesis

Urea

Pyruvate

Glucose

α-Amino acid

α-Ketoacid

transamination

deamination

Glutamate

α-Ketoglutarate

Aspartate

transamination

JWCL281_c26_1019-1087.qxd 6/21/10 10:39 AM Page 1022

cycle. Under these conditions both the amino group and the

pyruvate originate from muscle protein degradation, provid-

ing a pathway yielding glucose for other tissue use (recall that

muscle is not a gluconeogenic tissue; Section 23-1).

Nitrogen is also transported to the liver in the form of

glutamine, synthesized from glutamate and ammonia in a re-

action catalyzed by glutamine synthetase (Section 26-5Ab).

The ammonia is released for urea synthesis in liver mito-

chondria or for excretion in the kidney through the action

of glutaminase (Section 26-3D).

B. Oxidative Deamination: Glutamate

Dehydrogenase

Glutamate is oxidatively deaminated in the mitochondrial

matrix by glutamate dehydrogenase (GDH), the only

known enzyme that, in at least some organisms, can accept

either NAD

⫹

or NADP

⫹

as its redox coenzyme. Oxidation

is thought to occur with transfer of a hydride ion from gluta-

mate’s C

␣

to NAD(P)

⫹

, thereby forming ␣-iminoglutarate,

which is hydrolyzed to ␣-ketoglutarate and ammonia (Fig.

26-4). GDH is allosterically inhibited by GTP, NADH, and

nonpolar compounds such as palmitoyl-CoA and steroid

hormones. It is activated by ADP, NAD

⫹

, and leucine (the

most abundant amino acid in proteins;Table 4-1).

a. The X-Ray Structures of GDH Reveal its

Allosteric Mechanism

The X-ray structures of homohexameric GDH from

bovine and human liver mitochondria, determined by

Thomas Smith, reveal that each monomer has three domains,

a substrate domain, a coenzyme domain, and an antenna do-

main.The protein, which has D

symmetry, can be considered

to be a dimer of trimers, with the antenna domains of each

trimer wrapping around each other about the 3-fold axis

(Fig. 26-5a). Structural comparison of a 501-residue

monomer of the bovine GDH–glutamate–NADH–GTP

complex (Fig. 26-5b) with that of the 96% identical human

apoenzyme (no active site or regulatory ligands bound;

Fig. 26-5c) reveals that, on binding ligands, the coenzyme

binding domain rotates about the so-called pivot helix so as

to close the cleft between the coenzyme and substrate do-

mains. Simultaneously, the antenna domain twists in a way

that unwinds one turn of the antenna helix that is connected

to the pivot helix. Although the closed form is required for

catalysis, the open form favors the association and dissocia-

tion of substrates and products. In the open state, Arg 463

(human numbering) in the center of the pivot helix inter-

acts with the activator ADP (whose binding site in the

bovine complex is occupied by the ADP moiety of an

NADH; Fig. 26-5b), whereas in the closed state, the side

chain of His 454 hydrogen-bonds to the ␥-phosphate of the

inhibitor GTP. The GTP binding site is distorted and blocked

in the open state so that GTP binding favors the closed form

of the enzyme. This results in tight binding of substrates and

products and hence inhibition of the enzyme. ADP binding

favors the open form, allowing product dissociation, and

therefore activates the enzyme. Allosteric interactions ap-

pear to be communicated between subunits through the

interactions of the antenna domains. In fact, bacterial GDHs,

which lack allosteric regulation, differ from mammalian

GDHs mainly by the absence of antenna domains.

b. Hyperinsulinism/Hyperammonemia (HI/HA)

Is Caused by Uncontrolled GDH Activity

Charles Stanley has reported a new form of congenital

hyperinsulinism that is characterized by hypoglycemia and

hyperammonemia (HI/HA; hyperammonemia is elevated

levels of ammonia in the blood) and has shown that it is

caused by mutations in GDH at the N-terminal end of its

pivot helix in the GTP binding site or in the antenna do-

main near its joint with the pivot helix. The mutant en-

zymes have reduced sensitivity to GTP inhibition but re-

tain their ability to be activated by ADP. The GDH

mutants S448P, H454Y, and R463A, which were respec-

tively designed to affect the antenna region, the GTP bind-

ing site, and the ADP binding site (Fig. 26-5b), all have de-

creased sensitivity to GTP inhibition (Fig. 26-6), with

H454Y and S448P, which were previously known to be as-

sociated with HI/HA, conferring the most resistance to

GTP inhibition. The hypoglycemia and hyperammonemia

in HI/HA patients arises from the increased activity of the

GDH mutants in the breakdown direction, producing

increased amounts of ␣-ketoglutarate and NH

.The

increased levels of ␣-ketoglutarate stimulate the citric acid

cycle and oxidative phosphorylation, which has been

shown to lead to increased insulin secretion and hypo-

glycemia, thereby producing the symptoms of the disease.

The produced is usually converted to urea (Section

26-2) but can also be exported to the bloodstream.

If this scenario for the cause of HI/HA is correct, it

requires a reassessment of the role of GDH in ammonia

homeostasis.The equilibrium position of the GDH reaction

greatly favors the synthesis of Glu (⌬G°¿ ⬇ 30 kJ ⴢ mol

⫺1

for

the reaction as written in Fig. 26-4), but studies of cellular

⫹

Section 26-1. Amino Acid Deamination 1023

Figure 26-4 The oxidative deamination of glutamate by

glutamate dehydrogenase. This reaction involves the

intermediate formation of ␣-iminoglutarate.

C COO

––

OOC CH

+ NAD(P)

Glutamate

C COO

––

OOC CH

+ NAD(P)

␣-Iminoglutarate

H + H

C COO

––

OOC CH

␣-Ketoglutarate

JWCL281_c26_1019-1087.qxd 4/20/10 9:26 AM Page 1023

1024

Figure 26-6 Inhibition of human glutamate dehydrogenase

(GDH) by GTP. Human wild-type and mutant GDHs were

expressed in E. coli and assayed for sensitivity to GTP inhibition.

The midpoint of each curve corresponds to the concentration of

GTP causing 50% inhibition. [After Fang, J., Hsu, B.Y.L.,

MacMullen, C.M., Poncz, M., Smith,T.J., and Stanley, C.A.,

Biochem. J. 363, 81 (2002)].

0.01 0.1 1 10 10,0001000100 100,00

100

GDH activity (% basal)

GTP (μmol/L)

W/T

S448P

H454Y

R463A

Figure 26-5 X-ray structures of glutamate dehydrogenase (GDH).

(a) Bovine GDH in complex with glutamate, NADH, and GTP. The

homohexameric enzyme, which has D

symmetry, is viewed along one of its

2-fold axes with its 3-fold axis vertical. Each of its subunits is differently

colored.The bound substrates and ligands are shown in space-filling form

with glutamate orange, the substrate NADH pink, the NADH bound at the

ADP effector site brown, and the GTP effector gray. (b) One subunit of the

bovine GDH–glutamate–NADH–GTP complex drawn with the coenzyme

binding domain magenta, the substrate binding domain orange, the antenna

domain green, and the pivot helix cyan.The substrates and ligands are shown

in space-filling form colored according to atom type with glutamate C green,

substrate NADH C gold, ADP site-bound NADH C pink, GTP C cyan, N

blue, O red, and P magenta. The C

␣

atoms of Ser 448, His 454, and Arg 463

(human numbering) are represented by yellow spheres.

colored as and viewed similarly to Part b. [Based on X-ray

structures by Thomas Smith, Donald Danforth Plant

Science Center, St. Louis, Missouri. PDBids (a and b)

1HWX and (c) 1L1F.]

(a)

(b)

(c)

JWCL281_c26_1019-1087.qxd 4/20/10 9:26 AM Page 1024

substrate and product concentrations suggested that the

enzyme functions close to equilibrium (⌬G ⬇ 0) in vivo. It

was therefore widely accepted that increases in [NH

], high

levels of which are toxic, would cause GDH to act in re-

verse, removing NH

and hence preventing its buildup to

toxic levels. However,since HI/HA patients have increased

GDH activity yet have higher levels of NH

than normal,

this accepted role of GDH cannot be correct. Indeed, if

GDH functioned close to equilibrium,changes in its activity

resulting from allosteric interactions would not result in

significant flux changes.

C. Other Deamination Mechanisms

Two nonspecific amino acid oxidases,

L-amino acid oxidase

and

D-amino acid oxidase, catalyze the oxidation of L- and

D-amino acids, utilizing FAD as their redox coenzyme

[rather than NAD(P)

⫹

].The resulting FADH

is reoxidized

by O

D-Amino acid oxidase occurs mainly in kidney. Its function

is an enigma since

D-amino acids are associated mostly

with bacterial cell walls (Section 11-3Ba). A few amino

acids, such as serine and histidine, are deaminated nonox-

idatively (Sections 26-3B and 26-3D).

2 THE UREA CYCLE

Living organisms excrete the excess nitrogen resulting

from the metabolic breakdown of amino acids in one of

three ways. Many aquatic animals simply excrete ammonia.

Where water is less plentiful, however, processes have

evolved that convert ammonia to less toxic waste products

that therefore require less water for excretion. One such

product is urea, which is excreted by most terrestrial verte-

brates; another is uric acid, which is excreted by birds and

terrestrial reptiles:

Accordingly, living organisms are classified as being either

ammonotelic (ammonia excreting), ureotelic (urea excret-

ing), or uricotelic (uric acid excreting). Some animals can

shift from ammonotelism to ureotelism or uricotelism if

their water supply becomes restricted. Here we focus our

attention on urea formation. Uric acid biosynthesis is dis-

cussed in Section 28-4A.

Urea is synthesized in the liver by the enzymes of the

urea cycle. It is then secreted into the bloodstream and

mmonia Urea

Uric acid

NNH

FADH

⫹ O

FAD ⫹ H

␣-keto acid ⫹ NH

⫹ FADH

Amino acid ⫹ FAD ⫹ H

sequestered by the kidneys for excretion in the urine. The

urea cycle was elucidated in outline in 1932 by Hans Krebs

and Kurt Henseleit (the first known metabolic cycle; Krebs

did not elucidate the citric acid cycle until 1937). Its indi-

vidual reactions were later described in detail by Sarah

Ratner and Philip Cohen.The overall urea cycle reaction is

Thus, the two urea nitrogen atoms are contributed by NH

and aspartate, whereas the carbon atom comes from

HCO

⫺

. Five enzymatic reactions are involved in the urea

cycle, two of which are mitochondrial and three cytosolic

(Fig. 26-7). In this section, we examine the mechanisms of

these reactions and their regulation.

A. Carbamoyl Phosphate Synthetase: Acquisition

of the First Urea Nitrogen Atom

Carbamoyl phosphate synthetase (CPS) is technically not

a urea cycle enzyme. It catalyzes the condensation and ac-

tivation of NH

and to form carbamoyl phosphate,

the first of the cycle’s two nitrogen-containing substrates,

with the concomitant hydrolysis of two ATPs. Eukaryotes

have two forms of CPS:

1. Mitochondrial CPS I uses NH

as its nitrogen donor

and participates in urea biosynthesis.

2. Cytosolic CPS II uses glutamine as its nitrogen donor

and is involved in pyrimidine biosynthesis (Section 28-2A).

The reaction catalyzed by CPS I involves three steps

(Fig. 26-8):

1. Activation of by ATP to form carboxyphos-

phate and ADP.

2. Nucleophilic attack of NH

on carboxyphosphate,

displacing the phosphate to form carbamate and P

3. Phosphorylation of carbamate by the second ATP to

form carbamoyl phosphate and ADP.

The reaction is essentially irreversible and is the rate-

limiting step of the urea cycle. CPS I is subject to allosteric

activation by N-acetylglutamate as is discussed in Section

26-2F.

E. coli contains only one type of CPS, which is homolo-

gous to both CPS I and CPS II.The enzyme is a heterodimer

but when allosterically activated by ornithine (a urea cycle

intemediate), it forms a tetramer of heterodimers, (␣␤)

. Its

small subunit (382 residues) functions to hydrolyze gluta-

mine and deliver the resulting NH

to its large subunit (1073

residues). However, if the enzyme’s glutaminase (glutamine

HCO

⫺

HCO

⫺

Aspartate

COO

–

OOC

+ HCO

–

Urea Fumarate

CH CH

+ COO

––

OOC

3ATP

2ADP

+ 2P

+ AMP + PP

Section 26-2. The Urea Cycle 1025

JWCL281_c26_1019-1087.qxd 4/20/10 9:26 AM Page 1025

1026 Chapter 26. Amino Acid Metabolism

2ATP +

N NH

HCO

–

COO

–

(

)

CH NH

COO

–

(CH

)

CH NH

COO

–

COO

–

COO

–

COO

–

(CH

)

COO

–

C NH

COO

–

COO

–

(CH

)

COO

–

HC NH

+ NH

OPO

2–

+ 2ADP + P

+ ATP

ADP + P

Ornithine

Citrulline

ATP

AMP + PP

Arginino-

succinate

Fumarate

Arginine

Urea

Cytosol

Mitochondrion

Carbamoyl phosphate

Aspartate

Ornithine

Urea

cycle

Citrulline

COO

–

COO

–

Malate

fumarase

COO

–

COO

–

Oxaloacetate

NADH

NAD

Gluconeogenesis

NAD(P)H

NAD(P)

malate dehydrogenase

Glutamate

transaminase

trans-

aminase

pyruvate

carboxylase

Amino acid

COO

–

Alanine

CHH

␣-Keto acid

COO

–

Pyruvate

Oxaloacetate

Aspartate

␣-Ketoglutarate

glutamate

dehydrogenase

Glutamate

transaminase

+ H

JWCL281_c26_1019-1087.qxd 4/20/10 9:26 AM Page 1026

amidotransferase) activity is eliminated (e.g., by site-

directed mutagenesis), the large subunit can still produce

carbamoyl phosphate if NH

is supplied in high enough

concentration. The large subunit is composed of two nearly

superimposable halves that have 40% sequence identity.

The N-terminal half contains the carboxyphosphate syn-

thetic component and an oligomerization domain while the

C-terminal half contains the carbamoyl phosphate syn-

thetic component and an allosteric binding domain.

a. E. coli CPS Contains an Extraordinarily Long Tunnel

The X-ray structure of E. coli CPS in complex with

2⫹

,ADP, P

, and ornithine, determined by Hazel Holden

and Ivan Rayment, reveals that the active site for synthesis

of the carboxyphosphate intermediate is ⬃45 Å away from

the ammonia synthesis site and also ⬃35 Å away from the

carbamoyl phosphate synthesis active site. Astonishingly,

the three sites are connected by a narrow 96-Å-long molec-

ular tunnel that runs nearly the length of the elongated

protein molecule (Fig. 26-9). It therefore appears that CPS

guides its intermediate products from the active site in

which they are formed to that in which they are utilized.

This phenomenon, in which the intermediate of two reac-

tions is directly transferred from one enzyme active site to

Section 26-2. The Urea Cycle 1027

Figure 26-7 (Opposite) The urea cycle. Its five enzymes are

(1) carbamoyl phosphate synthetase, (2) ornithine

transcarbamoylase, (3) argininosuccinate synthetase,

(4) argininosuccinase, and (5) arginase. The reactions occur in

part in the mitochondrion and in part in the cytosol with

ornithine and citrulline being transported across the mitochondrial

membrane by specific transport systems (yellow circles). One of

the urea amino groups (green) originates as the NH

product of

the glutamate dehydrogenase reaction (top).The other amino

group (red) is obtained from aspartate through the transfer of an

amino acid to oxaloacetate via transamination (right).The

fumarate product of the argininosuccinase reaction is converted

to oxaloacetate for entry into gluconeogenesis via the same

reactions that occur in the citric acid cycle but take place in the

cytosol (bottom).The ATP utilized in Reactions 1 and 3 of the

cycle can be regenerated by oxidative phosphorylation from the

NAD(P)H produced in the glutamate dehydrogenase (top) and

malate dehydrogenase (bottom) reactions.

See the Animated

Figures

Figure 26-8 The mechanism of action of CPS I.

(1) Activation of HCO

⫺

by phosphorylation forms the

intermediate, carboxyphosphate; (2) nucleophilic attack on

carboxyphosphate by NH

forms the reaction’s second

intermediate, carbamate; and (3) phosphorylation of carbamate

by ATP yields the reaction product carbamoyl phosphate.

Figure 26-9 X-ray structure of E. coli carbamoyl phosphate

synthetase (CPS). The protein is represented by its C

␣

backbone.

The small subunit (magenta) contains the glutamine binding site

where NH

is produced or bound.The large subunit consists of

the carboxyphosphate domain (green), the oligomerization

domain (yellow), the carbamoyl phosphate domain (blue), and

the allosteric binding domain (orange).The 96-Å-long tunnel

connecting the three active sites is outlined in red. [Courtesy of

Hazel Holden and Ivan Rayment, University of Wisconsin.

PDBid 1JDB.]

ADP

–

ADP

Carboxyphosphate

–

ADP

–

HO +

Carbamate

ATP

–

Carbamoyl phosphate

2–

P C

OPO

–

:NH

JWCL281_c26_1019-1087.qxd 6/8/10 9:38 AM Page 1027

another, is called channeling (the term “tunneling” is re-

served for certain quantum mechanical phenomena).

Channeling increases the rate of a metabolic pathway by

preventing the loss of its intermediate products as well as

protecting the intermediate from degradation. NH

must

travel ⬃45 Å down the CPS tunnel to react with car-

boxyphosphate to form the next intermediate, carbamate.

The carbamate, in turn, must travel an additional ⬃35 Å to

the site where it is phosphorylated by ATP to form the final

product carbamoyl phosphate. The NH

transfer tunnel is

lined with polar groups capable of forming hydrogen bonds

with NH

, whereas the tunnel through which carbamate trav-

els is lined with backbone atoms and lacks charged groups

that might induce its hydrolysis as it diffuses between active

sites. Shielding and channeling are necessary because the in-

termediates carboxyphosphate and carbamate are extremely

reactive, having half-lives of 28 and 70 ms, respectively, at

neutral pH. Also, channeling allows the local concentration

of NH

to reach a higher value than is present in the cellular

medium.We shall encounter several other examples of chan-

neling in our studies of metabolic enzymes, but the CPS tun-

nel is far longer than that in any other known enzyme.

B. Ornithine Transcarbamoylase

Ornithine transcarbamoylase transfers the carbamoyl group

of carbamoyl phosphate to ornithine, yielding citrulline (Fig.

26-7, Reaction 2; note that both of these compounds are

“nonstandard” ␣-amino acids in that they do not occur in

proteins). The reaction occurs in the mitochondrion so that

ornithine, which is produced in the cytosol, must enter the

mitochondrion via a specific transport system. Likewise,

since the remaining urea cycle reactions occur in the cytosol,

citrulline must be exported from the mitochondrion.

C. Argininosuccinate Synthetase: Acquisition

of the Second Urea Nitrogen Atom

Urea’s second nitrogen atom is introduced in the urea cy-

cle’s third reaction by the condensation of citrulline’s urei-

do group with an aspartate amino group by argininosucci-

nate synthetase (Fig. 26-10). The ureido oxygen atom is

activated as a leaving group through formation of a

citrullyl–AMP intermediate, which is subsequently displaced

by the aspartate amino group. Support for the existence of

the citrullyl–AMP intermediate comes from experiments us-

ing

O-labeled citrulline (* in Fig. 26-10). The label was iso-

lated in the AMP produced by the reaction, demonstrating

that at some stage of the reaction, AMP and citrulline are

linked covalently through the ureido oxygen atom.

D. Argininosuccinase

With formation of argininosuccinate, all of the urea mole-

cule components have been assembled. However, the

amino group donated by aspartate is still attached to the

aspartate carbon skeleton.This situation is remedied by the

argininosuccinase-catalyzed elimination of arginine from

the aspartate carbon skeleton forming fumarate (Fig. 26-7,

Reaction 4). Arginine is urea’s immediate precursor. The

fumarate produced in the argininosuccinase reaction reacts

via the fumarase and malate dehydrogenase reactions to

form oxaloacetate (Fig. 26-7, bottom), which is then used in

gluconeogenesis (Section 23-1).

E. Arginase

The urea cycle’s fifth and final reaction is the arginase-

catalyzed hydrolysis of arginine to yield urea and regener-

ate ornithine (Fig. 26-7, Reaction 5). Ornithine is then re-

turned to the mitochondrion for another round of the cycle.

The urea cycle thereby converts two amino groups,one from

and one from aspartate, and a carbon atom from

to the relatively nontoxic excretion product urea at

the cost of four “high-energy” phosphate bonds (three ATP

hydrolyzed to two ADP, two P

, AMP, and PP

, followed by

rapid PP

hydrolysis). This energetic cost, together with that

of gluconeogenesis, is supplied by the oxidation of the

acetyl-CoA formed by the breakdown of amino acid carbon

skeletons (e.g., threonine, Fig. 26-12). Indeed, half the oxy-

gen that the liver consumes is used to provide this energy.

F. Regulation of the Urea Cycle

Carbamoyl phosphate synthetase I, the mitochondrial en-

zyme that catalyzes the first committed reaction of the urea

HCO

⫺

1028 Chapter 26. Amino Acid Metabolism

Figure 26-10 The mechanism of action of argininosuccinate

synthetase. The steps involved are (1) activation of the ureido

oxygen of citrulline through the formation of citrullyl–AMP and

P AMP

ATP

Citrulline

C H

COO

–

COO

–

NH C

COO

–

COO

–

O C

CNH

COO

–

(CH

)

Citrullyl–AMP

Aspartate

CNH

COO

–

(CH

)

AMP

Argininosuccinate

CNH

COO

–

(CH

)

(2) displacement of AMP by the ␣-amino group of aspartate. The

asterisk (*) traces the fate of

O originating in citrulline’s ureido

group.

JWCL281_c26_1019-1087.qxd 6/8/10 9:38 AM Page 1028

Alanine

Cysteine

Glycine

Serine

Threonine

Tryptophan

Isoleucine

Leucine

Lysine

Threonine

Pyruvate

Acetyl-CoA Acetoacetate

Leucine

Lysine

Phenylalanine

Tryptophan

Tyrosine

Citric

acid

cycle

Citrate

Isocitrate

Oxaloacetate

Fumarate

Succinyl-CoA α

-Ketoglutarate

Glucose

Asparagine

Aspartate

Phenylalanine

Tyrosine

Isoleucine

Methionine

Valine

Arginine

Glutamate

Glutamine

Histidine

Proline

cycle, is allosterically activated by N-acetylglutamate:

This metabolite is synthesized from glutamate and acetyl-

CoA by N-acetylglutamate synthase and hydrolyzed by a

specific hydrolase.The rate of urea production by the liver is,

in fact,correlated with the N-acetylglutamate concentration.

Increased urea synthesis is required when amino acid break-

down rates increase, generating excess nitrogen that must be

excreted. Increases in these breakdown rates are signaled by

an increase in glutamate concentration through transamina-

tion reactions (Section 26-1). This situation, in turn, causes

an increase in N-acetylglutamate synthesis, stimulating car-

bamoyl phosphate synthetase and thus the entire urea cycle.

The remaining enzymes of the urea cycle are controlled

by the concentrations of their substrates.Thus, inherited de-

ficiencies in urea cycle enzymes other than arginase do not

result in significant decreases in urea production (the total

lack of any urea cycle enzyme results in death shortly after

birth). Rather, the deficient enzyme’s substrate builds up,

increasing the rate of the deficient reaction to normal. The

anomalous substrate buildup is not without cost, however.

The substrate concentrations become elevated all the way

back up the cycle to NH

, resulting in hyperammonemia.

Although the root cause of NH

toxicity is not completely

understood, high [NH

] puts an enormous strain on the

-clearing system, especially in the brain (symptoms of

urea cycle enzyme deficiencies include mental retardation

and lethargy). This clearing system has been proposed to

involve glutamate dehydrogenase (working in reverse) and

glutamine synthetase, which decrease the ␣-ketoglutarate

and glutamate pools (Sections 26-1 and 26-5Ab).The brain

is most sensitive to the depletion of these pools. Depletion of

␣-ketoglutarate decreases the rate of the energy-generating

citric acid cycle, whereas decreasing the glutamate concen-

tration disturbs neuronal function, since it is both a neuro-

transmitter and a precursor to ␥-aminobutyrate (GABA),

another neurotransmitter (Section 20-5Cf). Glutamate de-

pletion would also decrease the functioning of the urea cy-

cle, since it is also the precursor to N-acetylglutamate, the

major regulator of the cycle. The involvement of GDH in

clearance is a subject of debate in light of the observa-

tion that HI/HA involves deinhibition of GDH (Section

26-1Bb), suggesting that increased GDH activity increases

the NH

concentration rather than decreasing it.

3 METABOLIC BREAKDOWN

OF INDIVIDUAL AMINO ACIDS

The degradation of amino acids converts them to citric acid

cycle intermediates or their precursors so that they can be

metabolized to CO

and H

O or used in gluconeogenesis.

N-Acetylglutamate

C CCH

–

OOC

COO

–

(CH

)

Indeed, oxidative breakdown of amino acids typically ac-

counts for 10 to 15% of the metabolic energy generated by

animals. In this section we consider how amino acid carbon

skeletons are catabolized. The 20 “standard” amino acids

(the amino acids of proteins) have widely differing carbon

skeletons, so their conversions to citric acid cycle intermedi-

ates follow correspondingly diverse pathways. We shall not

describe all of the many reactions involved in detail.Rather,

we shall consider how these pathways are organized and fo-

cus on a few reactions of chemical and/or medical interest.

A. Amino Acids Can Be Glucogenic,

Ketogenic, or Both

“Standard” amino acids are degraded to one of seven meta-

bolic intermediates: pyruvate, ␣-ketoglutarate, succinyl-

CoA, fumarate, oxaloacetate, acetyl-CoA, or acetoacetate

(Fig. 26-11).The amino acids may therefore be divided into

two groups based on their catabolic pathways (Fig. 26-11):

1. Glucogenic amino acids, whose carbon skeletons are

degraded to pyruvate, ␣-ketoglutarate, succinyl-CoA,

fumarate, or oxaloacetate and are therefore glucose pre-

cursors (Section 23-1A).

Section 26-3. Metabolic Breakdown of Individual Amino Acids 1029

Figure 26-11 Degradation of amino acids to one of seven

common metabolic intermediates. Glucogenic and ketogenic

degradations are indicated in green and red, respectively.

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COO

Pyruvate

COO

–

Alanine

Acetaldehyde

C COO

–

H NH

HO H

Threonine

C COO

–

HS H

Cysteine

Acetyl-CoA

SCoA

–

C COO

–

HO H

Serine

H C COO

–

Glycine

-Methylene-THF

THF

NADH + NH

+ CO

NAD

+ CH

Glycine

COO

C COO

–

NADH + H

CoA

NAD

␣-Amino-␤-ketobutyrate

–

α-Ketoglutarate

Glutamate

2–

S, , SCN

–

()

several

paths

CoA

2. Ketogenic amino acids, whose carbon skeletons are

broken down to acetyl-CoA or acetoacetate and can thus

be converted to ketone bodies or fatty acids (Sections 25-3

and 25-4).

For example, alanine is glucogenic because its transamina-

tion product, pyruvate (Section 26-1A), can be converted

to glucose via gluconeogenesis (Section 23-1A). Leucine,

on the other hand, is ketogenic; its carbon skeleton is con-

verted to acetyl-CoA and acetoacetate (Section 26-3F).

Since animals lack any metabolic pathway for the net con-

version of acetyl-CoA or acetoacetate to gluconeogenic

precursors, no net synthesis of carbohydrates is possible

from leucine, or from lysine, the only other purely keto-

genic amino acid. Isoleucine, phenylalanine, threonine,

tryptophan, and tyrosine,however,are both glucogenic and

ketogenic; isoleucine, for example, is broken down to

succinyl-CoA and acetyl-CoA and hence is a precursor of

both carbohydrates and ketone bodies (Section 26-3Ed).

The remaining 13 amino acids are purely glucogenic.

In studying the specific pathways of amino acid break-

down, we shall organize the amino acids into groups that

are degraded into each of the seven metabolic intermedi-

ates mentioned above: pyruvate, oxaloacetate, ␣-ketoglu-

tarate, succinyl-CoA, fumarate, acetyl-CoA, and acetoac-

etate. When acetoacetyl-CoA is a product in amino acid

degradation, it can, of course, be directly converted to

acetyl-CoA (Section 25-3).We also discuss the pathway by

which, in liver, it is converted instead to acetoacetate for

use as an alternative fuel source in peripheral tissues (Sec-

tion 25-3).

B. Alanine, Cysteine, Glycine, Serine, and

Threonine Are Degraded to Pyruvate

Five amino acids, alanine, cysteine, glycine, serine, and threo-

nine, are broken down to yield pyruvate (Fig. 26-12).Trypto-

phan should also be included in this group since one of its

breakdown products is alanine (Section 26-3G),which,as we

have seen (Section 26-1Ad), is transaminated to pyruvate.

1030 Chapter 26. Amino Acid Metabolism

Figure 26-12 The pathways converting

alanine, cysteine, glycine, serine, and threonine to

pyruvate. The enzymes involved are (1) alanine

aminotransferase, (2) serine dehydratase,

(3) glycine cleavage system, (4) and (5) serine

hydroxymethyltransferase, (6) threonine

dehydrogenase, and (7) ␣-amino-␤-ketobutyrate

lyase.

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