Download Amino Acid Metabolism 1 Key Concepts

Bioc 460 - Dr. Miesfeld Spring 2008
Amino Acid Metabolism 1
Key Concepts
- Nitrogen fixation and assimilation
- Protein and amino acid degradation
- The Urea cycle
- Glucogenic and ketogenic amino acids
Key Concept Questions In Amino Acid Metabolism
How is atmospheric nitrogen converted into ammonia as a
nitrogen source for plants?
Which three amino acids serve as the nitrogen donors in urea
synthesis?
Nitrogen fixation and assimilation by plants and bacteria
After carbon, nitrogen is the second most abundant element in
the biosphere, and in addition to its presence in amino acids and
nucleotides, it is also found in some carbohydrates (glucosamine) and lipids (sphingosine), as well
as, the enzyme cofactors thiamine, NAD+, and FAD. Nitrogen in biological compounds ultimately
comes from nitrogen gas (N2) which constitutes 80% of our atmosphere. However, N2 must first be
reduced to NH3 (the ionized form of ammonia in solution is NH4+) by the process of nitrogen
fixation, or oxidized to nitrate (NO3-) by atmospheric lightning, before it can be used by other liver
organisms. Nitrogen fixation in nature is carried out by certain types of soil bacteria that live in
both the soil and aquatic environments. Rhizobium is an example of a nitrogen-fixing soil bacteria
that lives symbiotically with leguminous plants such as beans and alfalfa and has an important
role in agriculture by reducing the need for commercial fertilizers. Plants cannot carry out nitrogen
fixation on their own, but they can incorporate NH4+ they obtain from the environment into the
amino acids glutamate and glutamine through a process called nitrogen assimilation. When
animals eat plants, amino acids and nucleotides provide the nitrogen needed to synthesize a
variety of biomolecules. Before looking at nitrogen metabolism in a little more detail, we need to
answer our four pathway questions about nitrogen fixation and assimilation.
1. What purpose does nitrogen fixation and assimilation serve in the biosphere?
• Nitrogen fixation takes place in bacteria and is the primary process by which atmospheric N2 gas
is converted to ammonia (NH4+) and nitrogen oxides (NO2- and NO3-) in the biosphere. Two other
nitrogen fixation processes are industrial (Haber process) and atmospheric (lightening).
• Nitrogen assimilation is the process by which plants and bacteria incorporate NH4+ into organic
compounds, most often the NH4+ is incorporated into the amino acids glutamate and glutamine.
2. What are the net reactions of nitrogen fixation and assimilation by plants and bacteria?
Nitrogen fixation in bacteria is mediated by the nitrogenase enzyme complex:
N2 + 8 H+ 8 e- + 16 ATP + 16 H2O ----> 2 NH3 + H2 + 16 ADP + 16 Pi
Nitrogen assimilation in plants using the enzymes glutamine synthetase and glutamate synthase:
α-ketoglutarate + NH4+ + ATP + NADPH + H+ --> Glutamate + ADP + Pi + NADP+
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3. What are the key enzymes in nitrogen fixation and assimilation in plants and bacteria?
Bacterial nitrogenase complex – is the enzyme that uses redox reactions coupled to ATP
hydrolysis to convert N2 gas into 2 NH3. The enzyme has two functional components,
dinitrogenase reductase that contains the binding site for ATP and 4Fe-4S redox center, and
dinitrogenase which carries out the N2 reduction reaction using Fe-Mo and FeS redox centers.
Glutamine synthetase - is found in all organisms and it incorporates NH4+ into glutamate to form
glutamine through an ATP coupled redox reaction. The activity of glutamine synthetase is
regulated by allosteric inhibitors (amino acids, AMP), and by covalent modification which is
mediated by adenylation.
Glutamate synthase - is found in bacteria, plants, and some insects, and it works in concert with
glutamine synthetase to replenish glutamate so that the glutamine synthetase reaction is not
substrate limited. Glutamate synthase converts α-ketoglutarate and glutamine to 2 glutamate.
Glutamate dehydrogenase - is found in all organisms and it interconverts glutamate, NH4+, and αketoglutarate in a redox reaction utilizing either NAD(P)+/NAD(P)H. Under conditions of high NH4+
concentrations in nature, e.g., fertilizer applications in crop fields, glutamate dehydrogenase can
assimilate NH4+ into glutamate, however, in animals, glutamate dehydrogenase most often
generates NH4+ from glutamate to initiate the process of nitrogen excretion as urea or uric acid.
4. What are examples of nitrogen fixation and assimilation in real life?
Natural fertilizers can be used in organic farming to reduce the dependence on industrial sources
of nitrogen. The two most common sources of natural fertilizers are manure, if livestock are readily
available, and crop rotation practices in which leguminous plants such as soy bean or clover,
are planted in alternate seasons with nonleguminous crop plants such as corn and wheat. By
plowing under the leguminous plants, the nitrogen
Figure 1.
contained in the plants is released into the soil and
processed by soil bacteria to provide nitrogenous
compounds for the corn and wheat plants.
Nitrogen fixation
In order to obtain nitrogen from the atmosphere for
incorporation into biomolecules, the triple bond of N2
must be broken. However, this is not easily done
considering that the bond energy of N2 is a
staggering 930 kJ/mol. To overcome this high
energy barrier, one of three processes are required,
1) biological fixation by bacteria that reduce N2 to
NH3 through an ATP-dependent process requiring
the multisubunit enzyme complex nitrogenase, 2)
industrial fixation by the Haber process in which N2
and H2 gases are heated to ~500ºC under a pressure
of ~250 atmospheres (~350 kilopascals) to produce
liquid ammonia which is used commercially to make
fertilizer (figure 1), or 3) atmospheric fixation as a
result of lightning which breaks the N2 triple bond and
allows nitrogen to combine with oxygen to form nitrogen oxides that are dissolved in rain and fall to
earth. It is estimated that ~90% of the nitrogen incorporated our biosphere comes from biological
and industrial fixation, of which almost half of this is ammonia contained in agricultural fertilizers.
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Fritz Haber, a German chemist, received the 1918 Nobel Prize in Chemistry for his development of
industrial ammonia synthesis which revolutionized agriculture.
Biological nitrogen
Figure 2.
fixation by bacteria requires the
activity of nitrogenase, a large
protein complex consisting of
two functional components.
One component is called
dinitrogenase reductase (Feprotein) which consists of two
identical subunits that each
contain a binding site for ATP,
and a single 4Fe-4S redox
center liganded to cysteine
residues in the two subunits (figure 2). The function of the dinitrogenase reductase is to obtain
electrons from ferredoxin (or flavodoxin depending on the bacterial species) and pass them onto
the second component of the complex called dinitrogenase (MoFe-protein) which catalyzes the
reduction of N2 to generate 2 NH3. The nitrogenase reaction requires 16 ATP to overcome the
large energy barrier in the N2 triple bond.
Bacterial
nitrogenase complex
N2 + 8 H+ 8 e- + 16 ATP + 16 H2O ----> 2 NH3 + H2 + 16 ADP + 16 Pi
The conformational change in dinitrogenase reductase requires the binding and hydrolysis
of 2 ATP for each electron transferred. The reduction
of N2 to 2 NH3 takes place in the Mo-Fe reaction center Figure 3.
of the dinitrogenase component, and as shown in
figure 3, requires three discrete steps that each utilize
2 H+ and 2 e, 1) reduction of N2 to form diimine (N2H2),
2) reduction of diimine to form hydrazine (N2H4), and
3) reduction of hydrazine to form two molecules of
NH3. Besides being energetically expensive, the
nitrogenase reaction is inhibited by O2. This means
that nitrogen-fixing bacteria need to either perform this
reaction under anaerobic conditions, or find a way to
reduce O2 levels locally within the cell. The free-living
facultative aerobe Klebsiella pneumoniae, for example,
only synthesizes the protein components of the
nitrogenase complex when it is living in an anaerobic
environment and nitrogen fixation is favorable.
Another mechanism to increase the efficiency of the
nitrogenase reaction is employed by nitrogen-fixing
bacteria that live as plant symbionts. One of these bacterial species is Rhizobium meiloti (also
known as Sinorhizobium meiloti) which invades the roots of leguminous plants through tubular
structures called infection threads (figure 4). Once in side the plant cell, the bacterium loses its cell
wall and becomes a bacteroid, containing an inner and outer membrane. The plant provides
citrate cycle intermediates such as fumarate and malate to the bacteroids which use them as
oxidizable energy sources to generate NADH. The NADH is used to generate ATP by the electron
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transport system and oxidative phosphorylation,
thereby providing chemical energy to the
Rhizobium bacteroid. In turn, the nitrogenase
complex of the bacteroid generates NH3 which is
used to synthesize amino acids such as glutamate
and aspartate that the plant can use as source of
nitrogen. Desert plants such as the palo verde tree
are leguminous which makes sense because the
desert soil is sandy and plants are few and far
between (figure 5). Non-leguminous plants obtain
nitrogen from the soil as a result of organic decay
mediated by bacteria. Of course, agricultural plants
get their nitrogen from chemical fertilizers such as
ammonium sulfate.
Figure 4.
Figure 5.
Nitrogen assimilation
Plants and bacteria, but not animals, use nitrogen assimilation reactions
to synthesize nitrogen-containing metabolites, primarily amino acids.
Nitrogen assimilation proceeds in one of two ways. First, if NH4+ levels
in the soil are high, plants can use the glutamate dehydrogenase
reaction to directly incorporate NH4+ into the amino acid glutamate using
α-ketoglutarate as the carbon skeleton (figure 6). Animals also contain
glutamate dehydrogenase, but because of low levels of free NH4+ in
cells (NH4+ is toxic), and correspondingly high levels of glutamate, the
reaction is run in the reverse direction as a way to deaminate glutamate
and form nitrogenous waste products such as urea and uric acid. A
second, and more common way that plants and bacteria incorporate
NH4+ into metabolites, is through a two reaction mechanism that
functions when NH4+ concentrations are low
Figure 6. Glutamate dehydrogenase
(most of the time for non-leguminous plants in
the wild). In this mechanism, the enzyme
glutamine synthetase uses ATP in a coupled
reaction to form glutamine from glutamate
using NH4+ as the source of nitrogen (figure 7).
Next, the glutamine is combined with αketoglutarate in a reaction catalyzed by the
enzyme glutamate synthase to form two
molecules of glutamate
(glutamine contains two
nitrogens) as shown in figure 8.
While all organisms contain the
enzyme glutamine synthetase,
only plants and animals, and
some insects, have the enzyme
glutamate synthase. These
three nitrogen assimilation
reactions are summarized in
Figure 7. Glutamine synthetase
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Figure 8. Glutamate synthase
Bioc 460 - Dr. Miesfeld Spring 2008
figure 9. Importantly,
the newly acquired
nitrogen in glutamate
and glutamine is used to
synthesize a variety of
other amino acids
through
aminotransferase
enzymes that convert
glutamate and α-keto acids into αFigure 9.
ketoglutarate and the corresponding αamino acid (figure 10).
As shown in figure 11, the Nitrogen
Cycle maintains nitrogen balance in our
biosphere. Atmospheric nitrogen is
converted to NH3 by biological, industrial
and atmospheric fixation processes.
Bacteria in the soil convert nitrogen to NH3
either as symbionts with leguminous plants
(e.g., Rhizobium), or as free-living
organisms (e.g., Azotobacter). The NH4+
in the soil derived from decomposition,
free-living soil bacteria, and man-made
fertilizers, is converted to NO2- (nitrite) and
NO3- (nitrate) by soil bacteria that carry out
the process of nitrification (e.g.,
Nitrosomonas and Nitrobacter). Plant roots
absorb NO2- and NO3- present in the soil
and convert them back into NH4+ using
nitrite and nitrate reductase enzymes. The
assimilation of NH4+ into amino acids by
plants provides a source of nitrogen for
animals (directly or indirectly). The
denitrification process carried out by
bacteria that reduce nitrites and nitrates
(e.g., Pseudomonas) releases N2 back into
the atmosphere, whereas other types of
bacteria anaerobically convert NH4+
Figure 10
directly to N2 gas to complete the
nitrogen cycle. The fixation and
cycling of nitrogen by bacteria and
plants, and the dependence of
animals on plants as their only
source of biological nitrogen, is
similar in many ways to the vital role
that plants play in providing
carbohydrates and O2 for aerobic
respiration in non-photosynthetic organisms such as ourselves.
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Figure 11.
Bioc 460 - Dr. Miesfeld Spring 2008
Protein and amino
acid degradation
Unlike glucose which
can be stored in the
body as glycogen, or
converted to acetyl CoA
and stored as fatty
acids, nitrogen cannot
be stored in a useable
form because NH4+ is
toxic. Therefore,
nitrogen lost as a result
of protein and nucleic
acid degradation, must
be replaced from the
diet. When an individual
is in nitrogen balance, it
means that their daily
intake of nitrogen,
primarily in the form of protein, equals the amount of nitrogen lost by excretion in the feces and
urine. A normal healthy adult needs about 400 grams of protein per day to maintain nitrogen
balance. In contrast, young children and pregnant women have a positive nitrogen balance
because they accumulate nitrogen in the
Figure 12.
form of new protein which is needed to
support tissue growth. Negative nitrogen
balance is a sign of disease or starvation
and occurs in individuals with elevated
rates of protein breakdown (loss of muscle
tissue) or an inability to obtain sufficient
amounts of amino acids in their diet.
Plants and bacteria have the
necessary enzymes to synthesize all 20
amino acids, however, animals depend on
protein in their diets to obtain the ten
essential amino acids they require for
growth and development. Protein digestion
in humans takes place in the stomach and
the small intestine where proteases cleave
the peptide bond to yield amino acids and
small oligopeptides. As seen in figure 12,
food enters the stomach through the
esophagus and protein digestion begins in
the stomach as a result of pepsin activation
by low pH. After leaving the stomach,
duodenumal hormones stimulate protease
secretion from the pancreas which leads to
protease activation and cleavage of protein
fragments into oligopeptides. Aminopeptidases and dipeptidases in the intestinal mucosal cells
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degrade the oligopeptides into single amino acids that are then absorbed and transported to the
liver. The highly acidic slurry of
Figure 13.
digested food, called chyme, leaves
the stomach by passing through
pyloric valve and into the duodenum,
resulting in the release of two
duodenumal hormones, secretin and
cholecystokinin (CCK). The
duodenum also secretes
enteropeptidase, a protease that
specifically activates several
protealytic zymogens released from
the pancreas. One of these
proteases is the pancreatic zymogen
trypsinogen which is cleaved to form
the endopeptidase trypsin. As shown in figure 13, trypsin cleaves numerous pancreatic
zymogens, including chymotrypsinogen, proelastase and procarboxypepetidases A and B, as
well as, trypsinogen itself to amplify the protealytic cascade. The combined activity of the
pancreatic endopeptidases (trypsin, chymotrypsin, elastase) and exopeptidases (carboxypeptidase
A and B), along with aminopeptidases and dipeptidases located on the membrane of intestinal
mucosal cells, further digest the protein fragments into individual amino acids that are transported
into intestinal epithelial cells and then exported to the blood. The complete degradation of dietary
proteins by these digestive proteases results from the distinct substrate specificities of the
enzymes.
Another source of free amino
acids in the body comes from
Figure 14.
degradation of cellular proteins
which occurs continuously in all
cells. Most eukaryotic cellular
proteins are degraded by one of two
pathways, 1) an ATP-independent
process that degrades proteins
inside cellular vesicles called
lysosomes, and 2) an ATPdependent pathway that targets
specific proteins for degradation in
proteasomes if they contain a
polymer of ubiquitin protein
covalently attached to lysine
residues. In order for proteins to be
degraded by the proteasome, they
must first be "tagged" on lysine
residues by covalent linkage of
ubiquitin (figure 14). Ubiquitin is a
highly conserved 76 amino acid
protein found in all eukaryotic cells
that is specifically attached to
proteins by ubiquitin ligating
enzymes. The signal for
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Figure 15.
ubiquination can be
specific residues within
the target protein, or a
physical or chemical
property of the protein,
such as a
phosphorylated residue
or abnormal
conformation, that are
recognized by the
ubiquitin ligase.
Ubiquinated proteins
enter the proteasome
one at a time where the
ubiquitin is removed and
recycled, and then
polypeptide is cleaved
into small oligopeptides
(6-10 amino acids long) that are released into the cytosol and degraded into individual amino
acids. As shown in figure 15, the proteasome consists of a 20S protealytic core, so named
because of its sedimentation properties in a density gradient (S is Svedberg units), and two 19S
regulatory complexes that serve as caps to regulate protein entry into the protealytic core. The
19S complexes contain binding sites for ubiquitinated proteins and encode ATP hydrolyzing
enzymes that function in the protein unfolding process required before the polypeptide can enter
the internal chamber and be
degraded. The intact
Figure 16.
proteasome is 26S and
has often been called the
"garbage disposer" of the
cell.
Since cells cannot
store amino acids that
accumulate as a result of
protein degradation, they
must either be recycled for
protein synthesis, or
deaminated in order to
salvage their carbon
skeletons for use in other
pathways. As shown in
figure 16, deamination of
amino acids results in
generation of NH4+ which is
used in the synthesis of
other nitrogen-containing
compounds, or excreted in
the form of urea or uric acid in terrestrial animals, whereas, aquatic animals can excrete NH4+
directly.
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The urea cycle removes toxic ammonia from the body
As described earlier, glutamate and glutamine function as the primary nitrogen carriers in most
organisms. In mammals, this nitrogen ends up in the liver where it is converted to urea. As
illustrated in figure 17, the two nitrogens in urea are derived from the NH4+ released when
glutamate or glutamine are
Figure 17.
deaminated, and from aspartate
which is formed when oxaloacetate is
transaminated by aspartate
aminotransferase. The carbon atom
in urea comes from CO2 (HCO3-) that
is produced in the mitochondrial
matrix by the citrate cycle (the oxygen
atom is derived from H2O in the final
reaction of the cycle).
Before we examine urea synthesis in
detail, let's answer the four metabolic
questions that pertain to the urea
cycle. Note that this is the last major
metabolic pathway we examine in this
course.
1. What does the urea cycle accomplish for the organism?
Urea synthesis provides an efficient mechanism for land animals to remove excess nitrogen from
the body. Urea is synthesized in the liver and exported to the kidneys where it enters the bladder.
2. What is the net reaction of the urea cycle?
NH4+ + HCO3- + aspartate + 3 ATP --->
urea + fumarate + 2 ADP + 2 Pi + AMP + PPi
3. What is the key regulated enzyme in urea synthesis?
Carbamoyl phosphate synthetase I – catalyzes the commitment step in the urea cycle; the
activity of this mitochondrial enzyme is activated by N-acetylglutamate in response to elevated
levels of glutamate and arginine.
4. What is an example of the urea cycle in real life?
A deficiency in the enzyme argininosuccinase inhibits flux through the urea cycle and causes
hyperammonemia and neurological symptoms. This metabolic disease can be treated with a low
protein diet that is supplemented with arginine, thereby resulting in argininosuccinate excretion a
substitute for urea.
As shown in figure 18, amino acids are transported to the liver where the nitrogen is
removed and used for urea synthesis. There are three sources of these amino acids 1) amino
acids derived from the digestion of dietary proteins, 2) the amino acid glutamine, which is
generated from glutamate and NH4+ in peripheral tissues by glutamine synthetase, and 3) the
amino acid alanine, which is formed by the alanine aminotransferase reaction as a way to remove
excess nitrogen from exercising (or starving) skeletal muscle. Dietary amino acids in the blood are
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taken up by the liver
Figure 18.
where aminotransferase
enzymes transfer the
amino group to αketoglutarate to form
glutamate. Amino acids
derived from the
degradation of cellular
proteins are also
deaminated to generate
glutamate. The glutamate
is imported into the
mitochondrial matrix
where it is metabolized by
the enzyme glutamate
dehydrogenase to
produce NH4+ which is
used to make the urea
cycle precursor
carbamoyl phosphate.
In addition, some of the
glutamate is converted to
aspartate by the aspartate
aminotransferase reaction
and fed into the urea cycle
as the second source of
nitrogen. Glutamine,
which carries excess two
nitrogen atoms to the liver
from peripheral tissues, is
deaminated by the
enzyme glutaminase to
generate NH4+ and
glutamate. The NH4+ is
used to make carbamoyl phosphate directly, and the glutamate, is deaminated by glutamate
dehydrogenase to liberate a second molecule of NH4+ for carbamoyl synthesis. Urea is
synthesized in the liver and transported through the blood to the kidneys where it is concentrated
and excreted in urine. As shown in figure 19, five enzymatic reactions are required for urea
synthesis, two of which occur inside mitochondria and three others in the cytosol. It can be seen
that two of the reactions require ATP hydrolysis (reactions 1 and 3), resulting in the expenditure of
four high energy phosphate bonds for every mole of urea produced. The urea cycle was
discovered in 1932 by Hans Krebs and a medical student who worked in his lab, Kurt Henseleit.
Krebs is the same biochemist who later described the citrate cycle and was co-recipient of the
1953 Noble Prize in Physiology or Medicine. In fact, elucidation of the urea cycle gave Krebs
insights into how cyclic pathways work and he exploited this knowledge to unravel the complexities
of the citrate cycle just five years later in 1937.
The starting point for urea synthesis is the formation of carbamoyl phosphate from NH4+
and HCO3- in a reaction catalyzed by the enzyme carbamoyl phosphate synthetase I. This
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Figure 19.
mitochondrial
enzyme is
distinct from
carbamoyl
phosphate
synthetase II
which is a
cytosolic
enzyme
involved in
pyrimidine
biosynthesis.
The three step
carbamoyl
phosphate
synthetase I
reaction
represents the
commitment
step in urea
synthesis and
requires the
hydrolysis of 2
ATP.
Carbamoyl
phosphate is
then
combined with
ornithine to
form citrulline in a mitochondrial reaction catalyzed by the enzyme ornithine transcarbamoylase.
The citrulline is then exported to the cytosol where it is first activated by AMP before being
converted to arginosuccinate when aspartate displaces the AMP. This reaction is catalyzed by the
cytosolic enzyme argininosuccinate synthetase and results in the incorporation of a second
nitrogen atom. Note that cleavage of PPi by pyrophosphatase means that this reaction
consumes two high energy phosphate bonds. Argininosuccinate is cleaved in the next reaction by
the enzyme argininosuccinase to yield fumarate and arginine which contains both nitrogens.
Lastly, the enzyme arginase converts arginine to urea and ornithine to complete the cycle.
Ornithine has the same role in the urea cycle as oxaloacetate does in the citrate cycle, namely, as
both the product of the last reaction and the substrate of the first reaction. By including the
pyrophosphatase reaction, it can be seen that four high energy phosphate bonds are required (4
ATP equivalents) for every molecule of urea that is synthesized, and moreover, that the C4 carbon
backbone of aspartate gives rise to fumarate:
NH4+ + CO2 + aspartate + 3 ATP ---> urea + fumarate + 2 ADP + AMP + 4 Pi
Since fumarate is an intermediate in the citrate cycle that can be used to form oxaloacetate,
and transamination of oxaloacetate using glutamate generates aspartate (via the aspartate
aminotransferase reaction), the urea cycle and citrate cycle are metabolically linked through
shared intermediates.
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As shown in figure 20, the aspartate-argininosuccinate shunt converts fumarate,
produced in the cytosol by the urea cycle, into malate that is used to make oxaloacetate in the
citrate cycle. Oxaloacetate combines with glutamate to generate aspartate and α-ketoglutarate,
Figure 20.
and then the aspartate is transported back into the cytosol where it is used as a substrate in the
argininosuccinate synthetase reaction of the urea cycle. In the simplest version of this bypass
reaction, sometimes called the "Krebs bicycle" pathway, fumarate is converted to malate in the
cytosol by an isozyme of fumarase. The malate can then be transported into the mitochondrial
matrix using the malate-aspartate shuttle and converted to oxaloacetate by malate
dehydrogenase. The resulting oxaloacetate is used as a substrate in the aspartate
aminotransferase reaction to generate aspartate which is transported to the cytosol where it serves
as a urea cycle substrate. Besides recycling fumarate to generate oxaloacetate for the aspartate
aminotransferase reaction, the aspartate-argininosuccinate shunt also produces an NADH in the
malate dehydrogenase reaction that can be used by the electron transport system to generate 2.5
ATP. This net yield of ATP helps offset the energetic cost of the urea cycle (4 ATP equivalents).
Inherited defects in many of the urea cycle enzymes have been observed clinically. While
complete loss of a urea cycle enzyme causes death shortly after birth, deficiencies in urea cycle
enzymes results in hyperammonemia (elevated ammonia levels in the blood). Most urea cycle
disorders also lead to a build-up of glutamine and glutamate which function as osmolites that can
cause brain swelling and associated neurological symptoms. Fortunately, it is possible to treat
some of the urea cycle disorders by restricting dietary protein as a means to limit nitrogen intake.
In addition, by providing metabolic substrates that increase the biosynthesis of nitrogen containing
compounds that can be excreted, it is often possible to decrease the severity of hyperammonemia.
For example, argininosuccinase deficiency can be treated effectively by putting patients on a
protein-depleted diet that is supplemented with high doses of L-arginine. As shown in figure 21,
this regimen increases flux through a "short-circuited" urea cycle by converting arginine to
ornithine, which combines with carbamoyl phosphate and aspartate to generate argininosuccinate.
Since argininosuccinate is soluble and can be excreted in the urine, it functions as a metabolic
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replacement for urea. Note that
supplementing the diet with ornithine would
also give the same result, but it is more
feasible to use arginine.
Figure 21.
Degradation of glucogenic and
ketogenic amino acids
The carbon backbones of eleven of the
twenty standard amino acids can be
converted into pyruvate or acetyl-CoA,
which can then be used for energy
conversion by the citrate cycle and
oxidative phosphorylation reactions. The
other nine amino acids are converted to the
citrate cycle intermediates α−
ketoglutarate, fumarate, succinyl-CoA,
and oxaloacetate, which can be used for
glucose synthesis by conversion of
oxaloacetate to phosphoenolypyruvate.
Under normal conditions, amino acid
degradation accounts for ~10-15% of the
metabolic fuel for animals, more so for
animals with high protein diets or during starvation when muscle protein is degraded. Alternatively,
when the energy charge of the cell is high, or metabolic fuel needs to be exported from the liver to
other parts of the body, amino
Figure 22.
acid degradation can be used to
provide precursors for glucose
and fatty acid synthesis.
Amino acid degradation
pathways are somewhat
complex, and therefore, it is
convenient to think about amino
acid degradation pathways in
terms of the metabolites they
produce, and whether these
metabolites are precursors to
glucose or ketone bodies. As
shown in figure 22, amino acids
that give rise to pyruvate, or any
of the citrate cycle intermediates,
are called glucogenic amino
acids because pyruvate and
oxaloacetate are precursors in
the gluconeogenic pathway (αketoglutarate, succinyl-CoA and
fumarate can be converted to
oxaloacetate). In contrast,
amino acids that are converted into acetyl-CoA or acetoacetyl-CoA are called ketogenic amino
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acids because they can give rise to ketone bodies. Notice in figure 22 that eight of the amino
acids (aspartate, isoleucine, leucine, lysine, phenylalanine, threonine, tryptophan, tyrosine), are
converted to more than one metabolite.
ANSWERS TO KEY CONCEPT QUESTIONS IN AMINO ACID METABOLISM:
Atmospheric nitrogen (N2) is converted into ammonia (NH3) as a nitrogen source for plants
by three distinct processes; 1) biological fixation by soil bacteria, 2) industrial fixation by
the Haber process, and 3) atmospheric fixation as a result of lightning. All organisms require
nitrogen for the synthesis of numerous biomolecules, the most abundant of which are amino acids
and nucleotides. However, since the N=N bond is so strong (930 kJ/mol), a large amount of
energy is required to overcome the energy barrier needed for this reduction reaction to go forward.
Biological fixation is carried out by two types of soil bacteria, free-living bacteria and symbiotic
bacteria, both of which use an ATP-dependent reaction mechanism catalyzed by the enzyme
nitrogenase to reduce N2 to NH3 (which becomes ammonium ion, NH4+). Nitrogenase consists of
two functional components, dinitrogenase reductase which accepts electrons from ferredoxin to be
used in the reduction reaction, and dinitrogenase which catalyzes the reduction reaction. ATP
binding and hydrolysis provides energy for conformational changes in the nitrogenase complex
that are needed to convert dinitrogenase reductase from an electron acceptor to an electron donor.
Since the nitrogenase reaction is inhibited by O2, nitrogen-fixing bacteria have evolved various
mechanisms to reduce O2 levels, one of which involves high local concentrations of the hemecontaining protein leghemoglobin that is produced by leguminous plants. Importantly, since much
of the NH4+ present in the soil is oxidized to nitrate (NO3-) and nitrite (NO2-) by nitrifying bacteria,
plants have nitrate and nitrite reductase enzymes to convert these compounds back into NH4+.
Urea is a waste product synthesized in terrestrial vertebrates (and some invertebrates) from
the NH4+ released from glutamine and glutamate, the nitrogen atom of aspartate, and CO2
produced by the citrate cycle in the mitochondrial matrix. Glutamine serves as a nitrogen
carrier in the body that transports NH4+ from the peripheral tissues to the liver where it is
deaminated by the enzyme glutaminase in the mitochondrial matrix to release NH4+ and
regenerate glutamate. Another source of NH4+ comes from amino acids in the blood (derived from
digestion or cellular degradation of proteins) which are taken up by the liver and used as
substrates in aminotransferase reactions that transfer the -amino group to -ketoglutarate to
form glutamate. The enzyme glutamate dehydrogenase has the important job in the liver of
releasing the NH4+ from glutamate and producing -ketoglutarate. The free NH4+ is then combined
with CO2 to form the urea cycle substrate carbamoyl phosphate in a mitochondrial reaction
catalyzed by the enzyme carbamoyl phosphate synthetase I. The second nitrogen in urea comes
from aspartate in a cytosolic reaction in which citrulline is converted to argininosuccinate by the
addition of aspartate. Since aspartate is formed from oxaloacetate by the aspartate
aminotransferase reaction in mitochondria, and urea cycle product fumarate can be converted to
oxaloacetate by the citrate cycle, the urea cycle and citrate cycle are metabolically linked through
their shared intermediates aspartate and fumarate (Krebs bicycle).
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