Chapter 22

22
Biosynthesis of Amino Acids,

Nucleotides, and Related Molecules
22.1 Overview of Nitrogen Metabolism 881 the metals molybdenum, selenium, and vanadium. The
effort also offers a practical dividend, especially for stu-
22.2 Biosynthesis of Amino Acids 891
dents of human or veterinary medicine. Many genetic
22.3 Molecules Derived from Amino Acids 902 diseases of humans and animals have been traced to an
absence of one or more enzymes of amino acid and nucle-
22.4 Biosynthesis and Degradation of Nucleotides 910
otide metabolism, and many pharmaceuticals in common
use to combat infectious diseases are inhibitors of enzymes
N
itrogen ranks behind only carbon, hydrogen, and in these pathways—as are a number of the most impor-
oxygen in its contribution to the mass of living sys- tant agents in cancer chemotherapy.
tems. Most of this nitrogen is bound up in amino Regulation is crucial in the biosynthesis of the
acids and nucleotides. In this chapter we address all nitrogen-containing compounds. Because each amino
aspects of the metabolism of these nitrogen-containing acid and each nucleotide is required in relatively small
compounds except amino acid catabolism, which is cov- amounts, the metabolic flow through most of these path-
ered in Chapter 18. ways is not nearly as great as the biosynthetic flow lead-
Discussing the biosynthetic pathways for amino acids ing to carbohydrate or fat in animal tissues. Because the
and nucleotides together is a sound approach, not only different amino acids and nucleotides must be made in
because both classes of molecules contain nitrogen (which the correct ratios and at the right time for protein and
arises from common biological sources) but because the nucleic acid synthesis, their biosynthetic pathways must
two sets of pathways are extensively intertwined, with be accurately regulated and coordinated with each
several key intermediates in common. Certain amino acids other. And because amino acids and nucleotides are
or parts of amino acids are incorporated into the structure charged molecules, their levels must be regulated to
of purines and pyrimidines, and in one case part of a maintain electrochemical balance in the cell. As dis-
purine ring is incorporated into an amino acid (histidine). cussed in earlier chapters, pathways can be controlled
The two sets of pathways also share much common chem- by changes in either the activity or the amounts of spe-
istry, in particular a preponderance of reactions involving cific enzymes. The pathways we encounter in this chap-
the transfer of nitrogen or one-carbon groups. ter provide some of the best-understood examples of the
The pathways described here can be intimidating to regulation of enzyme activity. Control of the amounts of
the beginning biochemistry student. Their complexity different enzymes in a cell (that is, of their synthesis and
arises not so much from the chemistry itself, which in degradation) is a topic covered in Chapter 28.
many cases is well understood, but from the sheer num-
ber of steps and variety of intermediates. These pathways
are best approached by maintaining a focus on metabolic
22.1 Overview of Nitrogen Metabolism
principles we have already discussed, on key intermedi- The biosynthetic pathways leading to amino acids and
ates and precursors, and on common classes of reactions. nucleotides share a requirement for nitrogen. Because
Even a cursory look at the chemistry can be rewarding, soluble, biologically useful nitrogen compounds are gen-
for some of the most unusual chemical transformations in erally scarce in natural environments, most organisms
biological systems occur in these pathways; for instance, maintain strict economy in their use of ammonia, amino
we find prominent examples of the rare biological use of acids, and nucleotides. Indeed, as we shall see, free
881
882 Biosynthesis of Amino Acids, Nucleotides, and Related Molecules
amino acids, purines, and pyrimidines formed during NH3-to-N2 conversion in the biosphere may occur
metabolic turnover of proteins and nucleic acids are through this pathway, undetected until the 1980s. The
often salvaged and reused. We first examine the path- obligate anaerobes that promote anammox are fasci-
ways by which nitrogen from the environment is intro- nating in their own right and are providing some useful
duced into biological systems. solutions to waste-treatment problems (Box 22–1).
Now let’s examine the processes that generate the
The Nitrogen Cycle Maintains a Pool ammonia that is incorporated into microorganisms,
plants, and the animals that eat them.
of Biologically Available Nitrogen More than 90% of the NH⫹4 generated by vascular
Although Earth’s atmosphere is four-fifths molecular plants, algae, and microorganisms comes from nitrate
nitrogen (N2), relatively few species can convert this assimilation, a two-step process. First NO3 is reduced to
atmospheric nitrogen into forms useful to living organ- NO2 by nitrate reductase, then the NO2 is reduced to
isms. In the biosphere, the metabolic processes of differ- NH⫹4 in a six-electron transfer catalyzed by nitrite
ent species function interdependently to salvage and reductase (Fig. 22–2). Both reactions involve chains
reuse biologically available nitrogen in a vast nitrogen of electron carriers and cofactors we have not yet
cycle (Fig. 22–1). The first step in the cycle is fixation encountered. Nitrate reductase is a large, soluble pro-
(reduction) of atmospheric nitrogen by nitrogen-fixing tein (Mr 220,000). Within the enzyme, a pair of elec-
bacteria to yield ammonia (NH3 or NH⫹4 ). Although trons, donated by NADH, flows through —SH groups of
ammonia can be used by most living organisms, soil bac- cysteine, FAD, and a cytochrome (cyt b557), then to a
teria that derive their energy by oxidizing ammonia to novel cofactor containing molybdenum, before reducing
nitrite (NO2 ) and ultimately nitrate (NO3 ) are so abun- the substrate NO 3 to NO2 .

dant and active that nearly all ammonia reaching the soil The nitrite reductase of plants is located in the
is oxidized to nitrate. This process is known as nitrifica- chloroplasts and receives its electrons from ferredoxin
tion. Plants and many bacteria can take up and readily (which is reduced in the light-dependent reactions of
reduce nitrate and nitrite to ammonia through the action photosynthesis; see Section 19.8). Six electrons, donat-
of nitrate and nitrite reductases. This ammonia is incor- ed one at a time by ferredoxin, pass through a 4S-4Fe
porated into amino acids by plants. Animals then use center in the enzyme, then through a novel heme-like
plants as a source of amino acids, both nonessential and molecule (siroheme) before reducing NO2 to NH⫹4
essential, to build their proteins. When organisms die, (Fig. 22–2). In nonphotosynthetic microbes, NADPH
microbial degradation of their proteins returns ammonia provides the electrons for this reaction.
to the soil, where nitrifying bacteria again convert it to
nitrite and nitrate. A balance is maintained between
fixed nitrogen and atmospheric nitrogen by bacteria that
Nitrogen Is Fixed by Enzymes of the
reduce nitrate to N2 under anaerobic conditions, a pro- Nitrogenase Complex
cess called denitrification (Fig. 22–1). These soil bac- Only certain bacteria and archaea can fix atmospheric
teria use NO3 rather than O2 as the ultimate electron N2. These organisms, called diazotrophs, include the
acceptor in a series of reactions that (like oxidative cyanobacteria of soils and fresh and salt waters, metha-
phosphorylation) generates a transmembrane proton nogenic archaea (strict anaerobes that obtain energy
gradient, which is used to synthesize ATP. and carbon by converting H2 and CO2 to methane), other
The nitrogen cycle is short-circuited by a group of kinds of free-living soil bacteria such as Azotobacter spe-
bacteria that promote anaerobic ammonia oxidation, or cies, and the nitrogen-fixing bacteria that live as symbi-
anammox (Fig. 22–1), a process that converts ammo- onts in the root nodules of leguminous plants. The first
nia and nitrite to N2. As much as 50% to 70% of the important product of nitrogen fixation is ammonia,
N2 (atmosphere) Denitrifying
Nitrogen-fixing bacteria, archaea,
bacteria and Oxidation and fungi
archaea state = 0
degradation
by animals and
Amino acids microorganisms
NH⫹ NO⫺
4 (ammonia) 3 (nitrate)
and other reduced
nitrogen-carbon Oxidation Oxidation
Anammox bacteria
compounds synthesis in state = ⴚ3 state = ⴙ5
plants and
microorganisms
NO⫺
2 (nitrite) Nitrifying
Oxidation bacteria
Nitrifying
bacteria and archaea state = ⴙ3
FIGURE 22–1 The nitrogen cycle. The total amount of nitrogen fixed annually in the biosphere exceeds 1011 kg. Reactions
with red arrows occur largely or entirely in anaerobic environments.
22.1 Overview of Nitrogen Metabolism 883
(a)
NO 3 FIGURE 22–2 Nitrate assimila-
tion by nitrate reductase and
O
nitrite reductase. (a) Nitrate
NADH
O S Mo O reductases of plants and bacteria
—SH FAD Cyt b557 H
N S catalyze the two-electron reduc-
HN tion of NO3 to NO 2 , in which a
MoCo 2e O O novel Mo-containing cofactor
NAD+
—
H2N N N O
—
Nitrate reductase H P plays a central role. NADH is the
—
—
—
Mo cofactor
O O electron donor. (b) Nitrite reduc-
tase converts the product of
NO 2 nitrate reductase into NH4 in a
six-electron, eight-proton trans-
(b) COOH
COOH fer process in which the metallic
H3C center in siroheme carries elec-
trons, and the carboxyl groups of
Fdred HOOC
siroheme may donate protons.
H3C N N COOH The initial source of electrons is
Fe-S Siroheme 6e
2
Fe reduced ferredoxin.
Fdox HOOC N N
Nitrite reductase
COOH
COOH COOH
NH 4 Siroheme
which can be used by all organisms either directly or binding sites for ATP/ADP (one site on each subunit).
after its conversion to other soluble compounds such as Dinitrogenase (Mr 240,000), an ␣2␤2 tetramer, has two
nitrites, nitrates, or amino acids. Fe-containing cofactors that transfer electrons (Fig.
The reduction of nitrogen to ammonia is an exer- 22–3b). One, the P cluster, has a pair of 4Fe-4S centers;
gonic reaction: these share a sulfur atom, making an 8Fe-7S center. The
second cofactor in dinitrogenase, the FeMo cofactor, is
N2 1 3H2 ¡ 2NH3 G98 5 233.5 kJ/mol
a novel structure composed of 7 Fe atoms, 9 inorganic S
The N;N triple bond, however, is very stable, with a atoms, a Cys side chain, and a single carbon atom in the
bond energy of 930 kJ/mol. Nitrogen fixation therefore center of the FeS cluster. Also part of the cofactor is a
has an extremely high activation energy, and atmo- molybdenum atom, with ligands that include three inor-
spheric nitrogen is almost chemically inert under nor- ganic S atoms, a His side chain, and two oxygen atoms
mal conditions. Ammonia is produced industrially by from a molecule of homocitrate that is an intrinsic part
the Haber process (named for its inventor, Fritz Haber), of the FeMo cofactor. There is also a form of nitrogenase
which requires temperatures of 400 to 500 8C and nitro- that contains vanadium rather than molybdenum, and
gen and hydrogen at pressures of tens of thousands of some bacterial species can produce both types. The
kilopascals (several hundred atmospheres) to provide vanadium-containing enzyme may be the primary
the necessary activation energy. Biological nitrogen nitrogen-fixing system under some conditions. The
fixation, however, must occur at biological tempera- vanadium nitrogenase of Azotobacter vinelandii has the
tures and at 0.8 atm of nitrogen, and the high activation remarkable capacity to catalyze the reduction of carbon
barrier is overcome by other means. This is accom- monoxide (CO) to ethylene (C2H4), ethane, and propane.
plished, at least in part, by the binding and hydrolysis of Nitrogen fixation is carried out by a highly reduced
ATP. The overall reaction can be written form of dinitrogenase and requires eight electrons: six
for the reduction of N2 and two to produce one molecule
N2 1 10H 1 1 8e 2 1 16ATP ¡
of H2. Production of H2 is an obligate part of the reaction
2NH 1
4 1 16ADP 1 16Pi 1 H2
mechanism, but its biological role in the process is not
Biological nitrogen fixation is carried out by a highly understood.
conserved complex of proteins called the nitrogenase Dinitrogenase is reduced by the transfer of elec-
complex; its central components are dinitrogenase trons from dinitrogenase reductase (Fig. 22–4). The
reductase and dinitrogenase (Fig. 22–3a). Dinitro- dinitrogenase tetramer has two binding sites for the
genase reductase (Mr 60,000) is a dimer of two identical reductase. The required eight electrons are transferred
subunits. It contains a single 4Fe-4S redox center (see from reductase to dinitrogenase one at a time: a reduced
Fig. 19–5), bound between the subunits, and can be reductase molecule binds to the dinitrogenase and
oxidized and reduced by one electron. It also has two transfers a single electron, then the oxidized reductase
BOX 22–1 Unusual Lifestyles of the Obscure but Abundant

Air-breathers that we are, we can easily overlook the organisms do for a living. Their metabolic pathways are
bacteria and archaea that thrive in anaerobic environ- replete with interesting reactions and highly specialized
ments. Although rarely featured in introductory bio- cofactors unknown in our own world of obligate aerobic
chemistry textbooks, these organisms constitute metabolism. Study of these organisms can yield practi-
much of the biomass of this planet, and their contri- cal dividends. It can also provide clues about the origins
butions to the balance of carbon and nitrogen in the of life on an early Earth, in an atmosphere that lacked
biosphere are essential to all forms of life. molecular oxygen.
As detailed in earlier chapters, the energy used to The nitrogen cycle depends on a wide range of spe-
maintain living systems relies on the generation of cialized bacteria. There are two groups of nitrifying
proton gradients across membranes. Electrons derived bacteria: those that oxidize ammonia to nitrites and
from a reduced substrate are made available to elec- those that oxidize the resulting nitrites to nitrates (see
tron carriers in membranes and pass through a series Fig. 22–1). Nitrate is second only to O2 as a biological
of electron transfers to a final electron acceptor. As a electron acceptor, and a great many bacteria and
byproduct of this process, protons are released on one archaea can catalyze the denitrification of nitrates to
side of the membrane, generating the transmembrane nitrogen, which the nitrogen-fixing bacteria then con-
proton gradient. The proton gradient is used to syn- vert back into ammonia. Ammonia is a major pollutant in
thesize ATP or to drive other energy-requiring pro- sewage and in farm animal waste, and is a byproduct of
cesses. For all eukaryotes, the reduced substrate is fertilizer manufacture and oil refining. Waste-treatment
generally a carbohydrate (glucose or pyruvate) or a plants have generally made use of communities of nitri-
fatty acid and the electron acceptor is oxygen. fying and denitrifying bacteria to convert ammonia
Many bacteria and archaea are much more versatile. waste to atmospheric nitrogen. The process is expen-
In anaerobic environments such as marine and freshwa- sive, requiring inputs of organic carbon and oxygen.
ter sediments, the variety of life strategies is extraordi- In the 1960s and 1970s, a few articles appeared in
nary. Almost any available redox pair can be an energy the research literature suggesting that ammonia
source for some specialized organism or group of organ- could be oxidized to nitrogen anaerobically, using
isms. For example, a large number of lithotrophic bacte- nitrite as an electron acceptor; this process was called
ria (a lithotroph is a chemotroph that uses inorganic anammox. The reports received little notice until bac-
energy sources; see Fig. 1–5) have a hydrogenase that teria promoting anammox were discovered in a waste-
uses molecular hydrogen to reduce NAD⫹: treatment system in Delft, the Netherlands, in the
mid-1980s. A team of Dutch microbiologists led by
hydrogenase "
H2 ⫹ NAD⫹ NADH ⫹ H⫹ Gijs Kuenen and Mike Jetten began to study these
bacteria, which were soon identified as belonging to
The NADH is a source of electrons for a variety of an unusual bacterial phylum, Planctomycetes. Some
membrane-bound electron acceptors, generating the surprises were to follow.
proton gradient needed for ATP synthesis. Other litho- The biochemistry underlying the anammox pro-
trophs oxidize sulfur compounds (H2S, elemental sul- cess was slowly unraveled (Fig. 1). Hydrazine (N2H4),
fur, or thiosulfate) or ferrous iron. A widespread group
of archaea called methanogens, all strict anaerobes,
extract energy from the reduction of CO2 to methane. b
And this is just a small sampling of what anaerobic a d a
b b
a
ATP
Cytosol
H2O NO2– ADP +
5H+
NH3 NH2OH e
FIGURE 1 The anammox reactions. Ammonia and hydroxy- Nitrite-
reducing
b2 g
lamine are converted to hydrazine and H2O by hydrazine enzyme
hydrolase, and the hydrazine is oxidized by hydrazine- Hydrazine 4e – a c10
oxidizing enzyme, generating N2 and protons. The pro- hydrolase
tons generate a proton gradient for ATP synthesis. On Hydrazine-
the anammoxosome exterior, protons are used by the oxidizing
enzyme 4H+
nitrite-reducing enzyme, producing hydroxylamine and
H2O N2H4
completing the cycle. All of the anammox enzymes are N2
embedded in the anammoxosome membrane. Anammoxosome
(a) O (b)
⫺
O C
HO CH2
HC O
HO CH2
HO CH2
HC O
H2C O C
O
FIGURE 2 (a) Ladderane lipids of the anammoxosome membrane. dense, impermeable, hydrophobic membrane structure, allowing
The mechanism for synthesis of the unstable fused cyclobutane ring sequestration of the hydrazine produced in the anammox reactions.
structures is unknown. (b) Ladderanes can stack to form a very
a highly reactive molecule used as a rocket fuel, was For now, the anammox bacteria offer a major
an unexpected intermediate. As a small molecule, advance in waste treatment, reducing the cost of
hydrazine is both highly toxic and difficult to con- ammonia removal by as much as 90% (the conven-
tain. It readily diffuses across typical phospholipid tional denitrification steps are eliminated completely,
membranes. The anammox bacteria solve this prob- and the aeration costs associated with nitrification are
lem by sequestering hydrazine in a specialized lower) and reducing the release of polluting byprod-
organelle, dubbed the anammoxosome. The mem- ucts. Clearly, a greater familiarity with the bacterial
brane of this organelle is composed of lipids known underpinnings of the biosphere can pay big dividends
as ladderanes (Fig. 2), never before encountered as we deal with the environmental challenges of the
in biology. The fused cyclobutane rings of ladder- twenty-first century.
anes stack tightly to form a very dense barrier,
greatly slowing the release of hydrazine. Cyclobu-
tane rings are strained and difficult to synthesize;
the bacterial mechanisms for synthesizing these
lipids are not yet known.
The anammoxosome was a surprising finding.
Bacterial cells generally do not have compartments, NE
and the lack of a membrane-enclosed nucleus is often
cited as the primary distinction between eukaryotes N
and bacteria. One type of organelle in a bacterium
was interesting enough, but planctomycetes also
have a nucleus: their chromosomal DNA is contained
within a membrane (Fig. 3). Discovery of this subcel-
lular organization has prompted further research to
trace the origin of the planctomycetes and the evolu-
tion of eukaryotic nuclei. Planctomycetes are an
ancient bacterial line with multiple genera, three of
which are known to carry out the anammox reac-
0.2 m
tions. Further study of this group may ultimately
bring us closer to a key goal of evolutionary biology: FIGURE 3 Transmission electron micrograph of a cross section
a description of the organism affectionately referred through Gemmata obscuriglobus, showing the DNA in a nucleus (N)
to as LUCA—the last universal common ancestor of with enclosing nuclear envelope (NE). Bacteria of the Gemmata genus
all life on our planet. (phylum Planctomycetes) do not promote the anammox reactions.
(a) (b)
a-Cys88
ADP
Pred
Mg
4Fe-4S
redox
center 6e
P cluster
6e Pox
FeMo cofactor
6e
N2 2NH3
Dinitrogenase FeMo cofactor

(a subunit)
Dinitrogenase Homocitrate
(b subunit)
Dinitrogenase a-His195
Mo
reductase
FIGURE 22–3 Enzymes and cofactors of the nitrogenase complex. (PDB make the crystal more stable. (b) The electron-transfer cofactors. A P
ID 1FP6 and PDB ID 1M1N) (a) The holoenzyme consists of two identi- cluster is shown here in its reduced (top) and oxidized (middle) forms.
cal dinitrogenase reductase molecules (green), each with a 4Fe-4S The FeMo cofactor (bottom) has a Mo atom with three S ligands, a His
redox center and binding sites for two ATP, and two identical dinitroge- ligand, and two oxygen ligands from a molecule of homocitrate. In some
nase heterodimers (purple and blue), each with a P cluster (Fe-S center) organisms, the Mo atom is replaced with a vanadium atom. (Fe is
and an FeMo cofactor. In this structure, ADP is bound in the ATP site, to shown in orange, S in yellow.)
dissociates from dinitrogenase, in a repeating cycle.

Each turn of the cycle requires the hydrolysis of two 4CoA + 4CO2 +
ATP molecules by the dimeric reductase. The immediate 4 pyruvate 4 acetyl-CoA
source of electrons to reduce dinitrogenase reductase
varies, with reduced ferredoxin (see Section 19.8), 8e–
reduced flavodoxin, and perhaps other sources playing a
role. In at least one species, the ultimate source of elec-
trons to reduce ferredoxin is pyruvate (Fig. 22–4). 8 Ferredoxin or 8 Ferredoxin or
8 flavodoxin 8 flavodoxin
The role of ATP in this process is somewhat unusual. (oxidized) (reduced)
Recall that ATP can contribute not only chemical energy,
through the hydrolysis of one or more of its phosphoan- 8e–
hydride bonds, but also binding energy (p. 195),

through noncovalent interactions that lower the activa-
tion energy. In the reaction carried out by dinitrogenase 8 Dinitrogenase 8 Dinitrogenase
reductase reductase
reductase, both ATP binding and ATP hydrolysis bring (reduced) (oxidized)
about protein conformational changes that help over-
16 ATP 16 ADP
come the high activation energy of nitrogen fixation. The + 16Pi
binding of two ATP molecules to the reductase shifts the
8 Dinitrogenase 8 Dinitrogenase
reduction potential (E98) of this protein from 2300 to reductase (reduced) reductase (oxidized)
2420 mV, an enhancement of its reducing power that is + 16ATP + 16ATP
FIGURE 22–4 Electron path in nitrogen fixation by the nitrogenase 8e–

complex. Electrons are transferred from pyruvate to dinitrogenase via
ferredoxin (or flavodoxin) and dinitrogenase reductase. Dinitrogenase
reductase reduces dinitrogenase one electron at a time, with at least six Dinitrogenase Dinitrogenase
electrons required to fix one molecule of N2. Two additional electrons (oxidized) (reduced)
are used to reduce 2H⫹ to H2 in a process that obligatorily accompanies
nitrogen fixation in anaerobes, making a total of eight electrons required 6e–
per N2 molecule. The subunit structures and metal cofactors of the dini-
trogenase reductase and dinitrogenase proteins are described in the text H2 2H+
2NH+4 N2
and in Figure 22–3.
required to transfer electrons through dinitrogenase to N

Dinitrogen N2
N2; the standard reduction potential for the half-reaction N
N2 1 6H⫹ 1 6e ¡ 2NH3 is 20.34 V. The ATP mole-
cules are then hydrolyzed just before the actual transfer M (FeMo cofactor)
of one electron to dinitrogenase.
NH
ATP binding and hydrolysis change the conforma- Diazenido
tion of nitrogenase reductase in two regions, which are N
structurally homologous with switch 1 and switch 2 M
regions of the GTP-binding proteins involved in biologi-
cal signaling (see Box 12–2). ATP binding produces a
conformational change that brings the 4Fe-4S center of NH2 NH
the reductase closer to the P cluster of dinitrogenase
N NH
(from 18 Å to 14 Å away), which facilitates electron
transfer between the reductase and dinitrogenase. The M M
details of electron transfer from the P cluster to the NH3 NH2
FeMo cofactor, and the means by which eight electrons
are accumulated by nitrogenase, are not known, nor are N NH
Nitrido Hydrazido
the intermediates in the reaction known with certainty; M M
two reasonable hypotheses are being tested, both
involving the Mo atom as a central player (Fig. 22–5). NH2
The nitrogenase complex is remarkably unstable in NH NH2
the presence of oxygen. The reductase is inactivated in Imido
air, with a half-life of 30 seconds; dinitrogenase has a M M
half-life of only 10 minutes in air. Free-living bacteria
that fix nitrogen cope with this problem in a variety of NH2 NH3
Amido
ways. Some live only anaerobically or repress nitroge- M
nase synthesis when oxygen is present. Some aerobic Amine
species, such as Azotobacter vinelandii, partially NH3
uncouple electron transfer from ATP synthesis so that M
oxygen is burned off as rapidly as it enters the cell (see
Box 19–1). When fixing nitrogen, cultures of these bac- NH3
teria actually increase in temperature as a result of their FIGURE 22–5 Two reasonable hypotheses for the intermediates involved
efforts to rid themselves of oxygen. in N2 reduction. In both scenarios, the FeMo cofactor (abbreviated as M
The symbiotic relationship between leguminous here) plays a central role, binding directly to one of the nitrogen atoms of
plants and the nitrogen-fixing bacteria in their root nod- N2 and remaining bound throughout the sequence of reduction steps.
ules (Fig. 22–6) takes care of both the energy require-
ments and the oxygen lability of the nitrogenase complex. reservoir of energy in the form of abundant carbohydrate
The energy required for nitrogen fixation was probably and citric acid cycle intermediates made available by the
the evolutionary driving force for this plant-bacteria asso- plant. This may allow the bacteria to fix hundreds of times
ciation. The bacteria in root nodules have access to a large more nitrogen than do their free-living cousins under
FIGURE 22–6 Nitrogen-fixing nodules. (a) Root nodules

of the common pea, Pisum saliva, a legume. The flower of
this plant is shown in the inset. (b) Artificially colorized
electron micrograph of a thin section through a pea root
nodule. Symbiotic nitrogen-fixing bacteria, or bacteroids
(red), live inside the nodule cell, surrounded by the peri-
bacteroid membrane (blue). Bacteroids produce the nitro-
genase complex that converts atmospheric nitrogen (N2)
to ammonium (NH⫹ 4); without the bacteroids, the plant is
unable to utilize N2. The infected root cell provides some
factors essential for nitrogen fixation, including leghemo-
globin; this heme protein has a very high binding affinity
for oxygen, which strongly inhibits nitrogenase. (The cell
nucleus is shown in yellow/green. Not visible in this
micrograph are other organelles of the infected root cell
that are normally found in plant cells.) (a) (b) 2 ␮m
conditions generally encountered in soils. To solve the provide the critical entry point. Recall that these same
oxygen-toxicity problem, the bacteria in root nodules are two amino acids play central roles in the catabolism of
bathed in a solution of the oxygen-binding heme protein ammonia and amino groups in amino acid oxidation
leghemoglobin, produced by the plant (although the (Chapter 18). Glutamate is the source of amino groups for
heme may be contributed by the bacteria). Leghemoglo- most other amino acids, through transamination reactions
bin binds all available oxygen so that it cannot interfere (the reverse of the reaction shown in Fig. 18–4). The
with nitrogen fixation, and efficiently delivers the oxygen amide nitrogen of glutamine is a source of amino groups
to the bacterial electron-transfer system. The benefit to in a wide range of biosynthetic processes. In most types of
the plant, of course, is a ready supply of reduced nitro- cells, and in extracellular fluids in higher organisms, one
gen. In fact, the bacterial symbionts typically produce far or both of these amino acids are present at higher concen-
more NH3 than is needed by their symbiotic partner; the trations—sometimes an order of magnitude or more
excess is released into the soil. The efficiency of the sym- higher—than other amino acids. An Escherichia coli cell
biosis between plants and bacteria is evident in the requires so much glutamate that this amino acid is one of
enrichment of soil nitrogen brought about by leguminous the primary solutes in the cytosol. Its concentration is
plants. This enrichment of NH3 in the soil is the basis of regulated not only in response to the cell’s nitrogen
crop rotation methods, in which plantings of nonlegumi- requirements but also to maintain an osmotic balance
nous plants (such as maize) that extract fixed nitrogen between the cytosol and the external medium.
from the soil are alternated every few years with plant- The biosynthetic pathways to glutamate and gluta-
ings of legumes such as alfalfa, peas, or clover. mine are simple, and all or some of the steps occur in most
Nitrogen fixation is energetically costly: 16 ATP and organisms. The most important pathway for the assimila-
8 electron pairs yield only 2 NH3. It is therefore not sur- tion of NH⫹4 into glutamate requires two reactions. First,
prising that the process is tightly regulated, so that NH3 is glutamine synthetase catalyzes the reaction of gluta-
produced only when needed. High [ADP], an indicator of mate and NH⫹4 to yield glutamine. This reaction takes
low [ATP], is a strong inhibitor of nitrogenase. NH⫹ 4 place in two steps, with enzyme-bound ␥-glutamyl phos-
represses the expression of the ⬃20 nitrogen fixation phate as an intermediate (see Fig. 18–8):
(nif ) genes, effectively shutting down the pathway. (1) Glutamate 1 ATP ¡
Covalent alteration of nitrogenase is also used in some ␥-glutamyl phosphate 1 ADP
diazotrophs to control nitrogen fixation in response to the
availability of NH⫹4 in the surroundings. Transfer of an (2) ␥-Glutamyl phosphate 1 NH⫹4 ¡
ADP-ribosyl group from NADH to a specific Arg residue in glutamine 1 Pi 1 H⫹
the nitrogenase reductase shuts down N2 fixation in Rho- Sum: Glutamate 1 NH⫹4 1 ATP ¡
dospirillum, for example. This is the same covalent glutamine 1 ADP 1 Pi 1 H⫹ (22–1)
modification that we saw in the case of G protein inhibi-
tion by the toxins of cholera and pertussis (see Box 12–2). Glutamine synthetase is found in all organisms. In addi-
Nitrogen fixation is the subject of intense study tion to its importance for NH⫹4 assimilation in bacteria, it
because of its immense practical importance. Industrial has a central role in amino acid metabolism in mammals,
production of ammonia for use in fertilizers requires a converting free NH⫹4, which is toxic, to glutamine for
large and expensive input of energy, and this has transport in the blood (Chapter 18).
spurred a drive to develop recombinant or transgenic In bacteria and plants, glutamate is produced from
organisms that can fix nitrogen. In principle, recombi- glutamine in a reaction catalyzed by glutamate syn-
nant DNA techniques (Chapter 9) might be used to thase. (An alternative name for this enzyme, glutamate:
transfer the DNA that encodes the enzymes of nitrogen oxoglutarate aminotransferase, yields the acronym
fixation into non-nitrogen-fixing bacteria and plants. GOGAT, by which the enzyme also is known.) ␣-Ketoglu-
However, those genes alone will not suffice. About 20 tarate, an intermediate of the citric acid cycle, undergoes
genes are essential to nitrogenase activity in bacteria, reductive amination with glutamine as nitrogen donor:
many of them needed for the synthesis, assembly, and ␣-Ketoglutarate 1 glutamine 1 NADPH 1 H⫹ ¡
insertion of the cofactors. There is also the problem of 2 glutamate 1 NADP⫹ (22–2)
protecting the enzyme in its new setting from destruc- The net reaction of glutamine synthetase and glutamate
tion by oxygen. In all, there are formidable challenges in synthase (Eqns 22–1 and 22–2) is
engineering new nitrogen-fixing plants. Success in
these efforts will depend on overcoming the problem of ␣-Ketoglutarate 1 NH⫹4 1 NADPH 1 ATP ¡
L-glutamate 1 NADP 1 ADP 1 Pi
⫹
oxygen toxicity in any cell that produces nitrogenase.
Glutamate synthase is not present in animals, which
Ammonia Is Incorporated into Biomolecules through instead maintain high levels of glutamate by processes
such as the transamination of ␣-ketoglutarate during
Glutamate and Glutamine amino acid catabolism.
Reduced nitrogen in the form of NH⫹4 is assimilated into Glutamate can also be formed in yet another, albeit
amino acids and then into other nitrogen-containing bio- minor, pathway: the reaction of ␣-ketoglutarate and
molecules. Two amino acids, glutamate and glutamine, NH⫹4 to form glutamate in one step. This is catalyzed by
L-glutamate dehydrogenase, an enzyme present in all Glutamate

organisms. Reducing power is furnished by NADPH:
NH3
␣-Ketoglutarate 1 NH⫹4 1 NADPH ¡
L-glutamate 1 NADP 1 H2O
⫹ glutamine
synthetase ATP
We encountered this reaction in the catabolism of amino
acids (see Fig. 18–7). In eukaryotic cells, L-glutamate
dehydrogenase is located in the mitochondrial matrix. ADP + Pi
The reaction equilibrium favors the reactants, and the Km Glycine
for NH⫹4 (⬃1 mM) is so high that the reaction probably Alanine
makes only a modest contribution to NH⫹ 4 assimilation
into amino acids and other metabolites. (Recall that the
glutamate dehydrogenase reaction, in reverse (see Fig.
18–10), is one source of NH⫹4 destined for the urea
cycle.) Concentrations of NH ⫹
4 high enough for the gluta-
mate dehydrogenase reaction to make a significant con- AMP Glutamine CTP
tribution to glutamate levels generally occur only when
NH3 is added to the soil or when organisms are grown in Tryptophan Histidine
a laboratory in the presence of high NH3 concentrations.
In general, soil bacteria and plants rely on the two-
enzyme pathway outlined above (Eqns. 22–1, 22–2). Carbamoyl phosphate Glucosamine 6-phosphate
FIGURE 22–8 Allosteric regulation of glutamine synthetase. The

Glutamine Synthetase Is a Primary Regulatory enzyme undergoes cumulative regulation by six end products of gluta-
Point in Nitrogen Metabolism mine metabolism. Alanine and glycine probably serve as indicators of
the general status of amino acid metabolism in the cell.
The activity of glutamine synthetase is regulated in vir-
tually all organisms—as expected, given its central met-
six end products of glutamine metabolism are allosteric
abolic role as an entry point for reduced nitrogen. In
inhibitors of the enzyme (Fig. 22–8). Each inhibitor
enteric bacteria such as E. coli, the regulation is unusu-
alone produces only partial inhibition, but the effects of
ally complex. Type I enzyme (from bacteria) has 12
multiple inhibitors are more than additive, and all eight
identical subunits of Mr 50,000 (Fig. 22–7) and is regu-
together virtually shut down the enzyme. This is an
lated both allosterically and by covalent modification.
example of cumulative feedback inhibition. This control
(Type II enzyme, from eukaryotes and some bacteria,
mechanism provides a constant adjustment of glutamine
has 10 identical subunits.) Alanine, glycine, and at least
levels to match immediate metabolic requirements.
Superimposed on the allosteric regulation is inhibi-
tion by adenylylation of (addition of AMP to) Tyr397,
located near the enzyme’s active site (Fig. 22–9). This
covalent modification increases sensitivity to the allo-
ADP
Glutamate
steric inhibitors, and activity decreases as more sub-
units are adenylylated. Both adenylylation and deade-
nylylation are promoted by adenylyltransferase (AT
in Fig. 22–9), part of a complex enzymatic cascade that
responds to levels of glutamine, ␣-ketoglutarate, ATP,
and Pi. The activity of adenylyltransferase is modulated
by binding to a regulatory protein called PII, and the
activity of PII, in turn, is regulated by covalent modifica-
tion (uridylylation), again at a Tyr residue. The adenyl-
yltransferase complex with uridylylated PII (PII-UMP)
stimulates deadenylylation, whereas the same complex
with deuridylylated PII stimulates adenylylation of gluta-
mine synthetase. Both uridylylation and deuridylylation
of PII are brought about by a single enzyme, uridylyl-
transferase. Uridylylation is inhibited by binding of
FIGURE 22–7 Subunit structure of bacterial type I glutamine synthetase. glutamine and Pi to uridylyltransferase and is stimulated
(PDB ID 2GLS) This view shows 6 of 12 the identical subunits; a second by binding of ␣-ketoglutarate and ATP to PII.
layer of 6 subunits lies directly beneath the six shown. Each of the 12 sub- The regulation does not stop there. The uridylylated
units has an active site, where ATP and glutamate are bound in orienta- PII also mediates the activation of transcription of the
tions that favor transfer of a phosphoryl group from ATP to the side-chain gene encoding glutamine synthetase, thus increasing the
carboxyl of glutamate. In this crystal structure, ADP occupies the ATP site. cellular concentration of the enzyme; the deuridylylated
(a) Figs 18–6, 18–17, and 18–18). Here we focus on amino

O group transfer involving the amide nitrogen of glutamine.
Tyr O P O CH2 O Adenine More than a dozen known biosynthetic reactions use
Enzyme glutamine as the major physiological source of amino
—
O
H H groups, and most of these occur in the pathways outlined
H H
in this chapter. As a class, the enzymes catalyzing these
OH OH reactions are called glutamine amidotransferases. All
(b) have two structural domains: one binding glutamine, the
AT other binding the second substrate, which serves as
amino group acceptor (Fig. 22–10). A conserved Cys
AT PII
PII Glutamine- NH3-
␣-Ketoglutarate binding domain acceptor domain
PPi ATP
UTP UMP
Glutamate COO–
adenylylation NH3 +
H3N CH
Glutamine Glutamine ATP
synthetase synthetase UT CH2
(inactive) (active) NH3
ADP uridylylation CH2
deadenylylation channel
AMP + Pi H+
:
Cys SH C
Pi ADP Glutamine PPi H2O O NH2
Glutamine
AT
PII
PII Glutamine
UMP amidotransferase
UMP AT
The g-amido nitrogen of R1
FIGURE 22–9 Second level of regulation of glutamine synthetase: cova- glutamine (red) is released as R OH or C O
lent modifications. (a) An adenylylated Tyr residue. (b) Cascade leading NH3 in a reaction that probably
involves a covalent glutamyl- 1 R2
to adenylylation (inactivation) of glutamine synthetase. AT represents ade- Acceptor
enzyme intermediate. The NH3
nylyltransferase; UT, uridylyltransferase. PII is a regulatory protein, itself travels via a channel to the
regulated by uridylylation. Details of this cascade are discussed in the text. second active site.
PII brings about a decrease in transcription of the same Activated

substrate
gene. This mechanism involves an interaction of PII
with additional proteins involved in gene regulation, of COO–
a type described in Chapter 28. The net result of this H3N
+
CH
R OX
elaborate system of controls is a decrease in glutamine or
CH2
synthetase activity when glutamine levels are high, and NH3 1
an increase in activity when glutamine levels are low CH2 R
and ␣-ketoglutarate and ATP (substrates for the syn- Cys S C C O
thetase reaction) are available. The multiple layers of O R2
regulation permit a sensitive response in which gluta-
mine synthesis is tailored to cellular needs.
Glutamyl-enzyme
intermediate H2O
Several Classes of Reactions Play Special Roles in the
NH3 reacts with any of several
Biosynthesis of Amino Acids and Nucleotides acceptors.
2 Glutamate
The pathways described in this chapter include a variety R NH2 + H OX
of interesting chemical rearrangements. Several of these
recur and deserve special note before we progress to the or
pathways themselves. These are (1) transamination reac- R1
tions and other rearrangements promoted by enzymes C NH + H2O
containing pyridoxal phosphate; (2) transfer of one-car- R2
bon groups, with either tetrahydrofolate (usually at the MECHANISM FIGURE 22–10 Proposed mechanism for glutamine amido-
—CHO and —CH2OH oxidation levels) or S-adenosylme- transferases. Each enzyme has two domains. The glutamine-binding domain
thionine (at the —CH3 oxidation level) as cofactor; and contains structural elements conserved among many of these enzymes,
(3) transfer of amino groups derived from the amide including a Cys residue required for activity. The NH3-acceptor (second-
nitrogen of glutamine. Pyridoxal phosphate (PLP), tetra- substrate) domain varies. Two types of amino acceptors are shown. X rep-
hydrofolate (H4 folate), and S-adenosylmethionine resents an activating group, typically a phosphoryl group derived from ATP,
(adoMet) are described in some detail in Chapter 18 (see that facilitates displacement of a hydroxyl group from R—OH by NH3.
residue in the glutamine-binding domain is believed to act

as a nucleophile, cleaving the amide bond of glutamine
22.2 Biosynthesis of Amino Acids
and forming a covalent glutamyl-enzyme intermediate. All amino acids are derived from intermediates in gly-
The NH3 produced in this reaction is not released, but colysis, the citric acid cycle, or the pentose phosphate
instead is transferred through an “ammonia channel” to a pathway (Fig. 22–11). Nitrogen enters these pathways
second active site, where it reacts with the second sub- by way of glutamate and glutamine. Some pathways
strate to form the aminated product. The covalent inter- are simple, others are not. Ten of the amino acids are
mediate is hydrolyzed to the free enzyme and glutamate. just one or several steps removed from the common
If the second substrate must be activated, the usual metabolite from which they are derived. The biosyn-
method is the use of ATP to generate an acyl phosphate thetic pathways for others, such as the aromatic
intermediate (R—OX in Fig. 22–10, with X as a phospho- amino acids, are more complex.
ryl group). The enzyme glutaminase acts in a similar Organisms vary greatly in their ability to synthe-
fashion but uses H2O as the second substrate, yielding size the 20 common amino acids. Whereas most bacte-
NH⫹ 4 and glutamate (see Fig. 18–8). ria and plants can synthesize all 20, mammals can
synthesize only about half of them—generally those
SUMMARY 22.1 Overview of Nitrogen Metabolism
The molecular nitrogen that makes up 80% of Glucose
Earth’s atmosphere is unavailable to most living
organisms until it is reduced. This fixation of
atmospheric N2 takes place in certain free-living
bacteria and in symbiotic bacteria in the root Glucose 6-phosphate
nodules of leguminous plants.
In soil bacteria and vascular plants, the sequential 4 steps
action of nitrate reductase and nitrite reductase

converts NO3 to NH3, which can be assimilated Ribose 5-
4 steps phosphate
into nitrogen-containing compounds.
The nitrogen cycle entails formation of ammonia by
bacterial fixation of N2, nitrification of ammonia to Histidine
nitrate by soil organisms, conversion of nitrate to
ammonia by higher plants, synthesis of amino acids
Erythrose 4-
from ammonia by all organisms, and conversion of phosphate
3-Phosphoglycerate Serine
nitrate to N2 by denitrifying soil bacteria. The
anammox bacteria anaerobically oxidize ammonia
to nitrogen, using nitrite as an electron acceptor.
Fixation of N2 as NH3 is carried out by the Glycine
nitrogenase complex, in a reaction that requires Phosphoenolpyruvate Cysteine
large investments of ATP and of reducing power. The

nitrogenase complex is highly labile in the presence of Alanine
Tryptophan
O2, and is subject to regulation by the supply of NH3. Phenylalanine Pyruvate Valine
Tyrosine Leucine
In living systems, reduced nitrogen is incorporated Isoleucine
first into amino acids and then into a variety of
other biomolecules, including nucleotides. The key Citrate
entry point is the amino acid glutamate. Glutamate
and glutamine are the nitrogen donors in a wide
range of biosynthetic reactions. Glutamine
Oxaloacetate -Ketoglutarate
synthetase, which catalyzes the formation of
glutamine from glutamate, is a main regulatory
enzyme of nitrogen metabolism.
Aspartate Glutamate
The amino acid and nucleotide biosynthetic
pathways make repeated use of the biological
cofactors pyridoxal phosphate, tetrahydrofolate, and
S-adenosylmethionine. Pyridoxal phosphate is Asparagine Glutamine
Methionine
required for transamination reactions involving Threonine
Proline
glutamate and for other amino acid transformations. Arginine
Lysine
One-carbon transfers require S-adenosylmethionine
and tetrahydrofolate. Glutamine amidotransferases FIGURE 22–11 Overview of amino acid biosynthesis. The carbon skele-
catalyze reactions that incorporate nitrogen derived ton precursors derive from three sources: glycolysis (light red), the citric
from glutamine. acid cycle (blue), and the pentose phosphate pathway (purple).
with simple pathways. These are the nonessential

amino acids, not needed in the diet (see Table 18–1). TABLE 22–1 Amino Acid Biosynthetic Families,
The remainder, the essential amino acids, must be Grouped by Metabolic Precursor
obtained from food. Unless otherwise indicated, the ␣-Ketoglutarate Pyruvate
pathways for the 20 common amino acids presented
Glutamate Alanine
below are those operative in bacteria.
A useful way to organize these biosynthetic pathways Glutamine Valine*
is to group them into six families corresponding to their Proline Leucine*
metabolic precursors (Table 22–1), and we use this Arginine Isoleucine*
approach to structure the detailed descriptions that follow.
3-Phosphoglycerate Phosphoenolpyruvate
In addition to these six precursors, there is a notable inter-
mediate in several pathways of amino acid and nucleotide Serine and erythrose
synthesis: 5-phosphoribosyl-1-pyrophosphate (PRPP): Glycine 4-phosphate
O Cysteine Tryptophan*

O P O CH2 O H Oxaloacetate Phenylalanine*
O O O Aspartate Tyrosine†
H H
H O P O P O Asparagine Ribose 5-phosphate
OH OH O O Methionine* Histidine*
PRPP is synthesized from ribose 5-phosphate derived Threonine*
from the pentose phosphate pathway (see Fig. 14–22), Lysine*
in a reaction catalyzed by ribose phosphate pyro-
*Essential amino acids in mammals.
phosphokinase: †
Derived from phenylalanine in mammals.
Ribose 5-phosphate 1 ATP ¡
5-phosphoribosyl-1-pyrophosphate 1 AMP
that avoid the problem of the spontaneous cyclization of
This enzyme is allosterically regulated by many of the glutamate ␥-semialdehyde (Fig. 22–12). In the first step,
biomolecules for which PRPP is a precursor. the ␣-amino group of glutamate is blocked by an acetyla-
tion requiring acetyl-CoA; then, after the transamination
␣-Ketoglutarate Gives Rise to Glutamate, step, the acetyl group is removed to yield ornithine.
The pathways to proline and arginine are somewhat
Glutamine, Proline, and Arginine different in mammals. Proline can be synthesized by the
-Ketoglutarate pathway shown in Figure 22–12, but it is also formed
from arginine obtained from dietary or tissue protein.
Arginase, a urea cycle enzyme, converts arginine to orni-
Glutamate thine and urea (see Figs 18–10, 18–26). The ornithine is
converted to glutamate ␥-semialdehyde by the enzyme
ornithine ␦-aminotransferase (Fig. 22–13). The
Glutamine Proline Arginine semialdehyde cyclizes to 1-pyrroline-5-carboxylate,
which is then converted to proline (Fig. 22–12). The
We have already described the biosynthesis of gluta- pathway for arginine synthesis shown in Figure 22–12 is
mate and glutamine. Proline is a cyclized derivative absent in mammals. When arginine from dietary intake
of glutamate (Fig. 22–12). In the first step of proline or protein turnover is insufficient for protein synthesis,
synthesis, ATP reacts with the ␥-carboxyl group of glu- the ornithine ␦-aminotransferase reaction operates in
tamate to form an acyl phosphate, which is reduced by the direction of ornithine formation. Ornithine is then
NADPH or NADH to glutamate ␥-semialdehyde. This converted to citrulline and arginine in the urea cycle.
intermediate undergoes rapid spontaneous cyclization
and is then reduced further to yield proline. Serine, Glycine, and Cysteine Are Derived
Arginine is synthesized from glutamate via ornithine from 3-Phosphoglycerate
and the urea cycle in animals (Chapter 18). In principle,
3-Phosphoglycerate
ornithine could also be synthesized from glutamate ␥-semi-
aldehyde by transamination, but the spontaneous cyc-
lization of the semialdehyde in the proline pathway pre-
Serine
cludes a sufficient supply of this intermediate for ornithine
synthesis. Bacteria have a de novo biosynthetic pathway
for ornithine (and thus arginine) that parallels some steps
of the proline pathway but includes two additional steps Glycine Cysteine
O
B O
⫹ CH3 OCOS-CoA CoA-SH B
O NH3 O HN O COCH3
M A M A
COCH2 OCH2 OCHOCOO COCH2 OCH2 OCHOCOO

D acetylglutamate synthase
D
O O
Glutamate N-Acetylglutamate
ATP ATP
glutamate kinase N-acetylglutamate
kinase
ADP ADP
⫹ O
O NH3 B
M A HNOCOCH3
COCH2 OCH2 OCHOCOO O A
D ␥ -Glutamyl M
P OO phosphate COCH2 OCH2 OCHOCOO
D N-Acetyl--glutamyl
P OO phosphate
NAD(P)H ⫹ H⫹ NAD(P)H ⫹ H⫹
-glutamyl N-acetylglutamate
phosphate NAD(P)⫹ dehydrogenase
reductase NAD(P)⫹
Pi Pi
⫹ O
O NH3 B
M A HNOCOCH3
COCH2 OCH2 OCHOCOO O A
D M
H Glutamate -semialdehyde COCH2 OCH2 OCHOCOO
D N-Acetylglutamate
H -semialdehyde
nonenzymatic -H2O
Glutamate
aminotransferase
H 2C CH2
␣ -Ketoglutarate
HOC ⫹ CH OCOO
N O
⌬1 -Pyrroline-5-carboxylate / Pyroline-5-Foramte B
H HNOCOCH3
(P5C)
⫹ A
NAD(P)H ⫹ H⫹ H3NOCH2 OCH2 OCH2 OCHOCOO
pyrroline carboxylate N-Acetylornithine
reductase H2O
NAD(P)⫹ N-acetylornithinase
CH3COO
H C CH2
H 2
H ⫹
Urea cycle
C ⫹ CHOCOO NH3
f N ⫹ A
H H2 H3NOCH2 OCH2 OCH2 OCHOCOO Ornithine
Proline Carbamoyl phosphate
ornithine
carbamoyl-
FIGURE 22–12 Biosynthesis of proline and arginine from glutamate in bac- transferase
Pi
teria. All five carbon atoms of proline arise from glutamate. In many organ-
isms, glutamate dehydrogenase is unusual in that it uses either NADH or L-Citrulline
NADPH as a cofactor. The same may be true of other enzymes in these path-
ways. The ␥-semialdehyde in the proline pathway undergoes a rapid, revers- ATP ⫹ aspartate
argininosuccinate
ible cyclization to D1-pyrroline-5-carboxylate (P5C), with the equilibrium synthetase
favoring P5C formation. Cyclization is averted in the ornithine/arginine path- AMP ⫹ PPi
way by acetylation of the ␣-amino group of glutamate in the first step and
4 Glu are
Argininosuccinate making 1
removal of the acetyl group after the transamination. Although some bacteria
Asp
lack arginase and thus the complete urea cycle, they can synthesize arginine
argininosuccinase
from ornithine in steps that parallel the mammalian urea cycle, with citrulline Fumarate
and argininosuccinate as intermediates (see Fig. 18–10). ⫹
H2N NH3
Here, and in subsequent figures in this chapter, the reaction arrows indi- G A
cate the linear path to the final products, without considering the reversibility CONOCH2 OCH2 OCH2 OCHOCOO
⫹J
H
of individual steps. For example, the step of the pathway leading to arginine H2N
Arginine
that is catalyzed by N-acetylglutamate dehydrogenase is chemically similar
to the glyceraldehyde 3-phosphate dehydrogenase reaction in glycolysis
(see Fig. 14–8), and is readily reversible.
FIGURE 22–13 Ornithine ␦-aminotransferase COO⫺ ␣ -Ketoglutarate COO⫺

reaction: a step in the mammalian pathway ⫹ ⫹
to proline. This enzyme is found in the mito- H3N CH Glutamate H3N CH
chondrial matrix of most tissues. Although the H2O H2C CH2
CH2 CH2
equilibrium favors P5C formation, the reverse H C CH COO⫺
ornithine ⫹
reaction is the only mammalian pathway for CH2 CH2 H2O N
␦-aminotransferase
synthesis of ornithine (and thus arginine) H
CH2 C
when arginine levels are insufficient for protein
synthesis. ⫹
NH3 H O
Ornithine Glutamate ⌬1-Pyrroline-5-

␥-semialdehyde carboxylate
(P5C)
The major pathway for the formation of serine is the Serine (three carbons) is the precursor of glycine
same in all organisms (Fig. 22–14). In the first step, the (two carbons) through removal of a carbon atom by
hydroxyl group of 3-phosphoglycerate is oxidized by a serine hydroxymethyltransferase (Fig. 22–14). Tet-
dehydrogenase (using NAD⫹) to yield 3-phosphohy- rahydrofolate accepts the ␤ carbon (C-3) of serine,
droxypyruvate. Transamination from glutamate yields which forms a methylene bridge between N-5 and
3-phosphoserine, which is hydrolyzed to free serine by N-10 to yield N 5,N10-methylenetetrahydrofolate (see
phosphoserine phosphatase. Fig. 18–17). The overall reaction, which is reversible,
also requires pyridoxal phosphate. In the liver of verte-
COO brates, glycine can be made by another route: the
A reverse of the reaction shown in Figure 18–20c, cata-
HOCOOH lyzed by glycine synthase (also called glycine cleavage
A 3-Phosphoglycerate
HOCO O O P enzyme):
A
H
CO2 1 NH⫹4 1 N5,N10-methylenetetrahydrofolate 1
NAD⫹ NADH 1 H⫹ ¡ glycine 1 tetrahydrofolate 1 NAD⫹
phosphoglycerate
dehydrogenase
NADH ⫹ H⫹ Plants and bacteria produce the reduced sulfur
required for the synthesis of cysteine (and methio-
COO
A nine, described later) from environmental sulfates; the
CP O 3-Phosphohydroxypyruvate pathway is shown on the right side of Figure 22–15.
A
CH2 OO O P
Sulfate is activated in two steps to produce 39-phospho-
adenosine 59-phosphosulfate (PAPS), which undergoes
Glutamate an eight-electron reduction to sulfide. The sulfide is
phosphoserine
aminotransferase then used in the formation of cysteine from serine in a
␣ -Ketoglutarate two-step pathway. Mammals synthesize cysteine from
COO
two amino acids: methionine furnishes the sulfur atom,
A
⫹ and serine furnishes the carbon skeleton. Methionine is
H3NOCOH 3-Phosphoserine first converted to S-adenosylmethionine (see Fig.
A
CH2 OO O P 18–18), which can lose its methyl group to any of a
number of acceptors to form S-adenosylhomocysteine
phosphoserine
H2O (adoHcy). This demethylated product is hydrolyzed to
phosphatase free homocysteine, which undergoes a reaction with
Pi
serine, catalyzed by cystathionine ␤-synthase, to
COO yield cystathionine (Fig. 22–16). Finally, cystathio-
A
⫹ nine ␥-lyase, a PLP-requiring enzyme, catalyzes
H3NOCOH Serine
A removal of ammonia and cleavage of cystathionine to
CH2OH yield free cysteine.
H4 folate
serine PLP
hydroxymethyl-
transferase N 5, N 10-Methylene H4 folate
H2O
COO FIGURE 22–14 Biosynthesis of serine from 3-phosphoglycerate and of

⫹ A glycine from serine in all organisms. Glycine is also made from CO2 and
H3NOCOH Glycine
A NH4+ by the action of glycine synthase, with N5,N10-methylenetetrahydro-
H folate as methyl group donor (see text).
COO⫺ ATP ⫹ SO42

⫹ ⫹
H3N C H ⌯
ATP sulfurylase
Serine PPi
CH2
O O Adenine
OH O ⫺
O S O P O CH2 O
H3C C
serine ⫺ ⫺ Adenosine
acetyltransferase O O H H
S-CoA 5⬘-phosphosulfate (APS)
CoA-SH H H
⫺
COO OH OH
⫹
H3N C H ATP
APS kinase
ADP
CH2
O-Acetylserine O O Adenine
O
⫺
O S O P O CH2 O
C O
⫺ ⫺
O O H H
CH3 H H
2⫺ ⫹
3⬘-Phosphoadenosine
O-acetylserine S ⫹H 5⬘-phosphosulfate (PAPS)
(thiol) lyase O OH
CH3COO⫺
⫺
O P O
COO⫺
⫺
⫹ O
H3N C H
Cysteine NADPH
CH2 PAPS reductase NADP⫹
SH 3⬘-Phosphoadenosine 5⬘-phosphate (PAP)
SO32⫺ Sulfite
FIGURE 22–15 Biosynthesis of cysteine from serine in bacteria and 3NADPH
plants. The origin of reduced sulfur is shown in the pathway on the right. sulfide reductase
3NADP⫹
S2⫺ Sulfide
⫹
Three Nonessential and Six Essential Amino Acids
NH3 Are Synthesized from Oxaloacetate and Pyruvate
⫺
Met--3steps-- OOC CH CH2 CH2 SH ⫹ HOCH2 CH COO⫺ Oxaloacetate
⫹
NH3
Homocysteine Serine Aspartate
PLP
cystathionine ␤-synthase
H2O
⫹ Asparagine Methionine Lysine Threonine
NH3
⫺
OOC CH CH2 CH2 S CH2 CH COO⫺
⫹
NH3 Alanine Valine Leucine Isoleucine
Cystathionine
H2O Pyruvate
cystathionine -lyase PLP
Alanine and aspartate are synthesized from pyruvate
NH⫹
4
and oxaloacetate, respectively, by transamination from
⫹
NH3
glutamate. Asparagine is synthesized by amidation of
aspartate, with glutamine donating the NH⫹4. These are
⫺
OOC C CH2 CH3 ⫹ HS CH2 CH COO⫺ nonessential amino acids, and their simple biosynthetic
O pathways occur in all organisms.
␣-Ketobutyrate Cysteine For reasons incompletely understood, the malig-
nant lymphocytes present in childhood acute
FIGURE 22–16 Biosynthesis of cysteine from homocysteine and serine lymphoblastic leukemia (ALL) require serum asparagine
in mammals. The homocysteine is formed from methionine, as for growth. The chemotherapy for ALL is administered
described in the text. together with an L-asparaginase derived from bacteria,
FIGURE 22–17 Biosynthesis of six essential amino acids from oxalo-

acetate and pyruvate in bacteria: methionine, threonine, lysine, iso-
⫹ leucine, valine, and leucine. Here, and in other multistep pathways,
O NH3
the enzymes are listed in the key. Note that L,L-␣,´-diaminopimelate,
Aspartate C CH2 CH COO⫺ the product of step 14 , is symmetric. The carbons derived from pyru-
⫺
O vate (and the amino group derived from glutamate) are not traced
beyond this point, because subsequent reactions may place them at
ATP either end of the lysine molecule.
aspartokinase 1
ADP NH3 ⫹
NH4 ⫹ H2O
PLP
⫹ Threonine CH3 CH CH COO⫺
O NH3 17 threonine
OH
dehydratase (serine
Aspartyl--phosphate C CH2 CH COO⫺
Pi dehydratase)
P O 5 PLP threonine synthase
H2O
NADPH ⫹ H⫹
aspartate-semialdehyde dehydrogenase 2
NH3
NADP⫹
P O CH2 CH2 CH COO⫺ Phosphohomoserine
Pi
⫹ ADP
O NH3 homoserine kinase
NADPH ⫹ H⫹ 4
C CH2 CH COO⫺ ATP
Pyruvate
H NADP
dihydropicolinate synthase
10 Aspartate -semialdehyde 3 ⫹
NH3
⫹ homoserine dehydrogenase
OH NH3 CH2 CH2 CH COO⫺ Homoserine
⫺ ⫺
OOC C CH2 C CH2 CH COO OH
O H Succinyl-CoA
homoserine acyltransferase 6
CoA
10 H2O
NADPH ⫹
H NH3
⫹ ⫹
H ⫹H NADP CH2 CH2 CH COO⫺ O-Succinylhomoserine
H
⫺
OOC N COO⫺ ⫺
OOC N COO⫺ O Succinate
11 1
Dihydropicolinate ⌬ -Piperidine-2,6- Cysteine
1-piperidine-2,6-dicarboxylate
dehydrogenase
dicarboxylate 7 PLP cystathionine y-synthase
Succinyl-CoA ⫹ H2O Succinate
N-succinyl-2-amino-6-
12 ⫹
ketopimelate synthase CoA NH3
H2C S CH2 CH2 CH COO⫺ Cystathionine
␣ -Ketoglutarate Glutamate ⫹
H
PLP H C NH3
⫺
OOC N COO⫺ ⫺
OOC O COO⫺
NH COO⫺
H2 NH 13
PLP
succinyl diaminopimelate Succinate
Succinate cystathionine B-lyase8 Pyruvate ⫹ NH3
aminotransferase N-Succinyl-2-amino-
N-Succinyl-L,L-
6-keto-L-pimelate ⫹
,-diamino- NH3
pimelate
HS CH2 CH2 CH COO⫺ Homocysteine
H2O
14 succinyl diaminopimelate desuccinylase N5-Methyl H4 folate
Succinate methionine synthase 9
H4 folate
COO⫺ COO⫺ COO⫺
⫹ ⫹ ⫹ ⫹ ⫹
H3N C H H3N C H H CO2 H3N C H NH3
diaminopimelate epimerase PLP
(CH2)3 (CH2)3 (CH2)3 CH3 S CH2 CH2 CH COO⫺
⫹ 15 ⫹ 16
H C NH3 H3N C H diaminopimelate CH2
Methionine
COO⫺ COO⫺ decarboxylase ⫹
NH3
L,L-,-Diamino- meso-,-Diamino-
pimelate pimelate Lysine
with the enzyme functioning to reduce serum aspara- 60% of cases). However, the asparaginase treatment has
gine. The combined treatment results in a greater than some deleterious side effects, and about 10% of patients
95% remission rate in cases of childhood ALL (L-aspara- who achieve remission eventually suffer relapse, with
ginase treatment alone produces remission in 40% to tumors resistant to drug therapy. Researchers are now
1 aspartokinase
2 aspartate -semialdehyde dehydrogenase
3 homoserine dehydrogenase
CH3 C COO⫺ 4 homoserine kinase
Pyruvate 5 threonine synthase
O
6 homoserine acyltransferase
acetolactate synthase 18
TPP 7 cystathionine ␥-synthase
CO2 8 cystathionine -lyase
⫺ 9 methionine synthase
CH3 C TPP 10 dihydropicolinate synthase
OH 11 ⌬1-piperidine-2,6-dicarboxylate dehydrogenase
12 N-succinyl-2-amino-6-ketopimelate synthase
CH3 CH2 C COO⫺ CH3 C COO⫺ 13 succinyl diaminopimelate aminotransferase
14 succinyl diaminopimelate desuccinylase
O O
15 diaminopimelate epimerase
-Ketobutyrate Pyruvate
16 diaminopimelate decarboxylase
18 17 threonine dehydratase (serine dehydratase)
18 18 acetolactate synthase
CH3 19 acetohydroxy acid isomeroreductase
CH2 CH3 20 dihydroxy acid dehydratase
-Aceto--
21 valine aminotransferase
CH3 C C COO⫺ hydroxybutyrate CH3 C C COO⫺ -Acetolactate
22 -isopropylmalate synthase
O OH O OH 23 isopropylmalate isomerase
24 -isopropylmalate dehydrogenase
19 acetohydroxy acid 25 leucine aminotransferase
19 isomeroreductase
CH3
CH2 CH3
CH3 C C COO⫺ CH3 C C COO⫺
HO O OH O
NAD(P)H ⫹ H⫹
NAD(P)H ⫹ H⫹ CH3 COO⫺
19
19 CH3 CH C CH2 COO⫺ -Isopropylmalate
NAD(P)⫹
NAD(P)⫹ OH
CH3
CH2 H CH3 H 23 isopropylmalate isomerase
,-Dihydroxy- ,-Dihydroxy-
CH3 C C COO ⫺ -methylvalerate CH3 C C COO ⫺ isovalerate
CH3 COO⫺
OH OH OH OH 22 CH3 CH CH CH COO⫺ -Isopropylmalate
CoA OH
20 H2O dihydroxy acid dehydratase a-isopropylmalate synthase
20 H2O NAD⫹
CH3 Acetyl -CoA
CH2 CH3 B-isopropylmalate 24
-Keto-- NADH ⫹ H⫹
CH3 C C COO⫺ methylvalerate CH3 C C COO⫺ -Keto- dehydrogenase
isovalerate
H O H O CO2
CH3
Glutamate Glutamate
21 CH3 CH CH2 C COO⫺ -Ketoisocaproate
PLP valine aminotransferase 21 PLP
-Ketoglutarate O
-Ketoglutarate
CH3
⫹ ⫹ Glutamate
CH2 NH3 CH3 NH3 leucine aminotransferase 25 PLP
CH3 CH CH COO⫺ CH3 CH CH COO⫺
␣ -Ketoglutarate
Isoleucine Valine ⫹
CH3 NH3
CH3 CH CH2 CH COO⫺
Leucine
developing inhibitors of human asparagine synthetase to synthesize them. Their biosynthetic pathways are com-
augment these therapies for childhood ALL. ■ plex and interconnected (Fig. 22–17). In some cases,
Methionine, threonine, lysine, isoleucine, valine, the pathways in bacteria, fungi, and plants differ signifi-
and leucine are essential amino acids; humans cannot cantly. Figure 22–17 shows the bacterial pathways.
Aspartate gives rise to methionine, threonine, Indole-3-glycerol phosphate

␣ subunit
and lysine. Branch points occur at aspartate ␤-semial-
dehyde, an intermediate in all three pathways, and at indole ⫹ glyceraldehyde 3-phosphate
homoserine, a precursor of threonine and methionine. Indole ⫹ serine tryptophan ⫹ H2O
␤2 subunit
Threonine, in turn, is one of the precursors of isoleu-
cine. The valine and isoleucine pathways share four The second part of the reaction requires pyridoxal
enzymes (Fig. 22–17, steps 18 to 21 ). Pyruvate gives phosphate (Fig. 22–20). Indole formed in the first part
rise to valine and isoleucine in pathways that begin with is not released by the enzyme, but instead moves
condensation of two carbons of pyruvate (in the form of through a channel from the ␣-subunit active site to one
hydroxyethyl thiamine pyrophosphate; see Fig. 14–15) of the ␤-subunit active sites, where it condenses with a
with another molecule of pyruvate (the valine path) Schiff base intermediate derived from serine and PLP.
or with ␣-ketobutyrate (the isoleucine path). The Intermediate channeling of this type may be a feature of
␣-ketobutyrate is derived from threonine in a reaction the entire pathway from chorismate to tryptophan.
that requires pyridoxal phosphate (Fig. 22–17, step 17 ). An Enzyme active sites catalyzing different steps (some-
intermediate in the valine pathway, ␣-ketoisovalerate, is times not sequential steps) of the pathway to trypto-
the starting point for a four-step branch pathway lead- phan are found on single polypeptides in some species
ing to leucine (steps 22 to 25 ). of fungi and bacteria, but are separate proteins in other
species. In addition, the activity of some of these
enzymes requires a noncovalent association with other
Chorismate Is a Key Intermediate in the Synthesis enzymes of the pathway. These observations suggest
of Tryptophan, Phenylalanine, and Tyrosine that all the pathway enzymes are components of a large,
multienzyme complex in both bacteria and eukaryotes.
Phosphoenolpyruvate Such complexes are generally not preserved intact
⫹ when the enzymes are isolated using traditional bio-
Erythrose 4-phosphate chemical methods, but evidence for the existence of
multienzyme complexes is accumulating for this and
other metabolic pathways (see Section 16.3).
Phenylalanine Tyrosine Tryptophan In plants and bacteria, phenylalanine and tyro-
sine are synthesized from chorismate in pathways
much less complex than the tryptophan pathway. The
Tyrosine common intermediate is prephenate (Fig. 22–21).
The final step in both cases is transamination with
Aromatic rings are not readily available in the environ- glutamate.
ment, even though the benzene ring is very stable. The Animals can produce tyrosine directly from phenyl-
branched pathway to tryptophan, phenylalanine, and alanine through hydroxylation at C-4 of the phenyl
tyrosine, occurring in bacteria, fungi, and plants, is the group by phenylalanine hydroxylase; this enzyme
main biological route of aromatic ring formation. It pro- also participates in the degradation of phenylalanine
ceeds through ring closure of an aliphatic precursor (see Figs 18–23, 18–24). Tyrosine is considered a con-
followed by stepwise addition of double bonds. The first ditionally essential amino acid, or as nonessential inso-
four steps produce shikimate, a seven-carbon molecule far as it can be synthesized from the essential amino
derived from erythrose 4-phosphate and phosphoenol- acid phenylalanine.
pyruvate (Fig. 22–18). Shikimate is converted to cho-
rismate in three steps that include the addition of three
more carbons from another molecule of phosphoenol- Histidine Biosynthesis Uses Precursors
pyruvate. Chorismate is the first branch point of the
pathway, with one branch leading to tryptophan, the
of Purine Biosynthesis
other to phenylalanine and tyrosine. Ribose 5-phosphate
In the tryptophan branch (Fig. 22–19), choris-
mate is converted to anthranilate in a reaction in which
glutamine donates the nitrogen that will become part of Histidine
the indole ring. Anthranilate then condenses with
PRPP. The indole ring of tryptophan is derived from the The pathway to histidine in all plants and bacteria dif-
ring carbons and amino group of anthranilate plus two fers in several respects from other amino acid biosyn-
carbons derived from PRPP. The final reaction in the thetic pathways. Histidine is derived from three precur-
sequence is catalyzed by tryptophan synthase. This sors (Fig. 22–22): PRPP contributes five carbons, the
enzyme has an ␣2␤2 subunit structure and can be dis- purine ring of ATP contributes a nitrogen and a carbon,
sociated into two ␣ subunits and a ␤2 unit that catalyze and glutamine supplies the second ring nitrogen. The
different parts of the overall reaction: key steps are condensation of ATP and PRPP, in which
P 3-Dehydroshikimate
O COO⫺ NADPH ⫹ H⫹
4
C NADP⫹
Phosphoenolpyruvate
CH2 (PEP)
COO⫺
⫹
O H
C HO 1 2-keto-3-deoxy-D-arabinoheptulosonate
OH 7-phosphate synthase
CHOH Erythrose 4-phosphate H H Shikimate
HO H 2 dehydroquinate synthase
CHOH 3 3-dehydroquinate dehydratase
ATP 4 shikimate dehydrogenase
CH2 O P
5 5 shikimate kinase
ADP 6 5-enolpyruvylshikimate 3-phosphate
H 2O
1 synthase
Pi COO⫺ 7 chorismate synthase
O COO⫺ P O
C OH Shikimate
H H 3-phosphate
CH2 HO H
HO C H PEP
2-Keto-3-deoxy-D- 6
H C OH arabinoheptulosonate Pi
7-phosphate
H C OH
COO⫺
CH2 O P
CH2
P O
NAD⫹
O C COO⫺
2
Pi H H
HO H 5-Enolpyruvylshikimate
3-phosphate
⫺
HO COO FIGURE 22–18 Biosynthesis of chorismate, an intermedi-
C 7 Pi ate in the synthesis of aromatic amino acids in bacteria
and plants. All carbons are derived from either erythrose
OH 4-phosphate (light purple) or phosphoenolpyruvate (light
O COO⫺
H 3-Dehydroquinate red). Note that the NAD⫹ required as a cofactor in step 2
HO H CH2 is released unchanged; it may be transiently reduced to
3 O C COO⫺ NADH during the reaction, with formation of an oxidized
H 2O
H reaction intermediate. Step 6 is competitively inhibited by
HO H
glyphosate ( 2 COOOCH2ONHOCH2OPO23 2 ) , the active
COO⫺ Chorismate ingredient in the widely used herbicide Roundup. The herbi-
cide is relatively nontoxic to mammals, which lack this bio-
OH synthetic pathway. The chemical names quinate, shikimate,
O H and chorismate are derived from the names of plants in
HO H 3-Dehydroshikimate which these intermediates have been found to accumulate.
N-1 of the purine ring is linked to the activated C-1 of Amino Acid Biosynthesis Is under Allosteric
the ribose of PRPP (step 1 in Fig. 22–22); purine ring
Regulation
opening that ultimately leaves N-1 and C-2 of adenine
linked to the ribose (step 3); and formation of the As detailed in Chapter 15, the control of flux through a
imidazole ring, a reaction in which glutamine donates a metabolic pathway often reflects the activity of multiple
nitrogen (step 5). The use of ATP as a metabolite enzymes in that pathway. In the case of amino acid syn-
rather than a high-energy cofactor is unusual—but not thesis, regulation takes place in part through feedback
wasteful, because it dovetails with the purine biosyn- inhibition of the first reaction by the end product of the
thetic pathway. The remnant of ATP that is released pathway. This first reaction is often catalyzed by an allo-
after the transfer of N-1 and C-2 is 5-aminoimidazole-4- steric enzyme that plays an important role in the overall
carboxamide ribonucleotide (AICAR), an intermediate control of flux through that pathway. As an example,
of purine biosynthesis (see Fig. 22–35) that is rapidly Figure 22–23 shows the allosteric regulation of isoleu-
recycled to ATP. cine synthesis from threonine (detailed in Fig. 22–17).
The end product, isoleucine, is an allosteric inhibitor of COO⫺

the first reaction in the sequence. In bacteria, such allo- CH2
steric modulation of amino acid synthesis contributes to B Chorismate
the minute-to-minute adjustment of pathway activity to ! O OCOCOO⫺
' H
cellular needs. HO H
Allosteric regulation of an individual enzyme can be Glutamine
considerably more complex. An example is the remark-
1
able set of allosteric controls exerted on glutamine Glutamate
synthetase of E. coli (Fig. 22–8). Six products derived Pyruvate
from glutamine serve as negative feedback modulators COO⫺
of the enzyme, and the overall effects of these and other NH2
Anthranilate
modulators are more than additive. Such regulation is
called concerted inhibition.
Additional mechanisms contribute to the regulation
PRPP
of the amino acid biosynthetic pathways. Because the 20 2
common amino acids must be made in the correct pro- PPi
portions for protein synthesis, cells have developed ways
not only of controlling the rate of synthesis of individual
H
amino acids but also of coordinating their formation. P OO OCH2 N
Such coordination is especially well developed in fast- O
COO⫺
growing bacterial cells. Figure 22–24 shows how E. coli H H
cells coordinate the synthesis of lysine, methionine, H H N-(5⬘-Phosphoribosyl)-
anthranilate
threonine, and isoleucine, all made from aspartate. Sev- OH OH
eral important types of inhibition patterns are evident.
The step from aspartate to aspartyl--phosphate is cata- 3
lyzed by three isozymes, each independently controlled
by different modulators. This enzyme multiplicity pre- COO⫺ HO OH
A A
vents one biosynthetic end product from shutting down HOOCOCOCOCH2 OO O P
key steps in a pathway when other products of the same B A A
C H H
pathway are required. The steps from aspartate -semial- G
H
N
dehyde to homoserine and from threonine to -ketobu- H
Enol-1-o-carboxyphenylamino-1-
tyrate (detailed in Fig. 22–17) are also catalyzed by dual, deoxyribulose phosphate
independently controlled isozymes. One isozyme for the 4 H2O ⫹ CO2
conversion of aspartate to aspartyl--phosphate is allo-
sterically inhibited by two different modulators, lysine OH OH
A A
and isoleucine, whose action is more than additive— CHOCHOCH2 O OO P
another example of concerted inhibition. The sequence
from aspartate to isoleucine undergoes multiple, overlap- Indole-3-glycerol phosphate
ping negative feedback inhibitions; for example, isoleucine N
inhibits the conversion of threonine to -ketobutyrate H
(as described above), and threonine inhibits its own for- Glyceraldehyde 3-phosphate
mation at three points: from homoserine, from aspartate 5 Serine
-semialdehyde, and from aspartate (steps 4, 3, and 1 PLP
in Fig. 22–17). This overall regulatory mechanism is H2O
called sequential feedback inhibition. ⫹
Similar patterns are evident in the pathways lead- NH3
A
ing to the aromatic amino acids. The first step of the CH2 OCHOCOO⫺
N
H
1 anthranilate synthase
FIGURE 22–19 Biosynthesis of tryp- 2 anthranilate phosphoribosyltransferase Tryptophan
tophan from chorismate in bacteria 3 N-(5'-phosphoribosyl)-anthranilate isomerase
and plants. In E. coli, enzymes cata- 4 indole-3-glycerol phosphate synthase
lyzing steps 1 and 2 are subunits of
5 tryptophan synthase
a single complex.
(a) OH OH Permanent figure # 2220

2nd pass
CH CH CH2 O P
Indole-3-glycerol phosphate
N
H
An aldol cleavage OH
tryptophan O
produces indole
synthase
and glyceraldehyde a subunits C CH CH2 O P
3-phosphate; PLP is
not required. 1 H
Glyceraldehyde
3-phosphate
Indole traverses
tunnel between
a and b subunits. N
Indole H
⫹
NH3
␤
Dehydration of CH2 CH COO⫺
tryptophan (b)
serine forms a synthase OH Serine
PLP-aminoacrylate b subunits PLP
intermediate. Glyceraldehyde
2 3-phosphate
H2O
␣ Indole
B
H
␤
␤ H2C COO⫺ Tunnel
N C
H
⫹
Indole NH
␤␤
HC
P O CH2 OH Indole
From serine
␤
⫹
N CH3
H
Quinonoid
PLP-aminoacrylate
Indole condenses adduct
with the aminoac-
rylate intermediate 3
(2 steps).
B HB
⫹
H H ⫹
CH2 COO⫺ NH3
CH2 CH COO⫺ Enzyme PLP
C H2O
⫹
N ⫹ CH2 CH COO⫺
H ⫹ N NH
NH
H
␤␤ HC ␤
HC
P O CH2 OH
4 P O CH2 OH 5 N Tryptophan
H
⫹ Imine linkage
N CH3 joining trypto-
N CH3 H
H phan to PLP is
hydrolyzed.
Quinonoid intermediate Aldimine with tryptophan
MECHANISM FIGURE 22–20 Tryptophan synthase reaction. (a) This enzyme Figure 18–6. The ␤ carbon of serine is attached to the indole ring system.
catalyzes a multistep reaction with several types of chemical rearrange- (b) (PDB ID 1KFJ) Indole generated on the ␣ subunit (white) moves
ments. The PLP-facilitated transformations occur at the ␤ carbon (C-3) through a tunnel to the ␤ subunit (blue), where it condenses with the
of the amino acid, as opposed to the ␣-carbon reactions described in serine moiety. Tryptophan Synthase Mechanism
COO⫺ the others are required in the diet (essential amino

CH2
acids).
O C COO⫺ Among the nonessential amino acids, glutamate is
H formed by reductive amination of ␣-ketoglutarate
HO H Chorismate
and serves as the precursor of glutamine, proline,
1 and arginine. Alanine and aspartate (and thus
⫺
OOC CH2 C COO⫺ asparagine) are formed from pyruvate and
O oxaloacetate, respectively, by transamination. The
Prephenate carbon chain of serine is derived from
3-phosphoglycerate. Serine is a precursor of
HO H
NAD⫹ glycine; the ␤-carbon atom of serine is transferred
NADH ⫹ H ⫹
CO2 ⫹ OH⫺
to tetrahydrofolate. In microorganisms, cysteine is
CO2 2 3 produced from serine and from sulfide produced
O O by the reduction of environmental sulfate.
CH2 C COO ⫺
CH2 C COO⫺
Mammals produce cysteine from methionine and
serine by a series of reactions requiring
S-adenosylmethionine and cystathionine.
4-Hydroxyphenyl-
pyruvate Phenylpyruvate Among the essential amino acids, the aromatic
OH amino acids (phenylalanine, tyrosine, and
tryptophan) form by a pathway in which
amino- Glutamate amino- Glutamate
transferase transferase
chorismate occupies a key branch point.
␣ -Ketoglutarate ␣ -Ketoglutarate Phosphoribosyl pyrophosphate is a precursor of
⫹ ⫹ tryptophan and histidine. The pathway to
NH3 NH3
histidine is interconnected with the purine
CH2 CH COO⫺ CH2 CH COO⫺ synthetic pathway. Tyrosine can also be formed
by hydroxylation of phenylalanine (and thus is
considered conditionally essential). The
pathways for the other essential amino acids are
OH complex.
Tyrosine Phenylalanine The amino acid biosynthetic pathways are subject
to allosteric end-product inhibition; the regulatory
1 chorismate mutase enzyme is usually the first in the sequence.
2 prephenate dehydrogenase Regulation of the various synthetic pathways is
3 prephenate dehydratase coordinated.
FIGURE 22–21 Biosynthesis of phenylalanine and tyrosine from

chorismate in bacteria and plants. Conversion of chorismate to pre- 22.3 Molecules Derived from Amino Acids
phenate is a rare biological example of a Claisen rearrangement.
In addition to their role as the building blocks of pro-
teins, amino acids are precursors of many specialized
biomolecules, including hormones, coenzymes, nucleo-
early pathway to the common intermediate chorismate
tides, alkaloids, cell wall polymers, porphyrins, antibi-
is catalyzed by the enzyme 2-keto-3-deoxy-D-arabino-
otics, pigments, and neurotransmitters. We describe
heptulosonate 7-phosphate (DAHP) synthase (1 in
here the pathways to a number of these amino acid
Fig. 22–18). Most microorganisms and plants have
derivatives.
three DAHP synthase isozymes. One is allosterically
inhibited (feedback inhibition) by phenylalanine,
another by tyrosine, and the third by tryptophan. This Glycine Is a Precursor of Porphyrins
scheme helps the overall pathway to respond to cellu- The biosynthesis of porphyrins, for which glycine is a
lar requirements for one or more of the aromatic amino major precursor, is our first example because of the cen-
acids. Additional regulation takes place after the path- tral importance of the porphyrin nucleus in heme pro-
way branches at chorismate. For example, the enzymes teins such as hemoglobin and the cytochromes. The
catalyzing the first two steps of the tryptophan branch porphyrins are constructed from four molecules of the
are subject to allosteric inhibition by tryptophan. monopyrrole derivative porphobilinogen, which itself is
derived from two molecules of ␦-aminolevulinate. There
SUMMARY 22.2 Biosynthesis of Amino Acids are two major pathways to ␦-aminolevulinate. In higher
Plants and bacteria synthesize all 20 common eukaryotes (Fig. 22–25a), glycine reacts with succinyl-
amino acids. Mammals can synthesize about half; CoA in the first step to yield ␣-amino-␤-ketoadipate,
22.3 Molecules Derived from Amino Acids 903
: NH2
P O CH2 O H
N
N
C H H C ⫹
HC
H C N N
C O P P
OH OH Rib P P P
5-Phosphoribosyl- ATP
1-pyrophosphate (PRPP)
1 PPi
N N Rib P P P N N Rib P
HN N PPi HN N
P O CH2 O N CH P O CH2 O N CH
C H H C 2 C H H C
H C C H H C C H
OH OH OH OH
N1-5⬘-Phosphoribosyl-ATP N1-5⬘-Phosphoribosyl-AMP
To purine biosynthesis 3 H2O
N N Rib P N N Rib P
N N Rib P O O
O H2N C N H2N C N
H2N C NH2 HN CH H N CH
P O CH2 O
5-Aminoimidazole- H C H
4-carboxamide
4
C H H C
ribonucleotide (AICAR) C O
H C C H
5 H C OH
OH OH
H C OH
Glutamine N1-5⬘-Phosphoribosylformimino-
H CH2O P
N 5-aminoimidazole-4-
HC carboxamide ribonucleotide
CH
Glutamate N1-5⬘-Phosphoribulosyl-
C formimino-5-amino-
N imidazole-4-carboxamide
H C OH ribonucleotide
H C OH
CH2O P
1 ATP phosphoribosyl transferase 5 glutamine amidotransferase
2 pyrophosphohydrolase 6 imidazole glycerol 3-phosphate dehydratase
Imidazole glycerol 3 phosphoribosyl-AMP cyclohydrolase 7 L-histidinol phosphate aminotransferase
3-phosphate 4 phosphoribosylformimino-5-aminoimidazole- 8 histidinol phosphate phosphatase
4-carboxamide ribonucleotide isomerase 9 histidinol dehydrogenase
6 H 2O
H H H H
N N N N
HC HC HC HC
CH CH CH CH
C Glutamate ␣ -Ketoglutarate C C 2NAD⫹ 2NADH ⫹ 2H⫹ C
N N Pi N N
CH2 CH2 CH2 CH2
7 ⫹ 8 ⫹ 9 ⫹
C O CH NH3 CH NH3 CH NH3
CH2O P CH2O P CH2OH COO⫺
Imidazole acetol L-Histidinol L-Histidinol Histidine

3-phosphate phosphate
FIGURE 22–22 Biosynthesis of histidine in bacteria and plants. Atoms (green). Note that the derivative of ATP remaining after step 5 (AICAR)
derived from PRPP and ATP are shaded light red and blue, respectively. is an intermediate in purine biosynthesis (see Fig. 22–35, step 9), so
Two of the histidine nitrogens are derived from glutamine and glutamate ATP is rapidly regenerated.
⫹ which is then decarboxylated to ␦-aminolevulinate. In

NH3
A plants, algae, and most bacteria, ␦-aminolevulinate is
CH3 OCHOCHOCOO⫺ Threonine formed from glutamate (Fig. 22–25b). The glutamate is
A
OH first esterified to glutamyl-tRNAGlu (see Chapter 27 on
the topic of transfer RNAs); reduction by NADPH con-
threonine dehydratase verts the glutamate to glutamate 1-semialdehyde, which
is cleaved from the tRNA. An aminotransferase converts
O
B the glutamate 1-semialdehyde to ␦-aminolevulinate.
CH3 OCH2OCOCOO⫺ -Ketobutyrate In all organisms, two molecules of ␦-aminolevu-
linate condense to form porphobilinogen and, through
5 steps
⫹ a series of complex enzymatic reactions, four molecules
NH3 of porphobilinogen come together to form protopor-
A
CH3 OCH2OCHOCHOCOO⫺ Isoleucine phyrin (Fig. 22–26). The iron atom is incorporated
A
CH3
after the protoporphyrin has been assembled, in a step
catalyzed by ferrochelatase. Porphyrin biosynthesis is
FIGURE 22–23 Allosteric regulation of isoleucine biosynthesis. The first regulated in higher eukaryotes by heme, which serves
reaction in the pathway from threonine to isoleucine is inhibited by the as a feedback inhibitor of early steps in the synthetic
end product, isoleucine. This was one of the first examples of allosteric pathway. Genetic defects in the biosynthesis of porphy-
feedback inhibition to be discovered. The steps from ␣-ketobutyrate to rins can lead to the accumulation of pathway interme-
isoleucine correspond to steps 18 through 21 in Figure 22–17 (five steps, diates, causing a variety of human diseases known
because 19 is a two-step reaction). collectively as porphyrias (Box 22–2).
Aspartate
Heme Is the Source of Bile Pigments
A1 A2 A3
The iron-porphyrin (heme) group of hemoglo-
bin, released from dying erythrocytes in the
spleen, is degraded to yield free Fe2⫹ and, ultimately,
Aspartyl-b-phosphate bilirubin. This pathway is arresting for its capacity to
inject color into human biochemistry.
The first step in the two-step pathway, catalyzed by
Aspartate b-semialdehyde heme oxygenase, converts heme to biliverdin, a linear
B1 B2 (open) tetrapyrrole derivative (Fig. 22–27). The other
products of the reaction are free Fe2⫹ and CO. The Fe2⫹
6 steps
is quickly bound by ferritin. Carbon monoxide is a poi-
Lysine son that binds to hemoglobin (see Box 5–1), and the
Homoserine production of CO by heme oxygenase ensures that,
even in the absence of environmental exposure, about
1% of an individual’s heme is complexed with CO.
3 steps
Biliverdin is converted to bilirubin in the second
Methionine step, catalyzed by biliverdin reductase. You can monitor
this reaction colorimetrically in a familiar in situ experi-
Threonine
ment. When you are bruised, the black and/or purple
C1 C2 color results from hemoglobin released from damaged
erythrocytes. Over time, the color changes to the green
of biliverdin, and then to the yellow of bilirubin. Biliru-
a-Ketobutyrate
bin is largely insoluble, and it travels in the bloodstream
5 steps as a complex with serum albumin. In the liver, bilirubin is
transformed to the bile pigment bilirubin diglucuronide.
Isoleucine
This product is sufficiently water-soluble to be secreted
FIGURE 22–24 Interlocking regulatory mechanisms in the biosynthesis with other components of bile into the small intestine,
of several amino acids derived from aspartate in E. coli. Three enzymes where microbial enzymes convert it to several products,
(A, B, C) have either two or three isozyme forms, indicated by numerical predominantly urobilinogen. Some urobilinogen is reab-
subscripts. In each case, one isozyme (A2, B1, and C2) has no allosteric sorbed into the blood and transported to the kidney,
regulation; these isozymes are regulated by changes in the amount of where it is converted to urobilin, the compound that gives
enzyme synthesized (Chapter 28). Synthesis of isozymes A2 and B1 is urine its yellow color (Fig. 22–27). Urobilinogen remain-
repressed when methionine levels are high, and synthesis of isozyme C2 ing in the intestine is converted (in another microbe-
is repressed when isoleucine levels are high. Enzyme A is aspartokinase; dependent reaction) to stercobilin (Fig. 22–27), which
B, homoserine dehydrogenase; C, threonine dehydratase. imparts the red-brown color to feces.
(a) COO⫺ COO⫺

COO⫺ CH2 CH2
CH2 CoA-SH CH2 CH2
⫹
CO2
CH2 ⫹ CH2 NH3 ␦-aminolevulinate C O C O
␦-aminolevulinate
synthase ⫹
synthase
C S-CoA COO⫺ CH NH3 CH2
⫹
O COO⫺ NH3
Succinyl-CoA Glycine -Amino-- ␦-Aminolevulinate
ketoadipate
hexanedioic acid
glutamate 1-semialdehyde
aminomutase
(b)
⫺
COO⫺ COO COO⫺
Glu ⫹ Glu
CH2 tRNA ATP AMP ⫹ PPi CH2 NADPH ⫹ H⫹ NADP tRNA CH2
CH2 glutamyl-tRNA
CH2 glutamyl-tRNA
CH2
⫹ synthetase ⫹ reductase ⫹
HC NH3 HC NH3 HC NH3
COO⫺ C O C O
H
Glutamate tRNAGlu
Glutamyl-tRNAGlu Glutamate 1-semialdehyde
FIGURE 22–25 Biosynthesis of ␦-aminolevulinate. (a) In most animals, succinyl-CoA. The atoms furnished by glycine are shown in red. (b) In
including mammals, ␦-aminolevulinate is synthesized from glycine and bacteria and plants, the precursor of ␦-aminolevulinate is glutamate.
Pr Ac Pr Ac
COO⫺ Ac Pr Ac Pr
COO⫺ ⫺
OOC NH HN NH HN
8 H2O 4 NH⫹
4
HO H2O
8 4 NH HN NH HN
O 1 2 Pr Ac 3 Ac Ac
N
H
H2N
NH2 Ac Pr Pr Pr
␦-Aminolevulinate Porphobilinogen Pre-uroporphyrinogen Uroporphyrinogen III
5-Amino-4-oxo-pentanoic acid
4 4 CO2
Vinyl group
CH3 CH3 CH3 Pr CH3
CH3 CH3 CH3 CH3 Pr

N N Fe2⫹ N HN NH HN 2 CO2 NH HN
Rearray of double bonds
Fe2⫹
N N
7 N HN 6 NH HN
5 NH HN
CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3
Pr Pr Pr Pr Pr Pr Pr Pr
Heme Protoporphyrin Protoporphyrinogen Coproporphyrinogen III
Conjugated double bonds~ Color 1 porphobilinogen synthase

Reducing agent which reduces the double bond can make blood Blue 2 uroporphyrinogen synthase
3 uroporphyrinogen III cosynthase
4 uroporphyrinogen decarboxylase
In one minute how many oxygens and Glucose are utilized or transported?
5 coproporphyrinogen oxidase
6 protoporphyrinogen oxidase
FIGURE 22–26 Biosynthesis of heme from ␦-aminolevulinate. Ac 7 ferrochelatase
represents acetyl (—CH2COO); Pr, propionyl (—CH2CH2COO).
BOX 22–2 MEDICINE On Kings and Vampires

Porphyrias are a group of genetic diseases that result This genetic condition may have given rise to the
from defects in enzymes of the biosynthetic pathway vampire myths of folk legend.
from glycine to porphyrins; specific porphyrin precur- The symptoms of most porphyrias are now readily
sors accumulate in erythrocytes, body fluids, and the controlled with dietary changes or the administration
liver. The most common form is acute intermittent of heme or heme derivatives.
porphyria. Most individuals inheriting this condition
are heterozygotes and are usually asymptomatic,
because the single copy of the normal gene provides ␦-Aminolevulinate
a sufficient level of enzyme function. However, certain Doss porphyria
nutritional or environmental factors (as yet poorly
Porphobilinogen
understood) can cause a buildup of ␦-aminolevulinate
and porphobilinogen, leading to attacks of acute Acute intermittent porphyria
abdominal pain and neurological dysfunction. King Pre-uroporphyrinogen
George III, British monarch during the American
Congenital erythropoietic porphyria
Revolution, suffered several episodes of apparent
madness that tarnished the record of this otherwise Urophorphyrinogen III
accomplished man. The symptoms of his condition Porphyria cutanea tarda
suggest that George III suffered from acute intermit- Coproporphyrinogen
tent porphyria.
Hereditary coproporphyria
One of the rarer porphyrias results in an accumu-
lation of uroporphyrinogen I, an abnormal isomer of a Protoporphyrinogen
protoporphyrin precursor. This compound stains the Variegate porphyria
urine red, causes the teeth to fluoresce strongly in
Protoporphyrin
ultraviolet light, and makes the skin abnormally sensi-
tive to sunlight. Many individuals with this porphyria Erythropoietic protoporphyria
are anemic because insufficient heme is synthesized. Heme
Impaired liver function or blocked bile secretion The cell toxicity associated with jaundice may be due to
causes bilirubin to leak from the liver into the blood, bilirubin levels in excess of the serum albumin needed
resulting in a yellowing of the skin and eyeballs, a condi- to solubilize it.
tion called jaundice. In cases of jaundice, determination Given these varied roles of heme degradation prod-
of the concentration of bilirubin in the blood may be use- ucts, the degradative pathway is subject to regulation,
ful in the diagnosis of underlying liver disease. Newborn mainly at the first step. Humans have at least three iso-
infants sometimes develop jaundice because they have zymes of heme oxygenase (HO). HO-1 is highly regu-
not yet produced enough glucuronyl bilirubin transfer- lated; the expression of its gene is induced by a wide
ase to process their bilirubin. A traditional treatment to range of stress conditions (shear stress, angiogenesis
reduce excess bilirubin, exposure to a fluorescent lamp, (uncontrolled development of blood vessels), hypoxia,
causes a photochemical conversion of bilirubin to com- hyperoxia, heat shock, exposure to ultraviolet light,
pounds that are more soluble and easily excreted. hydrogen peroxide, and many other metabolic insults).
These pathways of heme breakdown play signifi- HO-2 is found mainly in brain and testes, where it is
cant roles in protecting cells from oxidative damage and continuously expressed. The third isozyme, HO-3, is not
in regulating certain cellular functions. The CO pro- yet well characterized. ■
duced by heme oxygenase is toxic at high concentra-
tions, but at the very low concentrations generated
during heme degradation it seems to have some regula-
Amino Acids Are Precursors of Creatine
tory and/or signaling functions. It acts as a vasodilator, and Glutathione
much the same as (but less potent than) nitric oxide Phosphocreatine, derived from creatine, is an impor-
(discussed below). Low levels of CO also have some tant energy buffer in skeletal muscle (see Box 23–2).
regulatory effects on neurotransmission. Bilirubin is the Creatine is synthesized from glycine and arginine (Fig.
most abundant antioxidant in mammalian tissues and is 22–28); methionine, in the form of S-adenosylmethio-
responsible for most of the antioxidant activity in nine, acts as methyl group donor.
serum. Its protective effects seem to be especially Glutathione (GSH), present in plants, animals,
important in the developing brain of newborn infants. and some bacteria, often at high levels, can be thought
FIGURE 22–27 Bilirubin and its breakdown prod- Heme

ucts. M represents methyl; V, vinyl; Pr, propionyl; E,
ethyl. For ease of comparison, these structures are heme oxygenase CO
shown in linear form, rather than in their correct
stereochemical conformations. Fe2⫹
M V M Pr Pr M M V
O N N N N O Biliverdin
H H H
NADPH ⫹ H ⫹
biliverdin
reductase
NADP⫹
M V M Pr Pr M M V
O N N N N O
H H H H
H H
Bilirubin
glucuronyl- transport in blood
(in blood)
bilirubin as complex with
transferase (liver) serum albumin
Bilirubin diglucuronide Bilirubin

transport to intestine (in bile)
Urobilinogen Urobilinogen
transport to kidney
M E M Pr Pr M M E M E M Pr Pr M M E
H H H H
H H
O N N N N O O N N N N O
H H H H H H
Urobilin Stercobilin
of as a redox buffer. It is derived from glutamate, cysteine, D-Amino Acids Are Found Primarily in Bacteria
and glycine (Fig. 22–29). The ␥-carboxyl group of glu-
Although D-amino acids do not generally occur in
tamate is activated by ATP to form an acyl phosphate
proteins, they do serve some special functions in
intermediate, which is then attacked by the ␣-amino
the structure of bacterial cell walls and peptide antibiot-
group of cysteine. A second condensation reaction fol-
ics. Bacterial peptidoglycans (see Fig. 20–30) contain
lows, with the ␣-carboxyl group of cysteine activated to
both D-alanine and D-glutamate. D-Amino acids arise
an acyl phosphate to permit reaction with glycine. The
directly from the L isomers by the action of amino acid
oxidized form of glutathione (GSSG), produced in the
racemases, which have pyridoxal phosphate as cofactor
course of its redox activities, contains two glutathione
(see Fig. 18–6). Amino acid racemization is uniquely
molecules linked by a disulfide bond.
important to bacterial metabolism, and enzymes such as
Glutathione probably helps maintain the sulfhydryl
alanine racemase are prime targets for pharmaceutical
groups of proteins in the reduced state and the iron of
agents. One such agent, L-fluoroalanine, is being tested
heme in the ferrous (Fe2⫹) state, and it serves as a
as an antibacterial drug. Another, cycloserine, is used
reducing agent for glutaredoxin in deoxyribonucleotide
to treat tuberculosis. Because these inhibitors also
synthesis (see Fig. 22–41). Its redox function is also used
affect some PLP-requiring human enzymes, however,
to remove toxic peroxides formed in the normal course of
they have potentially undesirable side effects. ■
growth and metabolism under aerobic conditions:
COO᎐
2 GSH 1 R—O—O—H ¡ GSSG 1 H2O 1 R—OH ⫹ O
H3 N C H H2C NH
This reaction is catalyzed by glutathione peroxidase,
a remarkable enzyme in that it contains a covalently CH2 HC C
bound selenium (Se) atom in the form of selenocysteine F ⫹
NH3 O
(see Fig. 3–8a), which is essential for its activity. L-Fluoroalanine Cycloserine
NH2 (a) Glutamate

⫹ A ⫹
NH3 CPNH2
A A
Glycine CH2
A NH Cysteine
A Arginine
COO⫺ (CH2)3 ATP
A ⫹
CHONH3 ␥-glutamyl
A
cysteine synthetase
amidinotransferase COO⫺
ADP ⫹ Pi
Ornithine
NH2
A ⫹ ␥-Glu–Cys
CPNH2
A
NH Guanidinoacetate
A Glycine
CH2
A ATP
COO⫺
glutathione
synthetase
adoMet Methionine
methyltransferase
ADP ⫹ Pi
adoHcy
NH2
A ⫹ ␥-Glu Cys Gly
CPNH2 ⫹
A NH3 O O
NOCH3 Creatine ᎐
A OOC CH CH2 CH2 C N CH C N CH2 COO᎐
CH2
A H CH2 H
COO⫺
SH
ATP Glutathione (GSH)
creatine (reduced)
kinase
ADP
O⫺ (b) ␥-Glu–Cys–Gly
A
O P P O O⫺ S
A
NH S
A ⫹
CPNH2 ␥-Glu–Cys–Gly
A
NOCH3 Glutathione (GSSG)
A (oxidized)
CH2
A FIGURE 22–29 Glutathione metabolism. (a) Biosynthesis of glutathione.
COO⫺ (b) Oxidized form of glutathione.
Phosphocreatine
FIGURE 22–28 Biosynthesis of creatine and phosphocreatine. Creatine in the regulation of a wide range of biological processes
is made from three amino acids: glycine, arginine, and methionine. This
in plants.
pathway shows the versatility of amino acids as precursors of other
Phenylalanine and tyrosine also give rise to many
nitrogenous biomolecules.
commercially significant natural products, including
the tannins that inhibit oxidation in wines; alkaloids
such as morphine, which have potent physiological
Aromatic Amino Acids Are Precursors effects; and the flavoring of cinnamon oil (Fig. 22–30b),
of Many Plant Substances nutmeg, cloves, vanilla, cayenne pepper, and other
Phenylalanine, tyrosine, and tryptophan are converted products.
to a variety of important compounds in plants. The
rigid polymer lignin, derived from phenylalanine and
tyrosine, is second only to cellulose in abundance in
Biological Amines Are Products of Amino
plant tissues. The structure of the lignin polymer is Acid Decarboxylation
complex and not well understood. Tryptophan is also Many important neurotransmitters are primary
the precursor of the plant growth hormone indole-3- or secondary amines, derived from amino acids
acetate, or auxin (Fig. 22–30a), which is important in simple pathways. In addition, some polyamines that
⫹ mitter, serotonin, is derived from tryptophan in a two-

NH3
step pathway.
CH2 CH COO᎐ Histidine undergoes decarboxylation to histamine,
a powerful vasodilator in animal tissues. Histamine is
released in large amounts as part of the allergic response,
N
H and it also stimulates acid secretion in the stomach. A
growing array of pharmaceutical agents are being
Tryptophan
designed to interfere with either the synthesis or the
action of histamine. A prominent example is the hista-
amino- mine receptor antagonist cimetidine (Tagamet), a
transferase
structural analog of histamine:
O
CH3 CH2 S CH2 CH2 NH C NH CH3
CH2 C COO᎐
N C N
N NH
Indole-
3-pyruvate
N It promotes the healing of duodenal ulcers by inhibiting
H CH2 CH COO᎐
⫹ secretion of gastric acid.
NH3
Polyamines such as spermine and spermidine,
Phenylalanine involved in DNA packaging, are derived from methio-
decarboxylase CO2 nine and ornithine by the pathway shown in Figure
phenylalanine
22–32. The first step is decarboxylation of ornithine, a
ammonia precursor of arginine (Fig. 22–12). Ornithine decar-
CH2 COO ᎐
lyase NH3
boxylase, a PLP-requiring enzyme, is the target of
several powerful inhibitors used as pharmaceutical
agents (see Box 6–3). ■
N CH CH COO᎐
H
Indole-3-acetate
(auxin) Cinnamate Arginine Is the Precursor for Biological Synthesis
of Nitric Oxide
(a) (b) A surprise finding in the mid-1980s was the role of nitric
FIGURE 22–30 Biosynthesis of two plant substances from amino acids. oxide (NO)—previously known mainly as a component
(a) Indole-3-acetate (auxin) and (b) cinnamate (cinnamon flavor). of smog—as an important biological messenger. This
simple gaseous substance diffuses readily through
membranes, although its high reactivity limits its range
form complexes with DNA are derived from the amino of diffusion to about a 1 mm radius from the site of syn-
acid ornithine, a component of the urea cycle. A com- thesis. In humans NO plays a role in a range of physio-
mon denominator of many of these pathways is amino logical processes, including neurotransmission, blood
acid decarboxylation, another PLP-requiring reaction clotting, and the control of blood pressure. Its mode of
(see Fig. 18–6). action is described in Chapter 12 (see Section 12.4).
The synthesis of some neurotransmitters is illus- Nitric oxide is synthesized from arginine in an
trated in Figure 22–31. Tyrosine gives rise to a family NADPH-dependent reaction catalyzed by nitric oxide
of catecholamines that includes dopamine, norepi- synthase (Fig. 22–33), a dimeric enzyme structurally
nephrine, and epinephrine. Levels of catecholamines related to NADPH cytochrome P-450 reductase (see
are correlated with, among other things, changes in Box 21–1). The reaction is a five-electron oxidation.
blood pressure. The neurological disorder Parkinson Each subunit of the enzyme contains one bound mole-
disease is associated with an underproduction of dopa- cule of each of four different cofactors: FMN, FAD, tet-
mine, and it has traditionally been treated by adminis- rahydrobiopterin, and Fe3⫹ heme. NO is an unstable
tering L-dopa. Overproduction of dopamine in the brain molecule and cannot be stored. Its synthesis is stimu-
may be linked to psychological disorders such as lated by interaction of nitric oxide synthase with Ca2⫹-
schizophrenia. calmodulin (see Fig. 12–11).
Glutamate decarboxylation gives rise to ␥-amino-
butyrate (GABA), an inhibitory neurotransmitter. Its SUMMARY 22.3 Molecules Derived from Amino Acids
underproduction is associated with epileptic seizures. Many important biomolecules are derived from
GABA analogs are used in the treatment of epilepsy and amino acids. Glycine is a precursor of porphyrins.
hypertension. Levels of GABA can also be increased by Degradation of iron-porphyrin (heme) generates
administering inhibitors of the GABA-degrading enzyme bilirubin, which is converted to bile pigments, with
GABA aminotransferase. Another important neurotrans- several physiological functions.
FIGURE 22–31 Biosynthesis of some neurotransmitters from amino

⫹
acids. The key step is the same in each case: a PLP-dependent decar-
NH3
boxylation (shaded light red).
HO CH2 CH COO᎐
Tyrosine
Tetrahydrobiopterin
O2
tyrosine
hydroxylase
H 2O
Dihydrobiopterin
HO ⫹ ⫹ ⫹
NH3 NH3 NH3
᎐
HO CH2 CH COO᎐ OOC CH2 CH2 CH COO᎐ CH2 CH COO᎐
Dopa Glutamate
aromatic PLP N Tryptophan

amino acid glutamate PLP H
decarboxylase CO2 decarboxylase CO2
Tetrahydrobiopterin
HO ⫹ ⫹
NH3 NH3 O2
tryptophan
HO CH2 CH2 ᎐
OOC CH2 CH2 CH2 hydroxylase
H2O
Dopamine ␥-Aminobutyrate
Dihydrobiopterin
(GABA)
Ascorbate
⫹
O2 NH3
dopamine
⫹
-hydroxylase H 2O CH2 CH COO᎐
NH3
HO
Dehydroascorbate
CH2 CH COO᎐
HO ⫹
NH3 N 5-Hydroxy-
N NH H
tryptophan
HO CH CH2
Histidine aromatic PLP
OH amino acid
Norepinephrine decarboxylase CO2
histidine PLP
adoMet decarboxylase CO2
phenylethanolamine ⫹
N-methyltransferase NH3
adoHcy ⫹
NH3 CH2 CH2
HO ⫹
H2 N CH3 HO
CH2 CH2
HO CH CH2
N NH N
OH H
Epinephrine Histamine Serotonin
Glycine and arginine give rise to creatine and 22.4 Biosynthesis and Degradation
phosphocreatine, an energy buffer. Glutathione,
formed from three amino acids, is an important of Nucleotides
cellular reducing agent.
Bacteria synthesize D-amino acids from L-amino As discussed in Chapter 8, nucleotides have a variety of
acids in racemization reactions requiring pyridoxal important functions in all cells. They are the precursors
phosphate. D-Amino acids are commonly found in of DNA and RNA. They are essential carriers of chemical
certain bacterial walls and certain antibiotics. energy—a role primarily of ATP and to some extent
GTP. They are components of the cofactors NAD,
The aromatic amino acids give rise to many plant FAD, S-adenosylmethionine, and coenzyme A, as well
substances. The PLP-dependent decarboxylation as of activated biosynthetic intermediates such as
of some amino acids yields important biological UDP-glucose and CDP-diacylglycerol. Some, such as
amines, including neurotransmitters. cAMP and cGMP, are also cellular second messengers.
Arginine is the precursor of nitric oxide, a Two types of pathways lead to nucleotides: the de
biological messenger. novo pathways and the salvage pathways. De novo
COO
⫹
ATP PPi ⫹ Pi H 3N C H
CH2 S-Adenosylmethionine
Methionine
CH2
⫹
⫹
S Adenosine NH3
⫹
CH3 H3N CH2 CH2 CH2 CH COO Ornithine
adoMet ornithine
PLP PLP
decarboxylase decarboxylase
CO2 CO2
⫹ ⫹
⫹ H3N (CH2)4 NH3 Putrescine
H3N CH2
CH2 propylaminotransferase I
CH2 CH3 S Adenosine
⫹
S Adenosine Methylthioadenosine
CH3 ⫹ ⫹
Decarboxylated H3N (CH2)3 NH (CH2)4 NH3 Spermidine

adoMet
propylaminotransferase II
CH3 S Adenosine
FIGURE 22–32 Biosynthesis of spermidine and spermine. The PLP-
dependent decarboxylation steps are shaded light red. In these reactions, ⫹ ⫹
H3N (CH2)3 NH (CH2)4 NH (CH2)3 NH3
S-adenosylmethionine (in its decarboxylated form) acts as a source of
propylamino groups (shaded blue). Spermine
synthesis of nucleotides begins with their metabolic pyrimidine ring is synthesized as orotate, attached to
precursors: amino acids, ribose 5-phosphate, CO2, and ribose phosphate, and then converted to the common
NH3. Salvage pathways recycle the free bases and pyrimidine nucleotides required in nucleic acid synthe-
nucleosides released from nucleic acid breakdown. Both sis. Although the free bases are not intermediates in the
types of pathways are important in cellular metabolism de novo pathways, they are intermediates in some of the
and both are discussed in this section. salvage pathways.
The de novo pathways for purine and pyrimidine Several important precursors are shared by the de
biosynthesis seem to be nearly identical in all living novo pathways for synthesis of pyrimidines and purines.
organisms. Notably, the free bases guanine, adenine, Phosphoribosyl pyrophosphate (PRPP) is important in
thymine, cytidine, and uracil are not intermediates in both, and in these pathways the structure of ribose is
these pathways; that is, the bases are not synthesized retained in the product nucleotide, in contrast to its fate
and then attached to ribose, as might be expected. The in the tryptophan and histidine biosynthetic pathways
purine ring structure is built up one or a few atoms at a discussed earlier. An amino acid is an important precur-
time, attached to ribose throughout the process. The sor in each type of pathway: glycine for purines and
COO COO COO

⫹ ⫹ ⫹
H 3N C H H3N C H H3N C H
CH2 NADPH ⫹ H⫹, O2 CH2 1 CH2

2 NADPH, O2
1
CH2 NADP⫹, H2O CH2 2 NADP ,
⫹
H2O CH2
CH2 CH2 CH2

⫹ NO•
NH NH NH Nitric
⫹ oxide
C NH2 C N OH C O
NH2 NH2 NH2

Arginine Hydroxyarginine Citrulline
FIGURE 22–33 Biosynthesis of nitric oxide. Both steps are catalyzed by nitric oxide synthase. The nitrogen of the NO is derived from the
guanidinium group of arginine.
aspartate for pyrimidines. Glutamine again is the most CO2

important source of amino groups—in five different Glycine
steps in the de novo pathways. Aspartate is also used as Aspartate
the source of an amino group in the purine pathways, in C N
two steps. N C
C Formate
Two other features deserve mention. First, there is C C
evidence, especially in the de novo purine pathway, that N N
the enzymes are present as large, multienzyme complexes Formate
in the cell, a recurring theme in our discussion of metabo-
lism. Second, the cellular pools of nucleotides (other than Amide N
of glutamine
ATP) are quite small, perhaps 1% or less of the amounts
required to synthesize the cell’s DNA. Therefore, cells
FIGURE 22–34 Origin of the ring atoms of purines. This information
must continue to synthesize nucleotides during nucleic
was obtained from isotopic experiments with 14C- or 15N-labeled precur-
acid synthesis, and in some cases nucleotide synthesis
sors. Formate is supplied in the form of N10-formyltetrahydrofolate.
may limit the rates of DNA replication and transcription.
Because of the importance of these processes in dividing
cells, agents that inhibit nucleotide synthesis have become formyltetrahydrofolate 3, and a nitrogen is contributed
particularly important in medicine. by glutamine (step 4), before dehydration and ring
We examine here the biosynthetic pathways of closure yield the five-membered imidazole ring of the
purine and pyrimidine nucleotides and their regulation, purine nucleus, as 5-aminoimidazole ribonucleotide
the formation of the deoxynucleotides, and the degra- (AIR; step 5).
dation of purines and pyrimidines to uric acid and urea. At this point, three of the six atoms needed for
We end with a discussion of chemotherapeutic agents the second ring in the purine structure are in place.
that affect nucleotide synthesis. To complete the process, a carboxyl group is first
added (step 6). This carboxylation is unusual in that
it does not require biotin, but instead uses the bicar-
De Novo Purine Nucleotide Synthesis bonate generally present in aqueous solutions. A
Begins with PRPP rearrangement transfers the carboxylate from the
The two parent purine nucle- exocyclic amino group to position 4 of the imidazole
otides of nucleic acids are ring (step 7). Steps 6 and 7 are found only in bac-
adenosine 59-monophosphate teria and fungi. In higher eukaryotes, including
(AMP; adenylate) and guano- humans, the 5-aminoimidazole ribonucleotide prod-
sine 59-monophosphate uct of step 5 is carboxylated directly to carboxyami-
(GMP; guanylate), containing noimidazole ribonucleotide in one step instead of two
the purine bases adenine and (step 6a ). The enzyme catalyzing this reaction is AIR
guanine. Figure 22–34 carboxylase.
shows the origin of the car- Aspartate now donates its amino group in two steps
bon and nitrogen atoms of (8 and 9): formation of an amide bond, followed by
the purine ring system, as elimination of the carbon skeleton of aspartate (as
determined by John M. fumarate). (Recall that aspartate plays an analogous
John M. Buchanan, Buchanan using isotopic role in two steps of the urea cycle; see Fig. 18–10.) The
1917–2007 tracer experiments in birds. final carbon is contributed by N10-formyltetrahydrofo-
The detailed pathway of purine biosynthesis was worked late (step 10 ), and a second ring closure takes place to
out primarily by Buchanan and G. Robert Greenberg in yield the second fused ring of the purine nucleus (step
the 1950s. 11 ). The first intermediate with a complete purine ring
In the first committed step of the pathway, an is inosinate (IMP).
amino group donated by glutamine is attached at C-1 of As in the tryptophan and histidine biosynthetic
PRPP (Fig. 22–35). The resulting 5-phosphoribosyl- pathways, the enzymes of IMP synthesis seem to be
amine is highly unstable, with a half-life of 30 seconds organized as large, multienzyme complexes in the cell.
at pH 7.5. The purine ring is subsequently built up on Once again, evidence comes from the existence of sin-
this structure. The pathway described here is identical gle polypeptides with several functions, some catalyzing
in all organisms, with the exception of one step that dif- nonsequential steps in the pathway. In eukaryotic cells
fers in higher eukaryotes, as noted below. ranging from yeast to fruit flies to chickens, steps 1, 3,
The second step is the addition of three atoms from and 5 in Figure 22–35 are catalyzed by a multifunc-
glycine (Fig. 22–35, step 2). An ATP is consumed to tional protein. An additional multifunctional protein
activate the glycine carboxyl group (in the form of an catalyzes steps 10 and 11 . In humans, a multifunctional
acyl phosphate) for this condensation reaction. The enzyme combines the activities of AIR carboxylase
added glycine amino group is then formylated by N10- and SAICAR synthetase (steps 6a and 8). In bacteria,
P O CH2 O AIR
H
H H HCO ᎐3
H O P P
5-Phosphoribosyl 6
ATP
OH OH 1-pyrophosphate (PRPP)
6a ADP ⫹ Pi
Glutamine
1 N
Glutamate CO2 HC
CH N5-Carboxyaminoimidazole
PPi C ribonucleotide
O C N N
P O CH2 O (N5-CAIR)
NH2 ᎐ H
O R
H H 5-Phospho-␤-
H H D-ribosylamine
7
OH OH
Glycine ᎐
OOC N
C
2 CH Carboxyamino-
ATP
C imidazole ribonucleotide
H 2N N
ADP ⫹ Pi (CAIR)
R
⫹
NH3
H2C Aspartate
Glycinamide
O C ribonucleotide (GAR) 8
NH ATP
R ADP ⫹ Pi
10 COO᎐
N -Formyl H4 folate
3 CH2 O
H4 folate H N
H HC N C C
N CH N-Succinyl-5-aminoimidazole-4-
H 2C C H COO᎐ C carboxamide ribonucleotide (SAICAR)
Formylglycinamide H 2N N
O C O ribonucleotide (FGAR)
R
NH
R 9 Fumarate
Glutamine O
Glutamate C N
4 H 2N C
ATP CH 5-Aminoimidazole-4-carboxamide
C ribonucleotide (AICAR)
H 2N N
ADP ⫹ Pi
R
H
N N10-Formyl H4 folate
H 2C C H Formylglycinamidine 10
ribonucleotide (FGAM) H4 folate
HN C O
NH O
R C N
H 2N C
CH N-Formylaminoimidazole-
ATP C 4-carboxamide ribonucleotide (FAICAR)
O C N N
5 ADP ⫹ Pi H H
R
H2O 1 glutamine-PRPP
amidotransferase
11 H2O
N 2 GAR synthetase
HC 5-Aminoimidazole
CH
ribonucleotide (AIR)
O 3 GAR transformylase
C
H2N N C 4 FGAR amidotransferase
N
R HN C 5 FGAM cyclase
CH (AIR synthetase)
HC C
FIGURE 22–35 De novo synthesis of purine nucleotides: construction of the O᎐ N N 6 N5-CAIR synthetase
6a AIR carboxylase
purine ring of inosinate (IMP). Each addition to the purine ring is shaded to ᎐
O P O CH2 O
match Figure 22–34. After step 2, R symbolizes the 5-phospho-D-ribosyl
7 N5-CAIR mutase
O H H 8 SAICAR synthetase
group on which the purine ring is built. Formation of 5-phosphoribosylamine H H 9 SAICAR lyase
(step 1) is the first committed step in purine synthesis. Note that the prod-
OH OH 10 AICAR transformylase
uct of step 9, AICAR, is the remnant of ATP released during histidine biosyn-
Inosinate (IMP) 11 IMP synthase
thesis (see Fig. 22–22, step 5). Abbreviations are given for most intermedi-
ates to simplify the naming of the enzymes. Step 6a is the alternative path
from AIR to CAIR occurring in higher eukaryotes.
H
᎐
OOC CH2 C COO᎐
NH Fumarate NH2
N N
GDP ⫹ Pi N N
GTP adenylosuccinate
Aspartate N N lyase N N
O adenylosuccinate Rib P Rib P

synthetase
N Adenylosuccinate Adenylate (AMP)
HN
H2O
N N NAD⫹
NADH ⫹ H⫹
Rib P
IMP O Gln Glu AMP ⫹ PPi O
Inosinate dehydrogenase ATP
N N
(IMP) HN HN
XMP-glutamine
O N N H2O amidotransferase H2N N N
H
Rib P Rib P
Xanthylate (XMP) Guanylate (GMP)
FIGURE 22–36 Biosynthesis of AMP and GMP from IMP.
these activities are found on separate proteins, but out affecting the formation of AMP. Conversely, an
the proteins may form a large noncovalent complex. accumulation of adenylate inhibits formation of adeny-
The channeling of reaction intermediates from one losuccinate by adenylosuccinate synthetase, without
enzyme to the next permitted by these complexes is affecting the biosynthesis of GMP. When both products
probably especially important for unstable intermedi- are present in sufficient quantities, IMP builds up, and
ates such as 5-phosphoribosylamine. it inhibits an earlier step in the pathway; this regulatory
Conversion of inosinate to adenylate requires the strategy is called sequential feedback inhibition. In
insertion of an amino group derived from aspartate the third mechanism, GTP is required in the conversion
(Fig. 22–36); this takes place in two reactions similar
to those used to introduce N-1 of the purine ring (Fig.
22–35, steps 8 and 9). A crucial difference is that GTP Ribose 5-phosphate
ADP ATP
rather than ATP is the source of the high-energy phos- ribose phosphate
pyrophosphokinase ADP
phate in synthesizing adenylosuccinate. Guanylate is (PRPP synthetase)
formed by the NAD⫹-requiring oxidation of inosinate at
PRPP
C-2, followed by addition of an amino group derived
from glutamine. ATP is cleaved to AMP and PPi in the AMP
glutamine-PRPP
final step (Fig. 22–36). amidotransferase GMP
IMP
Purine Nucleotide Biosynthesis Is Regulated 5-Phosphoribosylamine
by Feedback Inhibition
9 steps
Three major feedback mechanisms cooperate in regulat-
ing the overall rate of de novo purine nucleotide synthe-
sis and the relative rates of formation of the two end IMP
products, adenylate and guanylate (Fig. 22–37). The adenylosuccinate
synthetase
IMP
dehydrogenase
first mechanism is exerted on the first reaction that is
AMP GMP
unique to purine synthesis: transfer of an amino group to
PRPP to form 5-phosphoribosylamine. This reaction is XMP
catalyzed by the allosteric enzyme glutamine-PRPP ami- XMP-glutamine
dotransferase, which is inhibited by the end products amidotransferase
Adenylosuccinate
IMP, AMP, and GMP. AMP and GMP act synergistically GMP
adenylosuccinate
in this concerted inhibition. Thus, whenever either lyase
AMP or GMP accumulates to excess, the first step in its
biosynthesis from PRPP is partially inhibited. AMP
In the second control mechanism, exerted at a later FIGURE 22–37 Regulatory mechanisms in the biosynthesis of adenine
stage, an excess of GMP in the cell inhibits formation of and guanine nucleotides in E. coli. Regulation of these pathways differs
xanthylate from inosinate by IMP dehydrogenase, within other organisms.
of IMP to AMP, whereas ATP is required for conver- Aspartate

sion of IMP to GMP (Fig. 22–36), a reciprocal arrange-
ment that tends to balance the synthesis of the two Carbamoyl
phosphate
ribonucleotides. aspartate
trans-
The final control mechanism is the inhibition of carbamoylase
PRPP synthesis by the allosteric regulation of ribose Pi ᎐
O O
C
phosphate pyrophosphokinase. This enzyme is inhibited H2N CH2
N-Carbamoylaspartate
by ADP and GDP, in addition to metabolites from other C CH COO᎐
pathways for which PRPP is a starting point. O N
H
dihydroorotase
O
H2O
Pyrimidine Nucleotides Are Made from Aspartate, C
HN CH2
PRPP, and Carbamoyl Phosphate L-Dihydroorotate
C CH COO᎐
The common pyrimidine ribonucleotides are cytidine O N
H
59-monophosphate (CMP; cytidylate) and uridine NAD⫹
dihydroorotate
59-monophosphate (UMP; uridylate), which contain the dehydrogenase
pyrimidines cytosine and uracil. De novo pyrimidine NADH ⫹ H⫹
O
nucleotide biosynthesis (Fig. 22–38) proceeds in a C
somewhat different manner from purine nucleotide syn- Orotate
HN CH
thesis; the six-membered pyrimidine ring is made first O

C
N
C
COO᎐
and then attached to ribose 5-phosphate. Required in H
PRPP
this process is carbamoyl phosphate, also an intermediate orotate O
in the urea cycle. However, as we noted in Chapter 18, in phosphoribosyl-
transferase
C
animals the carbamoyl phosphate required in urea syn- PPi
HN CH
thesis is made in mitochondria by carbamoyl phosphate O

C
N
C
COO᎐
synthetase I, whereas the carbamoyl phosphate Orotidylate P OOCH2
O
required in pyrimidine biosynthesis is made in the cyto- H H
sol by a different form of the enzyme, carbamoyl H H
phosphate synthetase II. In bacteria, a single enzyme orotidylate

OH OH O
supplies carbamoyl phosphate for the synthesis of argi- decarboxylase C
nine and pyrimidines. The bacterial enzyme has three CO2 HN CH
separate active sites, spaced along a channel nearly 100 O

C
N
CH
Uridylate (UMP)
Å long (Fig. 22–39). Bacterial carbamoyl phosphate P OOCH2
O
synthetase provides a vivid illustration of the channel- H H
ing of unstable reaction intermediates between active 2 ATP H H
sites. kinases OH OH
Carbamoyl phosphate reacts with aspartate to yield
2ADP
N-carbamoylaspartate in the first committed step of
pyrimidine biosynthesis (Fig. 22–38). This reaction is Uridine 5⬘-triphosphate (UTP)
catalyzed by aspartate transcarbamoylase. In bacte- Gln
ria, this step is highly regulated, and bacterial aspartate
transcarbamoylase is one of the most thoroughly stud-
ied allosteric enzymes (see below). By removal of water cytidylate
Glu
from N-carbamoylaspartate, a reaction catalyzed by synthetase
ATP
dihydroorotase, the pyrimidine ring is closed to form NH2
L-dihydroorotate. This compound is oxidized to the C
pyrimidine derivative orotate, a reaction in which NAD⫹ ADP ⫹ Pi N CH
is the ultimate electron acceptor. In eukaryotes, the O

C
N
CH
first three enzymes in this pathway—carbamoyl phos- P P P OOCH2

O
phate synthetase II, aspartate transcarbamoylase, and Cytidine 5⬘-triphosphate (CTP) H H
dihydroorotase—are part of a single trifunctional pro- H H
tein. The protein, known by the acronym CAD, contains OH OH
three identical polypeptide chains (each of Mr 230,000), FIGURE 22–38 De novo synthesis of pyrimidine nucleotides: biosynthesis
each with active sites for all three reactions. This sug- of UTP and CTP via orotidylate. The pyrimidine is constructed from car-
gests that large, multienzyme complexes may be the bamoyl phosphate and aspartate. The ribose 5-phosphate is then added
rule in this pathway. to the completed pyrimidine ring by orotate phosphoribosyltransferase.
Once orotate is formed, the ribose 5-phosphate The first step in this pathway (not shown here; see Fig. 18–11a) is the syn-
side chain, provided once again by PRPP, is attached to thesis of carbamoyl phosphate from CO2 and NH⫹4. In eukaryotes, the
yield orotidylate (Fig. 22–38). Orotidylate is then first step is catalyzed by carbamoyl phosphate synthetase II.
Vmax
Normal
activity
Glutamine-binding (no CTP) CTP ⫹ ATP
site
V0 (␮M/min)
1
2 Vmax
CTP
Tunnel
10 20 30
ATP-binding site K0.5 ⫽ 12 mM K0.5 ⫽ 23 mM

[Aspartate] (mM)
FIGURE 22–40 Allosteric regulation of aspartate transcarbamoylase by

CTP and ATP. Addition of 0.8 mM CTP, the allosteric inhibitor of ATCase,
ATP-binding site increases the K0.5 for aspartate (lower curve) thereby reducing the rate
of conversion of aspartate to N-carbamoylaspartate. ATP at 0.6 mM fully
reverses this inhibition by CTP (middle curve).
FIGURE 22–39 Channeling of intermediates in bacterial carbamoyl ATP prevents the changes induced by CTP. Figure
phosphate synthetase. (Derived from PDB ID 1M6V) The reaction cata- 22–40 shows the effects of the allosteric regulators on
lyzed by this enzyme (and its mitochondrial counterpart) is illustrated in
the activity of ATCase.
Figure 18–11a. The small and large subunits are shown in tan and blue, re-
spectively; the tunnel between active sites (almost 100 Å long) is shown
as white. In this reaction, a glutamine molecule binds to the small subunit, Nucleoside Monophosphates Are Converted
donating its amido nitrogen as NH⫹4 in a glutamine amidotransferase–type to Nucleoside Triphosphates
reaction. The NH⫹4 enters the tunnel, which takes it to a second active Nucleotides to be used in biosynthesis are generally
site, where it combines with bicarbonate in a reaction requiring ATP. The
converted to nucleoside triphosphates. The conversion
carbamate then reenters the tunnel to reach the third active site, where it
pathways are common to all cells. Phosphorylation of
is phosphorylated by ATP to carbamoyl phosphate. To solve this structure,
AMP to ADP is promoted by adenylate kinase, in the
the enzyme was crystallized with ornithine bound to the glutamine-binding
reaction
site and ADP bound to the ATP-binding sites.
ATP 1 AMP ∆ 2 ADP
decarboxylated to uridylate, which is phosphorylated
to UTP. CTP is formed from UTP by the action of cyti- The ADP so formed is phosphorylated to ATP by the
dylate synthetase, by way of an acyl phosphate inter- glycolytic enzymes or through oxidative phosphorylation.
mediate (consuming one ATP). The nitrogen donor is ATP also brings about the formation of other nucle-
normally glutamine, although the cytidylate synthetas- oside diphosphates by the action of a class of enzymes
es in many species can use NH⫹4 directly. called nucleoside monophosphate kinases. These
enzymes, which are generally specific for a particular
base but nonspecific for the sugar (ribose or deoxyri-
Pyrimidine Nucleotide Biosynthesis Is Regulated bose), catalyze the reaction
by Feedback Inhibition
ATP 1 NMP ∆ ADP 1 NDP
Regulation of the rate of pyrimidine nucleotide synthe-
sis in bacteria occurs in large part through aspartate The efficient cellular systems for rephosphorylating
transcarbamoylase (ATCase), which catalyzes the first ADP to ATP tend to pull this reaction in the direction of
reaction in the sequence and is inhibited by CTP, the products.
end product of the sequence (Fig. 22–38). The bacterial Nucleoside diphosphates are converted to triphos-
ATCase molecule consists of six catalytic subunits and phates by the action of a ubiquitous enzyme, nucleo-
six regulatory subunits (see Fig. 6–33). The catalytic side diphosphate kinase, which catalyzes the reaction
subunits bind the substrate molecules, and the alloste-
NTPD 1 NDPA ∆ NDPD 1 NTPA
ric subunits bind the allosteric inhibitor, CTP. The
entire ATCase molecule, as well as its subunits, exists in This enzyme is notable in that it is not specific for the
two conformations, active and inactive. When CTP is base (purines or pyrimidines) or the sugar (ribose or
not bound to the regulatory subunits, the enzyme is deoxyribose). This nonspecificity applies to both phos-
maximally active. As CTP accumulates and binds to the phate acceptor (A) and donor (D), although the donor
regulatory subunits, they undergo a change in confor- (NTPD) is almost invariably ATP because it is present in
mation. This change is transmitted to the catalytic sub- higher concentration than other nucleoside triphos-
units, which then also shift to an inactive conformation. phates under aerobic conditions.
(a) (b)
Ribonucleotides Are the Precursors
of Deoxyribonucleotides NADPH ⫹ H⫹ NADP⫹ NADPH ⫹ H⫹ NADP⫹
glutathione
Deoxyribonucleotides, the building blocks of DNA, are reductase
derived from the corresponding ribonucleotides by
direct reduction at the 29-carbon atom of the D-ribose
to form the 29-deoxy derivative. For example, adenos- GSSG 2GSH FAD FADH2
ine diphosphate (ADP) is reduced to 29-deoxyadenosine glutaredoxin
reductase
thioredoxin
reductase
diphosphate (dADP), and GDP is reduced to dGDP.
This reaction is somewhat unusual in that the reduc-
tion occurs at a nonactivated carbon; no closely analo- SH
S
SH
Glutaredoxin Glutaredoxin Thioredoxin Thioredoxin

gous chemical reactions are known. The reaction is SH
S S
SH
catalyzed by ribonucleotide reductase, best charac-
terized in E. coli, in which its substrates are ribonucle-
oside diphosphates.
The reduction of the D-ribose portion of a ribonu- HS
Ribonucleotide S Ribonucleotide
cleoside diphosphate to 29-deoxy-D-ribose requires a reductase S reductase
HS
pair of hydrogen atoms, which are ultimately donated
by NADPH via an intermediate hydrogen-carrying pro-
tein, thioredoxin. This ubiquitous protein serves a
similar redox function in photosynthesis (see Fig.
20–19) and other processes. Thioredoxin has pairs of
H2O
—SH groups that carry hydrogen atoms from NADPH
dNDP NDP
to the ribonucleoside diphosphate. Its oxidized (disul-
fide) form is reduced by NADPH in a reaction catalyzed FIGURE 22–41 Reduction of ribonucleotides to deoxyribonucleotides by
by thioredoxin reductase (Fig. 22–41), and reduced ribonucleotide reductase. Electrons are transmitted (red arrows) to the
thioredoxin is then used by ribonucleotide reductase to enzyme from NADPH via (a) glutaredoxin or (b) thioredoxin. The sul-
reduce the nucleoside diphosphates (NDPs) to deoxy- fide groups in glutaredoxin reductase are contributed by two molecules of
ribonucleoside diphosphates (dNDPs). A second source bound glutathione (GSH; GSSG indicates oxidized glutathione). Note that
of reducing equivalents for ribonucleotide reductase is thioredoxin reductase is a flavoenzyme, with FAD as prosthetic group.
glutathione (GSH). Glutathione serves as the reductant
for a protein closely related to thioredoxin, glutare- Ribonucleotide reductase is notable in that its
doxin, which then transfers the reducing power to reaction mechanism provides the best-characterized
ribonucleotide reductase (Fig. 22–41). example of the involvement of free radicals in bio-
chemical transformations, once thought to be rare in
biological systems. The enzyme in E. coli and most
Regulatory Allosteric eukaryotes is an ␣2␤2 dimer, with catalytic subunits ␣2
sites effectors
and radical-generation subunits ␤2 (Fig. 22–42). Each
Substrate catalytic subunit contains two kinds of regulatory sites,
specificity ATP, dATP,
site dGTP, dTTP
Primary
regulation ATP,
site
␣ ␣ Cys439
dATP
subunit subunit Tyr730
Substrates
Active HS SH ADP,
HS SH a
site CDP, a Tyr731
XH HX
UDP, Tyr356
GDP Trp48
␤ subunit b b Tyr122
O F e3⫹ Fe3⫹ O
3⫹ 3⫹
Fe O O Fe
a2b2 docking model Radical pathway

(a) (b) (c)
FIGURE 22–42 Ribonucleotide reductase. (a) A schematic diagram of ␣2␤2 (derived from PDB ID 3UUS). (c) The likely path of radical
of the subunit structures. The catalytic subunit (␣; also called R1) formation from the initial Tyr122 in a ␤ subunit to the active-site Cys439,
contains the two regulatory sites described in Fig. 22–44 and two Cys which is used in the mechanism shown in Figure 22–43. Several aromatic
residues central to the reaction mechanism. The radical-generation amino acid residues participate in long-range radical transfer from the
subunits ␤ (also called R2) contain the critical Tyr122 residue and the point of its formation at Tyr 1 22 to the active site, where the nucleotide
binuclear iron center. (b) Structures of ␣2 and ␤2 give the likely structure substrate is bound.
as described below. The two active sites of the enzyme enzyme (class I) requires oxygen to regenerate the tyro-
are formed at the interface between the catalytic (␣2) syl radical if it is quenched, so this enzyme functions only
and radical-generation (␤2) subunits. At each active site, in an aerobic environment. Class II enzymes, found in
an ␣ subunit contributes two sulfhydryl groups required other microorganisms, have 59-deoxyadenosylcobalamin
for activity and the ␤2 subunits contribute a stable tyrosyl (see Box 17–2) rather than a binuclear iron center. Class
radical. The ␤2 subunits also have a binuclear iron (Fe3⫹) III enzymes have evolved to function in an anaerobic
cofactor that helps generate and stabilize the Tyr122 environment. E. coli contains a separate class III ribo-
radical (Fig. 22–42). The tyrosyl radical is too far from nucleotide reductase when grown anaerobically; this
the active site to interact directly with the site, but sev- enzyme contains an iron-sulfur cluster (structurally dis-
eral aromatic residues form a long-range radical transfer tinct from the binuclear iron center of the class I enzyme)
pathway to the active site (Fig. 22–42c). A likely mech- and requires NADPH and S-adenosylmethionine for
anism for the ribonucleotide reductase reaction is illus- activity. It uses nucleoside triphosphates rather than
trated in Figure 22–43. In E. coli, the sources of the nucleoside diphosphates as substrates. The evolution of
required reducing equivalents for this reaction are thio- different classes of ribonucleotide reductase for produc-
redoxin and glutaredoxin, as noted above. tion of DNA precursors in different environments reflects
Three classes of ribonucleotide reductase have been the importance of this reaction in nucleotide metabolism.
reported. Their mechanisms (where known) generally Regulation of E. coli ribonucleotide reductase is
conform to the scheme in Figure 22–43, but they differ in unusual in that not only its activity but its substrate
the identity of the group supplying the active-site radical specificity is regulated by the binding of effector mol-
and in the cofactors used to generate it. The E. coli ecules. Each ␣ subunit has two types of regulatory
a subunit
Base Base
P P O CH2 O HS P P O CH2 O HS
H H HS 1 H HS
H H H H
3⬘ 2⬘
OH OH A 3⬘-ribonucleotide OH OH
NDP radical is formed. H
X X
Ribonucleotide
reductase b subunit
Thioredoxin S S
The enzyme (glutaredoxin)
NDP
dithiol is reduced The 2⬘-hydroxyl is
to complete the
6 2 protonated.
dNDP Thioredoxin (SH)2
cycle. (glutaredoxin)
Base Base
P P O CH2 O S P P O CH2 O HS
H H S H S
H H H H
⫹
OH H OH O H
dNDP H
H
X X
H2O is eliminated
Step 1 is reversed, 3 to form a
O
regenerating a tyrosyl 5 H H radical-stabilized
radical on the enzyme. carbocation.
S
Base Base
P P O CH2 O S P P O CH2 O H
H
H
H
S 4 H
H
H
S
⫹
OH H Dithiol is oxidized on OH
the enzyme; two electrons
X H are transferred to the X H
2⬘-carbon.
MECHANISM FIGURE 22–43 Proposed mechanism for ribonucleotide groups are on the ␣ subunit; the active-site radical (—X•) is on the ␤
reductase. In the enzyme of E. coli and most eukaryotes, the active thiol subunit and in E. coli is probably a thiyl radical of Cys439 (see Fig. 22–42).
Regulation at primary Regulation at substrate-

regulatory sites specificity sites
ATP (d)ATP
dCTP dCDP CDP dCDP dCTP
dTTP dUDP UDP dUDP dTTP
dGTP dGDP GDP dGDP dGTP
dATP dADP ADP dADP dATP
Products Substrates Products
FIGURE 22–44 Regulation of ribonucleotide reductase by deoxynucleo- the second type of regulatory site, the substrate-specificity site (right).
side triphosphates. The overall activity of the enzyme is affected by The diagram indicates inhibition or stimulation of enzyme activity with
binding at the primary regulatory site (left). The substrate specificity of the four different substrates. The pathway from dUDP to dTTP is
the enzyme is affected by the nature of the effector molecule bound at described later (see Figs. 22–46, 22–47).
site (Fig. 22–42). One type affects overall enzyme dNTPs is signaled by high levels of dTTP, which shifts
activity and binds either ATP, which activates the the specificity to favor reduction of GDP. High levels
enzyme, or dATP, which inactivates it. The second of dGTP, in turn, shift the specificity to ADP reduc-
type alters substrate specificity in response to the tion, and high levels of dATP shut the enzyme down.
effector molecule—ATP, dATP, dTTP, or dGTP—that These effectors are thought to induce several distinct
is bound there (Fig. 22–44). When ATP or dATP is enzyme conformations with altered specificities.
bound, reduction of UDP and CDP is favored. When These regulatory effects are accompanied by, and
dTTP or dGTP is bound, reduction of GDP or ADP, presumably mediated by, large structural rearrange-
respectively, is stimulated. The scheme is designed to ments in the enzyme. When the active form of the
provide a balanced pool of precursors for DNA synthe- E. coli enzyme (␣2␤2) is inhibited by the addition of
sis. ATP is also a general activator for biosynthesis the allosteric inhibitor dATP, a ringlike ␣4␤4 structure
and ribonucleotide reduction. The presence of dATP forms, with alternating ␣2 and ␤2 subunits (Fig. 22–45).
in small amounts increases the reduction of pyrimi- In this altered structure, the radical-forming path from
dine nucleotides. An oversupply of the pyrimidine ␤ to ␣ is disrupted and the residues in the path are
a2
b2 a2b2 a4b4
FIGURE 22–45 Oligomerization of ribonucleotide reductase induced by the residues in the radical-forming path are exposed to the solvent,
the allosteric inhibitor dATP. In high concentrations of dATP (50 ␮M), blocking the radical reaction and inhibiting the enzyme. The oligomeriza-
ring-shaped ␣4␤4 structures form (PDB ID 3UUS). In this conformation, tion is reversed at lower dATP concentrations.
exposed to solvent, effectively preventing radical trans- H

O O
fer and thus inhibiting the reaction. The formation of
C H
ringlike ␣4␤4 structures is reversed when dATP levels dUMP HN dTMP HN
are reduced. The yeast ribonucleotide reductase also H
undergoes oligomerization in the presence of dATP, O N O N
P O CH2 P O CH2
forming a hexameric ring structure, ␣6␤6. O O
H H H H
Thymidylate Is Derived from dCDP and dUMP H H H H
DNA contains thymine rather than uracil, and the de OH H OH H
novo pathway to thymine involves only deoxyribonucle-
otides. The immediate precursor of thymidylate (dTMP) thymidylate
is dUMP. In bacteria, the pathway to dUMP begins with synthase
formation of dUTP, either by deamination of dCTP or by

phosphorylation of dUDP (Fig. 22–46). The dUTP is
converted to dUMP by a dUTPase. The latter reaction
must be efficient to keep dUTP pools low and prevent H H
H2N N N H2N N N
incorporation of uridylate into DNA.
Conversion of dUMP to dTMP is catalyzed by thy- H
HN HN
midylate synthase. A one-carbon unit at the hydroxy- N CH2 N CH2
methyl (—CH2OH) oxidation level (see Fig. 18–17) is O H C N R O
HN R
transferred from N5,N10-methylenetetrahydrofolate to H
dUMP, then reduced to a methyl group (Fig. 22–47).
N5,N10-Methylene- 7,8-Dihydrofolate
The reduction occurs at the expense of oxidation of tetrahydrofolate
tetrahydrofolate to dihydrofolate, which is unusual in
tetrahydrofolate-requiring reactions. (The mechanism Glycine NADPH ⫹ H⫹
serine
of this reaction is shown in Fig. 22–53.) The dihydro- PLP hydroxymethyl-
dihydrofolate
reductase
folate is reduced to tetrahydrofolate by dihydrofolate transferase
Serine NADP⫹
reductase—a regeneration that is essential for the
many processes that require tetrahydrofolate. In plants H
and at least one protist, thymidylate synthase and H2N N N
dihydrofolate reductase reside on a single bifunctional H
protein. HN
CH2
N
About 10% of the human population (and up to O H
50% of people in impoverished communities) HN R
suffers from folic acid deficiency. When the deficiency Tetrahydrofolate
is severe, the symptoms can include heart disease, can- FIGURE 22–47 Conversion of dUMP to dTMP by thymidylate synthase
cer, and some types of brain dysfunction. At least some and dihydrofolate reductase. Serine hydroxymethyltransferase is
of these symptoms arise from a reduction of thymi- required for regeneration of the N5,N10-methylene form of tetrahydrofo-
dylate synthesis, leading to an abnormal incorporation late. In the synthesis of dTMP, all three hydrogens of the added methyl
of uracil into DNA. Uracil is recognized by DNA repair group are derived from N5,N10-methylenetetrahydrofolate (light red
pathways (described in Chapter 25) and is cleaved from and gray).
the DNA. The presence of high levels of uracil in DNA
CDP dCDP dCTP leads to strand breaks that can greatly affect the func-
ribonucleotide
nucleoside tion and regulation of nuclear DNA, ultimately causing
diphosphate deaminase
reductase kinase the observed effects on the heart and brain, as well as
UDP dUDP dUTP increased mutagenesis that leads to cancer. ■
dUTPase
Degradation of Purines and Pyrimidines Produces
dUMP Uric Acid and Urea, Respectively
thymidylate
synthase Purine nucleotides are degraded by a pathway in which
they lose their phosphate through the action of
dTMP
59-nucleotidase (Fig. 22–48). Adenylate yields
FIGURE 22–46 Biosynthesis of thymidylate (dTMP). The pathways are adenosine, which is deaminated to inosine by adenos-
shown beginning with the reaction catalyzed by ribonucleotide reduc- ine deaminase, and inosine is hydrolyzed to hypoxan-
tase. Figure 22–47 gives details of the thymidylate synthase reaction. thine (its purine base) and D-ribose. Hypoxanthine is
Excreted by:
AMP
O
H2O
59-nucleotidase C N
Pi HN C Primates, birds,
C OH Uric acid
C C reptiles, insects
GMP Adenosine N
HO N H
H2O H2O
adenosine
59-nucleotidase
deaminase 1
O2 ⫹ H2O
Pi NH3 2
urate oxidase
Guanosine Inosine CO2
H2O H2O O
nucleosidase nucleosidase H
N
Ribose Ribose NH2 C
C O Allantoin Most mammals
O O O C C
N H N
N N H H
HN HN Hypoxanthine
(keto form) H2O
H2N N N N N allantoinase
H H
Guanine
H2O H2O ⫹ O2
xanthine
oxidase NH2 COO NH2
NH3 H2O2
C C C Allantoate Bony fishes
O O N H N O
guanine H H
deaminase N
HN Xanthine
(keto form) H2O
N allantoicase
HO N
H COO
H2O ⫹ O2 CHO
xanthine
oxidase Glyoxylate
H2O2
O Amphibians,
O
Urea cartilaginous
N 2 H2N C NH2 fishes
HN
OH
2H2O
HO N N urease
H
2CO2
Uric acid Marine
4NH⫹
4
invertebrates
FIGURE 22–48 Catabolism of purine nucleotides. Note that primates as uric acid from purine degradation. Similarly, fish excrete much more
excrete much more nitrogen as urea via the urea cycle (Chapter 18) than nitrogen as NH⫹4 than as urea produced by the pathway shown here.
oxidized successively to xanthine and then uric acid by purine nucleotides of nucleic acids. In most mammals
xanthine oxidase, a flavoenzyme with an atom of and many other vertebrates, uric acid is further degraded
molybdenum and four iron-sulfur centers in its pros- to allantoin by the action of urate oxidase. In other
thetic group. Molecular oxygen is the electron acceptor organisms the pathway is further extended, as shown in
in this complex reaction. Figure 22–48.
GMP catabolism also yields uric acid as end prod- The pathways for degradation of pyrimidines
uct. GMP is first hydrolyzed to guanosine, which is generally lead to NH⫹4 production and thus to urea
then cleaved to free guanine. Guanine undergoes synthesis. Thymine, for example, is degraded to
hydrolytic removal of its amino group to yield xan- methylmalonylsemialdehyde (Fig. 22–49), an inter-
thine, which is converted to uric acid by xanthine oxi- mediate of valine catabolism. It is further degraded
dase (Fig. 22–48). through propionyl-CoA and methylmalonyl-CoA to
Uric acid is the excreted end product of purine succinyl-CoA (see Fig. 18–27).
catabolism in primates, birds, and some other animals. Genetic aberrations in human purine metabolism
A healthy adult human excretes uric acid at a rate of have been found, some with serious consequences.
about 0.6 g/24 h; the excreted product arises in part For example, adenosine deaminase (ADA) defi-
from ingested purines and in part from turnover of the ciency leads to severe immunodeficiency disease in
O Purine and Pyrimidine Bases Are Recycled

HN
C
C CH3
by Salvage Pathways
Thymine
Free purine and pyrimidine bases are constantly
C CH
O N released in cells during the metabolic degradation of
H nucleotides. Free purines are in large part salvaged and
reused to make nucleotides, in a pathway much simpler
NADPH ⫹ H⫹ than the de novo synthesis of purine nucleotides
dihydrouracil
dehydrogenase described earlier. One of the primary salvage pathways
NADP⫹ consists of a single reaction catalyzed by adenosine
O phosphoribosyltransferase, in which free adenine
reacts with PRPP to yield the corresponding adenine
C H
HN C nucleotide:
CH3 Dihydrothymine
C CH2 Adenine 1 PRPP ¡ AMP 1 PPi
O N
H Free guanine and hypoxanthine (the deamination prod-
uct of adenine; Fig. 22–48) are salvaged in the same way
H2O by hypoxanthine-guanine phosphoribosyltransfer-
dihydropyrimidinase ase. A similar salvage pathway exists for pyrimidine
bases in microorganisms, and possibly in mammals.
A genetic lack of hypoxanthine-guanine phospho-
O
ribosyltransferase activity, seen almost exclusive-
H2N C NH CH2 CH C -Ureidoisobutyrate
ly in male children, results in a bizarre set of symptoms
O⫺
O H3 called Lesch-Nyhan syndrome. Children with this
H2O genetic disorder, which becomes manifest by the age of 2
-ureidopropionase
years, are sometimes poorly coordinated and have intel-
⫹ ⫺
lectual deficits. In addition, they are extremely hostile and
NH4 ⫹ HCO3
show compulsive self-destructive tendencies: they muti-
O late themselves by biting off their fingers, toes, and lips.
The devastating effects of Lesch-Nyhan syndrome
⫹
H3N CH2 CH C -Aminoisobutyrate
O⫺ illustrate the importance of the salvage pathways.
CH3
Hypoxanthine and guanine arise constantly from the
␣ -Ketoglutarate breakdown of nucleic acids. In the absence of hypoxan-
aminotransferase thine-guanine phosphoribosyltransferase, PRPP levels
Glutamate rise and purines are overproduced by the de novo path-
way, resulting in high levels of uric acid production and
O O goutlike damage to tissue (see below). The brain is
C CH C especially dependent on the salvage pathways, and this
H CH3 O ⫺
may account for the central nervous system damage in
Methylmalonyl- children with Lesch-Nyhan syndrome. This syndrome is
semialdehyde another potential target for gene therapy. ■
FIGURE 22–49 Catabolism of a pyrimidine. Shown here is the pathway
for thymine. The methylmalonylsemialdehyde is further degraded to Excess Uric Acid Causes Gout
succinyl-CoA. Long thought (erroneously) to be due to “high
living,” gout is a disease of the joints caused by
which T lymphocytes and B lymphocytes do not develop an elevated concentration of uric acid in the blood and
properly. Lack of ADA leads to a 100-fold increase in the tissues. The joints become inflamed, painful, and arthritic,
cellular concentration of dATP, a strong inhibitor of ribo- owing to the abnormal deposition of sodium urate crys-
nucleotide reductase (Fig. 22–44). High levels of dATP tals. The kidneys are also affected, as excess uric acid is
produce a general deficiency of other dNTPs in T lym- deposited in the kidney tubules. Gout occurs predomi-
phocytes. The basis for B-lymphocyte toxicity is less nantly in males. Its precise cause is not known, but it
clear. Individuals with ADA deficiency lack an effective often involves an underexcretion of urate. A genetic
immune system and do not survive unless isolated in a deficiency of one or another enzyme of purine metabo-
sterile “bubble” environment. ADA deficiency was one of lism may also be a factor in some cases.
the first targets of human gene therapy trials (in 1990), Gout is effectively treated by a combination of
which yielded mixed results. In more recent trials, some nutritional and drug therapies. Foods especially rich in
ADA-deficient individuals regained normal immune nucleotides and nucleic acids, such as liver or glandular
function after being given a functional gene for ADA. ■ products, are withheld from the diet. Major alleviation
OH OH Many Chemotherapeutic Agents Target Enzymes

C H C
N C
C
N C
N in the Nucleotide Biosynthetic Pathways
N CH The growth of cancer cells is not controlled in
HC C HC C
N N N N the same way as cell growth in most normal tis-
H H
Allopurinol Hypoxanthine
sues. Cancer cells have greater requirements for
(enol form) nucleotides as precursors of DNA and RNA, and con-
xanthine
oxidase
sequently are generally more sensitive than normal
cells to inhibitors of nucleotide biosynthesis. A grow-
OH ing array of important chemotherapeutic agents—for
C H cancer and other diseases—act by inhibiting one or
C
N C more enzymes in these pathways. We describe here
N
C C several well-studied examples that illustrate produc-
HO N N
H tive approaches to treatment and help us understand
Oxypurinol how these enzymes work.
The first set of agents includes compounds that
FIGURE 22–50 Allopurinol, an inhibitor of xanthine oxidase. Hypoxan- inhibit glutamine amidotransferases. Recall that gluta-
thine is the normal substrate of xanthine oxidase. Only a slight alteration
mine is a nitrogen donor in at least half a dozen sepa-
in the structure of hypoxanthine (shaded light red) yields the medically
rate reactions in nucleotide biosynthesis. The binding
effective enzyme inhibitor allopurinol. At the active site, allopurinol is
sites for glutamine and the mechanism by which NH⫹4 is
converted to oxypurinol, a strong competitive inhibitor that remains
tightly bound to the reduced form of the enzyme.
extracted are quite similar in many of these enzymes.
Most are strongly inhibited by glutamine analogs such
as azaserine and acivicin (Fig. 22–51). Azaserine,
of the symptoms is provided by the drug allopurinol characterized by John Buchanan in the 1950s, was one
(Fig. 22–50), which inhibits xanthine oxidase, the of the first examples of a mechanism-based enzyme
enzyme that catalyzes the conversion of purines to uric inactivator (suicide inactivator; p. 210 and Box 6–3).
acid. Allopurinol is a substrate of xanthine oxidase, Acivicin shows promise as a cancer chemotherapeutic
which converts allopurinol to oxypurinol (alloxanthine). agent.
Oxypurinol inactivates the reduced form of the enzyme Other useful targets for pharmaceutical agents are
by remaining tightly bound in its active site. When xan- thymidylate synthase and dihydrofolate reductase,
thine oxidase is inhibited, the excreted products of enzymes that provide the only cellular pathway for
purine metabolism are xanthine and hypoxanthine, thymine synthesis (Fig. 22–52). One inhibitor that
which are more water-soluble than uric acid and less acts on thymidylate synthase, fluorouracil, is an
likely to form crystalline deposits. Allopurinol was important chemotherapeutic agent. Fluorouracil itself
developed by Gertrude Elion and George Hitchings, is not the enzyme inhibitor. In the cell, salvage path-
who also developed acyclovir, used in treating people ways convert it to the deoxynucleoside monophos-
with genital and oral herpes infections, and other purine phate FdUMP, which binds to and inactivates the
analogs used in cancer chemotherapy. ■ enzyme. Inhibition by FdUMP (Fig. 22–53) is a classic
example of mechanism-based enzyme inactivation.
Another prominent chemotherapeutic agent, metho-
trexate, is an inhibitor of dihydrofolate reductase.
⫹ ⫹
NH3 NH3
N
CH2 C COO N⫹ CH2 C COO
NH2 CH
H H
C CH2 C O
O O
Glutamine Azaserine
⫹
NH3
O
N CH C COO
C CH2 H
Cl
Acivicin
FIGURE 22–51 Azaserine and acivicin, inhibitors of glutamine amido-

Gertrude Elion, 1918–1999, and George Hitchings, transferases. These analogs of glutamine interfere in several amino acid
1905–1998 and nucleotide biosynthetic pathways.
FdUMP dUMP FdUMP

dUMP dTMP
N5,N10-Methylene
CH2 CH2
H
H4 folate H
5
:N C :N C
thymidylate 10
CH2 CH2
H C H C
synthase O N O N
H R⬘ H R⬘
HN C H + HN C F +
H H
5 10 7,8-Dihydrofolate C H :B C H :B
N ,N -Methylene O N O N
H4 folate R
–S
R
–S
NADPH 1 H1 Enzyme thiolate

adds at C-6 of
Glycine dUMP, a Michael-
dihydrofolate
serine
reductase 1 H+ H+ type addition; N10 1 H+ H+
hydroxymethyl- Methotrexate is protonated and
transferase Aminopterin
PLP N5-iminium ion is
Trimethoprim formed from
H4 folate CH2 methylene-H4 CH2
H H
NADP1
+
N
C folate. +
N
C
Serine CH2 CH2
H C H C
O NH O NH
(a) H R⬘ H R⬘
HN C H ᎐
HN C᎐ F
O CH3 C H :B C H :B
O O N O N
H3CO N NH2 S S
F R R
HN
N
H3CO
O N
H NH2 C-5 carbanion
Fluorouracil Trimethoprim 2 H+ adds to 2 H+
N5-iminium ion.
CH2
H CH2
H2N N N + C H H
NH + C
NH
CH2 CH2
H C
N 5 O NH O
H C
NH
N CH2 O COO⫺ H R⬘ H
10 HN C H R⬘
NH2 ⫺ HN C F
N C NH CHCH2CH2COO C H :B
O N C H :B
O N
CH3 S
R S
R
Methotrexate
(b) Methylidene is
formed at C-5 of Dead-end
FIGURE 22–52 Thymidylate synthesis and folate metabo- 3 pyrimidine; N5 is covalent
lism as targets of chemotherapy. (a) During thymidylate syn- eliminated to form complex
H4 folate.
thesis, N5,N10-methylenetetrahydrofolate is converted to 7,8-dihydrofo-
late; the N5,N10-methylenetetrahydrofolate is regenerated in two steps
CH2
H
C
:
(see Fig. 22–47). This cycle is a major target of several chemotherapeu- N

H CH2
H
tic agents. (b) Fluorouracil and methotrexate are important chemother- O NH
C H
apeutic agents. In cells, fluorouracil is converted to FdUMP, which inhib- HN C
R⬘
its thymidylate synthase. Methotrexate, a structural analog of C H HB

O N
tetrahydrofolate, inhibits dihydrofolate reductase; the shaded amino and R S
methyl groups replace a carbonyl oxygen and a proton, respectively, in
folate (see Fig. 22–47). Another important folate analog, aminopterin, is
identical to methotrexate except that it lacks the shaded methyl group.
Trimethoprim, a tight-binding inhibitor of bacterial dihydrofolate reduc-
4 Enzyme ⫹
dihydrofolate
tase, was developed as an antibiotic. O
CH3
HN 1,3 hydride shift
generates dTMP
O N
and dihydrofolate.
R
dTMP
MECHANISM FIGURE 22–53 Conversion of dUMP to dTMP and its inhi- gray. The 1,3 hydride shift (step 4), moves a hydride ion (shaded light
bition by FdUMP. The left side is the normal reaction mechanism of red) from C-6 of H4 folate to the methyl group of thymidine, resulting
thymidylate synthase. The nucleophilic sulfhydryl group contributed by in the oxidation of tetrahydrofolate to dihydrofolate. This hydride shift
the enzyme in 1 and the ring atoms of dUMP taking part in the reac- is blocked when FdUMP is the substrate (right). Steps 1 and 2 pro-
tion are shown in red; :B denotes an amino acid side chain that acts as ceed normally, but result in a stable complex—consisting of FdUMP
a base to abstract a proton after step 3. The hydrogens derived from linked covalently to the enzyme and to tetrahydrofolate—that inacti-
the methylene group of N5,N10-methylenetetrahydrofolate are shaded in vates the enzyme. Thymidylate Synthase Mechanism
Further Reading 925
This folate analog acts as a competitive inhibitor; the

enzyme binds methotrexate with about 100 times Key Terms
higher affinity than dihydrofolate. Aminopterin is a Terms in bold are defined in the glossary.
related compound that acts similarly.
The medical potential of inhibitors of nucleotide nitrogen cycle 882 spermidine 909
biosynthesis is not limited to cancer treatment. All nitrogen fixation 882 ornithine
fast-growing cells (including bacteria and protists) are anammox 882 decarboxylase 909
potential targets. Trimethoprim, an antibiotic devel- symbionts 882 de novo pathway 910
oped by Hitchings and Elion, binds to bacterial dihy- nitrogenase complex 883 salvage pathway 910
drofolate reductase nearly 100,000 times better than P cluster 883 inosinate (IMP) 912
to the mammalian enzyme. It is used to treat certain FeMo cofactor 883 carbamoyl phosphate
urinary and middle-ear bacterial infections. Parasitic leghemoglobin 888 synthetase II 915
protists, such as the trypanosomes that cause African glutamine synthetase 888 aspartate
sleeping sickness (African trypanosomiasis), lack glutamate synthase 888 transcarbamoylase 915
pathways for de novo nucleotide biosynthesis and are glutamine nucleoside
particularly sensitive to agents that interfere with their amidotransferases 890 monophosphate
scavenging of nucleotides from the surrounding envi- 5-phosphoribosyl-1- kinase 916
ronment using salvage pathways. Allopurinol (Fig. pyrophosphate nucleoside diphosphate
22–50) and several similar purine analogs have shown (PRPP) 892 kinase 916
promise for the treatment of African trypanosomiasis tryptophan synthase 898 ribonucleotide
and related afflictions. See Box 6–3 for another porphyrin 902 reductase 917
approach to combating African trypanosomiasis, made porphyria 904 thioredoxin 917
possible by advances in our understanding of metabo- bilirubin 904 thymidylate synthase 920
lism and enzyme mechanisms. ■ phosphocreatine 906 dihydrofolate
creatine 906 reductase 920
glutathione (GSH) 906 adenosine deaminase
SUMMARY 22.4 Biosynthesis and Degradation of auxin 908 (ADA) deficiency 921
Nucleotides dopamine 909 Lesch-Nyhan
The purine ring system is built up step-by-step norepinephrine 909 syndrome 922
beginning with 5-phosphoribosylamine. The amino epinephrine 909 allopurinol 923
acids glutamine, glycine, and aspartate furnish all ␥-aminobutyrate azaserine 923
the nitrogen atoms of purines. Two ring-closure (GABA) 909 acivicin 923
steps form the purine nucleus. serotonin 909 fluorouracil 923
histamine 909 methotrexate 923
Pyrimidines are synthesized from carbamoyl cimetidine 909 aminopterin 925
phosphate and aspartate, and ribose 5-phosphate spermine 909
is then attached to yield the pyrimidine
ribonucleotides.
Nucleoside monophosphates are converted to their Further Reading
triphosphates by enzymatic phosphorylation
reactions. Ribonucleotides are converted to Nitrogen Fixation
deoxyribonucleotides by ribonucleotide reductase, Arp, D.J. & Stein, L.Y. (2003) Metabolism of inorganic N
an enzyme with novel mechanistic and regulatory compounds by ammonia-oxidizing bacteria. Crit. Rev. Biochem. Mol.
Biol. 38, 491–495.
characteristics. The thymine nucleotides are
Burris, R.H. (1995) Breaking the N–N bond. Annu. Rev. Plant
derived from dCDP and dUMP.
Physiol. Plant Mol. Biol. 46, 1–19.
Uric acid and urea are the end products of purine Eisenberg, D.S., Harindarpal, S., Gill, G., Pfluegl, M.U., &
and pyrimidine degradation. Rothstein, H. (2000) Structure-function relationships of glutamine
synthetases. Biochim. Biophys. Acta 1477, 122–145.
Free purines can be salvaged and rebuilt into
Fuerst, J.A. (2005) Intracellular compartmentation in
nucleotides. Genetic deficiencies in certain salvage
planctomycetes. Annu. Rev. Microbiol. 59, 299–328.
enzymes cause serious disorders such as Lesch- Advanced discussion of the structure and biochemistry of anammox.
Nyhan syndrome and ADA deficiency. Igarishi, R.Y. & Seefeldt, L.C. (2003) Nitrogen fixation: the
Accumulation of uric acid crystals in the joints, mechanism of the Mo-dependent nitrogenase. Crit. Rev. Biochem.
possibly caused by another genetic deficiency, Mol. Biol. 38, 351–384.
results in gout. Lin, J.T. & Stewart, V. (1998) Nitrate assimilation in bacteria. Adv.
Microbiol. Physiol. 39, 1–30.
Enzymes of the nucleotide biosynthetic pathways Patriarca, E.J., Tate, R., & Iaccarino, M. (2002) Key role of
are targets for an array of chemotherapeutic bacterial NH⫹4 metabolism in rhizobium-plant symbiosis. Microbiol.
agents used to treat cancer and other diseases. Mol. Biol. Rev. 66, 203–222.
A good overview of ammonia assimilation in bacterial systems Cotruvo, J.A., Jr. & Stubbe, J. (2011) Class I ribonucleotide
and its regulation. reductases: metallocofactor assembly and repair in vitro and in vivo.
Prell, J. & Poole, P. (2006) Metabolic changes of rhizobia in Annu. Rev. Biochem. 80, 733–767.
legume nodules. Trends Microbiol. 14, 161–168. Fairman, J.W., Wijerathna, S.R., Ahmad, M.F., Xu, H., Nakano,
A good summary of the intricate symbiotic relationship between R., Jha, S., Prendergast, J., Welin, M., Flodin, S., Roos, A., et al.
rhizobial bacteria and their hosts. (2011) Structural basis for allosteric regulation of human
Reese, D.C. & Howard, J.B. (2000) Nitrogenase: standing at the ribonucleotide reductase by nucleotide-induced oligomerization. Nat.
crossroads. Curr. Opin. Chem. Biol. 4, 559–566. Struct. Mol. Biol. 18, 316–322.
Schwarz, G., Mendel, R.R., & Ribbe, M.W. (2009) Molybdenum Herrick, J. & Sclavi, B. (2007) Ribonucleotide reductase and the
cofactors, enzymes and pathways. Nature 460, 839–847. regulation of DNA replication: an old story and an ancient heritage.
Mol. Microbiol. 63, 22–34.
Seefeldt, L.C., Hoffman, B.M., & Dean, D.R. (2009) Mechanism
of Mo-dependent nitrogenase. Annu. Rev. Biochem. 78, 701–722. Holmgren, A. (1989) Thioredoxin and glutaredoxin systems. J.
Biol. Chem. 264, 13,963–13,966.
Sinha, S.C. & Smith, J.L. (2001) The PRT protein family. Curr.
Opin. Struct. Biol. 11, 733–739. Kappock, T.J., Ealick, S.E., & Stubbe, J. (2000) Modular evolution
Description of a protein family that includes many amidotrans- of the purine biosynthetic pathway. Curr. Opin. Chem. Biol. 4,
ferases, with channels for the movement of NH3 from one active site 567–572.
to another. Kornberg, A. & Baker, T.A. (1991) DNA Replication, 2nd edn, W. H.
Freeman and Company, New York.
Amino Acid Biosynthesis This text includes a good summary of nucleotide biosynthesis.
Frey, P.A. & Hegeman, A.D. (2007) Enzymatic Reaction Licht, S., Gerfen, G.J., & Stubbe, J. (1996) Thiyl radicals in
Mechanisms, Oxford University Press, New York. ribonucleotide reductases. Science 271, 477–481.
An updated summary of reaction mechanisms, including one-
carbon metabolism and pyridoxal phosphate enzymes. Nordlund, P. & Reichard, P. (2006) Ribonucleotide reductases.
Annu. Rev. Biochem. 75, 681–706.
Neidhardt, F.C. (ed.). (1996) Escherichia coli and Salmonella:
Cellular and Molecular Biology, 2nd edn, ASM Press, Washington, DC. Schachman, H.K. (2000) Still looking for the ivory tower. Annu.
Volume 1 of this two-volume set has 13 chapters devoted to Rev. Biochem. 69, 1–29.
detailed descriptions of amino acid and nucleotide biosynthesis in A lively description of research on aspartate transcarbamoylase,
bacteria. The Web-based version at www.ecosal.org is updated accompanied by delightful tales of science and politics.
regularly. A valuable resource. Weeks, A., Lund, L., & Raushel, F.M. (2006) Tunneling of
Pan P., Woehl, E., & Dunn, M.F. (1997) Protein architecture, intermediates in enzyme-catalyzed reactions. Curr. Opin. Chem.
dynamics and allostery in tryptophan synthase channeling. Trends Biol. 10, 465–472.
Biochem. Sci. 22, 22–27. Genetic Diseases
Richards, N.G.J. & Kilberg, M.S. (2006) Asparagine synthetase Löffler, M., Fairbanks, L.D., Zameitat, E., Marinaki, A.M., &
chemotherapy. Annu. Rev. Biochem. 75, 629–654. Simmonds, H.A. (2005) Pyrimidine pathways in health and disease.
Stadtman, E.R. (2001) The story of glutamine synthetase Trends Mol. Med. 11, 430-437.
regulation. J. Biol. Chem. 276, 44,357–44,364. Valle, D., Beaudet, A.L., Vogelstein, B., Kinzler, K.W.,
Antonarakis, S.E., & Ballabio, A. (eds). Scriver's Online
Compounds Derived from Amino Acids
Metabolic and Molecular Bases of Inherited Disease, www.
Ajioka R.S., Phillips, J.D., & Kushner, J.P. (2006) Biosynthesis ommbid.com. Published January 2006. Updated March 28, 2011.
of heme in mammals. Biochim. Biophys. Acta Mol. Cell Res. 1763, This classic medical encyclopedia, last published in print in 2001
723–736. as a four-volume set, is now maintained online. It has good chapters
Bredt, D.S. & Snyder, S.H. (1994) Nitric oxide: a physiologic on disorders of amino acid, porphyrin, and heme metabolism. See
messenger molecule. Annu. Rev. Biochem. 63, 175–195. also the chapters on inborn errors of purine and pyrimidine
Meister, A. & Anderson, M.E. (1983) Glutathione. Annu. Rev. metabolism.
Biochem. 52, 711–760.
Morse, D. & Choi, A.M.K. (2002) Heme oxygenase-1—the “emerging
molecule” has arrived. Am. J. Respir. Cell Mol. Biol. 27, 8–16.
Rondon, M.R., Trzebiatowski, J.R., & Escalante-Semerena, J.C.
(1997) Biochemistry and molecular genetics of cobalamin
Problems
biosynthesis. Prog. Nucleic Acid Res. Mol. Biol. 56, 347–384. 1. ATP Consumption by Root Nodules in Legumes
Stadtman, T.C. (1996) Selenocysteine. Annu. Rev. Biochem. 65, Bacteria residing in the root nodules of the pea plant consume
83–100. more than 20% of the ATP produced by the plant. Suggest why
Nucleotide Biosynthesis these bacteria consume so much ATP.
Aiuti, A., Cattaneo, R., Galimberti, S., Benninghoff, U., Cassani, 2. Glutamate Dehydrogenase and Protein Synthesis
B., Callegaro, L., Scaramuzza, S., Andolfi, G., Mirolo, M.,
Brigida, I., et al. (2009) Gene therapy for immunodeficiency due to
The bacterium Methylophilus methylotrophus can synthe-
adenosine deaminase deficiency. N. Engl. J. Med. 360, 447–458. size protein from methanol and ammonia. Recombinant DNA
Ando, N., Brignole, E.J., Zimanyi, C.M., Funk, M.A., techniques have improved the yield of protein by introducing
Yokoyama, K., Asturias, F.J., Stubbe, J., & Drennan, C.L. into M. methylotrophus the glutamate dehydrogenase gene
(2011) Structural interconversions modulate activity of Escherichia from E. coli. Why does this genetic manipulation increase the
coli, ribonucleotide reductase. Proc. Natl. Acad. Sci. USA 108, protein yield?
21,046–21,051.
Carreras, C.W. & Santi, D.V. (1995) The catalytic mechanism and 3. PLP Reaction Mechanisms Pyridoxal phosphate can
structure of thymidylate synthase. Annu. Rev. Biochem. 64, 721–762. help catalyze transformations one or two carbons removed
Problems 927
from the ␣ carbon of an amino acid. The enzyme threonine 11. Nucleotide Biosynthesis in Amino Acid Auxo-
synthase (see Fig. 22–17) promotes the PLP-dependent con- trophic Bacteria Wild-type E. coli cells can synthesize
version of phosphohomoserine to threonine. Suggest a mecha- all 20 common amino acids, but some mutants, called
nism for this reaction. amino acid auxotrophs, are unable to synthesize a specific
amino acid and require its addition to the culture medium
4. Transformation of Aspartate to Asparagine There
for optimal growth. Besides their role in protein synthesis,
are two routes for transforming aspartate to asparagine at
some amino acids are also precursors for other nitroge-
the expense of ATP. Many bacteria have an asparagine syn-
nous cell products. Consider the three amino acid auxo-
thetase that uses ammonium ion as the nitrogen donor. Mam-
trophs that are unable to synthesize glycine, glutamine,
mals have an asparagine synthetase that uses glutamine as the
and aspartate, respectively. For each mutant, what nitrog-
nitrogen donor. Given that the latter requires an extra ATP (for
enous products other than proteins would the cell fail to
the synthesis of glutamine), why do mammals use this route?
synthesize?
5. Equation for the Synthesis of Aspartate from Glucose
Write the net equation for the synthesis of aspartate (a nones- 12. Inhibitors of Nucleotide Biosynthesis Suggest
sential amino acid) from glucose, carbon dioxide, and ammonia. mechanisms for the inhibition of (a) alanine racemase by
L-fluoroalanine and (b) glutamine amidotransferases by
6. Asparagine Synthetase Inhibitors in Leukemia azaserine.
Therapy Mammalian asparagine synthetase is a
glutamine-dependent amidotransferase. Efforts to identify an 13. Mode of Action of Sulfa Drugs Some bacteria
effective inhibitor of human asparagine synthetase for use in require p-aminobenzoate in the culture medium for
chemotherapy for patients with leukemia has focused not on normal growth, and their growth is severely inhibited by the
the amino-terminal glutaminase domain but on the carboxyl- addition of sulfanilamide, one of the earliest sulfa drugs. More-
terminal synthetase active site. Explain why the glutaminase over, in the presence of this drug, 5-aminoimidazole-4-carbox-
domain is not a promising target for a useful drug. amide ribonucleotide (AICAR; see Fig. 22–35) accumulates in
the culture medium. These effects are reversed by addition of
7. Phenylalanine Hydroxylase Deficiency and Diet excess p-aminobenzoate.
Tyrosine is normally a nonessential amino acid, but individuals
O
with a genetic defect in phenylalanine hydroxylase require O
H2N C H2N S NH2
tyrosine in their diet for normal growth. Explain.
O᎐ O
8. Cofactors for One-Carbon Transfer Reactions p-Aminobenzoate Sulfanilamide
Most one-carbon transfers are promoted by one of three
(a) What is the role of p-aminobenzoate in these bacteria?
cofactors: biotin, tetrahydrofolate, or S-adenosylmethio-
(Hint: See Fig. 18–16.)
nine (Chapter 18). S-Adenosylmethionine is generally used
(b) Why does AICAR accumulate in the presence of sulfa-
as a methyl group donor; the transfer potential of the
nilamide?
methyl group in N5-methyltetrahydrofolate is insufficient
(c) Why are the inhibition and accumulation reversed by
for most biosynthetic reactions. However, one example of
addition of excess p-aminobenzoate?
the use of N 5-methyltetrahydrofolate in methyl group
transfer is in methionine formation by the methionine syn- 14. Pathway of Carbon in Pyrimidine Biosynthesis Pre-
thase reaction (step 9 of Fig. 22–17); methionine is the dict the locations of 14C in orotate isolated from cells grown on
immediate precursor of S-adenosylmethionine (see Fig. a small amount of uniformly labeled [14C]succinate. Justify
18–18). Explain how the methyl group of S-adenosylme- your prediction.
thionine can be derived from N5-methyltetrahydrofolate,
15. Nucleotides as Poor Sources of Energy Under starva-
even though the transfer potential of the methyl group in
tion conditions, organisms can use proteins and amino acids as
N 5-methyltetrahydrofolate is one-thousandth of that in
sources of energy. Deamination of amino acids produces car-
S-adenosylmethionine.
bon skeletons that can enter the glycolytic pathway and the
9. Concerted Regulation in Amino Acid Biosynthesis citric acid cycle to produce energy in the form of ATP. Nucle-
The glutamine synthetase of E. coli is independently modulated otides, on the other hand, are not similarly degraded for use as
by various products of glutamine metabolism (see Fig. 22–8). energy-yielding fuels. What observations about cellular physi-
In this concerted inhibition, the extent of enzyme inhibition is ology support this statement? What aspect of the structure of
greater than the sum of the separate inhibitions caused by each nucleotides makes them a relatively poor source of energy?
product. For E. coli grown in a medium rich in histidine, what
16. Treatment of Gout Allopurinol (see Fig. 22–50),
would be the advantage of concerted inhibition?
an inhibitor of xanthine oxidase, is used to treat chron-
10. Relationship between Folic Acid Deficiency ic gout. Explain the biochemical basis for this treatment.
and Anemia Folic acid deficiency, believed to be the Patients treated with allopurinol sometimes develop xanthine
most common vitamin deficiency, causes a type of anemia in stones in the kidneys, although the incidence of kidney dam-
which hemoglobin synthesis is impaired and erythrocytes do age is much lower than in untreated gout. Explain this obser-
not mature properly. What is the metabolic relationship vation in the light of the following solubilities in urine: uric
between hemoglobin synthesis and folic acid deficiency? acid, 0.15 g/L; xanthine, 0.05 g/L; and hypoxanthine, 1.4 g/L.
17. Inhibition of Nucleotide Synthesis by Azaserine acyl carrier proteins, the majority molecular species had an
The diazo compound O-(2-diazoacetyl)-L-serine, known also Mr of 12,153.
as azaserine (see Fig. 22–51), is a powerful inhibitor of (b) How would you use these data to argue that BtrI can
glutamine amidotransferases. If growing cells are treated with function as an acyl carrier protein with a CoA prosthetic
azaserine, what intermediates of nucleotide biosynthesis will group?
accumulate? Explain. Using standard terminology, Li and coauthors called the
form of the protein lacking CoA apo-BtrI and the form with
CoA (linked as in Fig. 21–5) holo-BtrI. When holo-BtrI was
incubated with glutamine, ATP, and purified BtrJ protein, the
Data Analysis Problem holo-BtrI species of Mr 12,153 was replaced with a species of
Mr 12,281, corresponding to the thioester of glutamate and
18. Use of Modern Molecular Techniques to Determine
holo-BtrI. Based on these data, the authors proposed the fol-
the Synthetic Pathway of a Novel Amino Acid Most of
lowing structure for the Mr 12,281 species (␥-glutamyl-S-BtrI):
the biosynthetic pathways described in this chapter were
determined before the development of recombinant DNA ⫹
technology and genomics, so the techniques were quite differ- NH3

O O
ent from those that researchers would use today. Here we C C
explore an example of the use of modern molecular tech-
O S
niques to investigate the pathway of synthesis of a novel
amino acid, (2S)-4-amino-2-hydroxybutyrate (AHBA). The BtrI
techniques mentioned here are described in various places in -Glutamyl-S-BtrI
the book; this problem is designed to show how they can be
(c) What other structure(s) is (are) consistent with the
integrated in a comprehensive study.
data above?
AHBA is a ␥-amino acid that is a component of some
(d) Li and coauthors argued that the structure shown
aminoglycoside antibiotics, including the antibiotic butirosin.
here (␥-glutamyl-S-BtrI) is likely to be correct because the
Antibiotics modified by the addition of an AHBA residue are
␣-carboxyl group must be removed at some point in the syn-
often more resistant to inactivation by bacterial antibiotic-
thetic process. Explain the chemical basis of this argument.
resistance enzymes. As a result, understanding how AHBA is
(Hint: See Fig. 18–6, reaction C.)
synthesized and added to antibiotics is useful in the design of
The BtrK protein showed significant homology to PLP-
pharmaceuticals.
dependent amino acid decarboxylases, and BtrK isolated from
In an article published in 2005, Li and coworkers describe
E. coli was found to contain tightly bound PLP. When
how they determined the synthetic pathway of AHBA from
␥-glutamyl-S-BtrI was incubated with purified BtrK, a molecu-
glutamate.
lar species of Mr 12,240 was produced.
⫹
NH3 OH
(e) What is the most likely structure of this species?
O O ⫹ O (f) Interestingly, when the investigators incubated gluta-
C C H3N C mate and ATP with purified BtrI, BtrJ, and BtrK, they found a
O O O molecular species of Mr 12,370. What is the most likely struc-
Glutamate AHBA ture of this species? Hint: Remember that BtrJ can use ATP to
␥-glutamylate nucleophilic groups.
(a) Briefly describe the chemical transformations needed
Li and colleagues found that BtrO is homologous to
to convert glutamate to AHBA. At this point, don’t be con-
monooxygenase enzymes (see Box 21–1) that hydroxylate
cerned about the order of the reactions.
alkanes, using FMN as a cofactor, and BtrV is homologous to
Li and colleagues began by cloning the butirosin biosyn-
an NAD(P)H oxidoreductase. Two other genes in the cluster,
thetic gene cluster from the bacterium Bacillus circulans,
btrG and btrH, probably encode enzymes that remove the
which makes large quantities of butirosin. They identified five
␥-glutamyl group and attach AHBA to the target antibiotic
genes that are essential for the pathway: btrI, btrJ, btrK, btrO,
molecule.
and btrV. They cloned these genes into E. coli plasmids that
(g) Based on these data, propose a plausible pathway for
allow overexpression of the genes, producing proteins with
the synthesis of AHBA and its addition to the target antibiotic.
“histidine tags” fused to their amino termini to facilitate purifi-
Include the enzymes that catalyze each step and any other
cation (see Section 9.1).
substrates or cofactors needed (ATP, NAD, etc.).
The predicted amino acid sequence of the BtrI protein
showed strong homology to known acyl carrier proteins Reference
(see Fig. 21–5). Using mass spectrometry, Li and colleagues Li, Y., Llewellyn, N.M., Giri, R., Huang, F., & Spencer,
found a molecular mass of 11,812 for the purified BtrI protein J.B. (2005) Biosynthesis of the unique amino acid side chain
(including the His tag). When the purified BtrI was incubated of butirosin: possible protective-group chemistry in an acyl
with coenzyme A and an enzyme known to attach CoA to other carrier protein–mediated pathway. Chem. Biol. 12, 665–675.

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22

Biosynthesis of Amino Acids,

BOX 22–1 Unusual Lifestyles of the Obscure but Abundant

Dinitrogenase FeMo cofactor

dissociates from dinitrogenase, in a repeating cycle.

hydride bonds, but also binding energy (p. 195),

FIGURE 22–4 Electron path in nitrogen fixation by the nitrogenase 8e–

required to transfer electrons through dinitrogenase to N

FIGURE 22–6 Nitrogen-fixing nodules. (a) Root nodules

L-glutamate dehydrogenase, an enzyme present in all Glutamate

FIGURE 22–8 Allosteric regulation of glutamine synthetase. The

(a) Figs 18–6, 18–17, and 18–18). Here we focus on amino

PII brings about a decrease in transcription of the same Activated

residue in the glutamine-binding domain is believed to act

action of nitrate reductase and nitrite reductase

large investments of ATP and of reducing power. The

with simple pathways. These are the nonessential

FIGURE 22–13 Ornithine ␦-aminotransferase COO⫺ ␣ -Ketoglutarate COO⫺

Ornithine Glutamate ⌬1-Pyrroline-5-

COO FIGURE 22–14 Biosynthesis of serine from 3-phosphoglycerate and of

COO⫺ ATP ⫹ SO42

FIGURE 22–17 Biosynthesis of six essential amino acids from oxalo-

Aspartate gives rise to methionine, threonine, Indole-3-glycerol phosphate

The end product, isoleucine, is an allosteric inhibitor of COO⫺

(a) OH OH Permanent ﬁgure # 2220

Quinonoid intermediate Aldimine with tryptophan

COO⫺ the others are required in the diet (essential amino

FIGURE 22–21 Biosynthesis of phenylalanine and tyrosine from

To purine biosynthesis 3 H2O

Imidazole acetol L-Histidinol L-Histidinol Histidine

⫹ which is then decarboxylated to ␦-aminolevulinate. In

(a) COO⫺ COO⫺

CH3 CH3 CH3 CH3 Pr

Conjugated double bonds~ Color 1 porphobilinogen synthase

BOX 22–2 MEDICINE On Kings and Vampires

FIGURE 22–27 Bilirubin and its breakdown prod- Heme

Bilirubin diglucuronide Bilirubin

NH2 (a) Glutamate

⫹ mitter, serotonin, is derived from tryptophan in a two-

FIGURE 22–31 Biosynthesis of some neurotransmitters from amino

aromatic PLP N Tryptophan

Decarboxylated H3N (CH2)3 NH (CH2)4 NH3 Spermidine

COO COO COO

CH2 NADPH ⫹ H⫹, O2 CH2 1 CH2

CH2 CH2 CH2

NH2 NH2 NH2

aspartate for pyrimidines. Glutamine again is the most CO2

O adenylosuccinate Rib P Rib P

FIGURE 22–36 Biosynthesis of AMP and GMP from IMP.

Purine Nucleotide Biosynthesis Is Regulated 5-Phosphoribosylamine

of IMP to AMP, whereas ATP is required for conver- Aspartate

thesis; the six-membered pyrimidine ring is made first O

thesis is made in mitochondria by carbamoyl phosphate O

phosphate synthetase II. In bacteria, a single enzyme orotidylate

separate active sites, spaced along a channel nearly 100 O

is the ultimate electron acceptor. In eukaryotes, the O

first three enzymes in this pathway—carbamoyl phos- P P P OOCH2

ATP-binding site K0.5 ⫽ 12 mM K0.5 ⫽ 23 mM

FIGURE 22–40 Allosteric regulation of aspartate transcarbamoylase by

Glutaredoxin Glutaredoxin Thioredoxin Thioredoxin