Bioc 460 - Dr.

Miesfeld Fall 2008

Lecture 38 - Amino Acid Synthesis

Albinism is a defect in tyrosine metabolism.

Key Concepts
- Overview of amino acid biosynthetic pathways
- Amino acids are precursors of other biomolecules
- Inborn errors of metabolism: Phenylketonuria, albinism,
and porphyria
Key Concept Questions In Nitrogen Metabolism
What is the biochemical basis for the most common form of
phenylketonuria?
Overview of amino acid biosynthetic pathways
The carbon skeletons of all twenty amino acids are derived from just seven metabolic
intermediates, that together, are found in three metabolic pathways. As shown in figure 1, these
include 1) three glycolytic pathway intermediates; 3-phosphoglycerate, phosphoenolypyruvate,
and pyruvate, 2) two pentose phosphate pathway intermediates; ribose 5-phosphate and
erythrose 4-phosphate, and 3) two citrate cycle
Figure 1.
intermediates; -ketoglutarate and oxaloacetate. Note
that while plants and bacteria are capable of
synthesizing all twenty of the amino acids illustrated in
figure 1, amino acid biosynthesis in animals is much
more restricted because they lack many of the required
enzymes. This is thought to be a result of evolutionary
adaptation in animals, which because of their
dependence on food for chemical energy, have diets that
are rich in protein, and therefore don't need to commit
energy reserves to synthesizing amino acids they obtain
in their diet.
Based on the principles of nutritional
biochemistry, it was determined that humans (and most
animals) require ten of the twenty amino acids in their
diet in order to thrive. As shown in figure 2, these ten
amino acids are called essential amino acids, whereas,
the other ten amino
Figure 2.
acids which humans
can synthesize on
their own, are called
nonessential amino
acids. For example,
alanine and
aspartate are
nonessential amino
acids because
humans can make
them from pyruvate
and oxaloacetate,
respectively, using
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transamination reactions. In contrast, essential amino acids, such as tryptophan and methionine,
must be obtained from the diet because humans lack the enzymes necessary to synthesize them
de novo. In general, the structures of the essential amino acids are more complex than the
nonessential amino acids which is reflected in the number of enzymatic reactions required to
synthesize them. As shown in figure 3, the nonessential amino acids alanine, serine and aspartate
are synthesized by all organisms using simple reaction pathways, whereas, plants and bacteria
synthesize the essential amino acids tryptophan, histidine and methionine using multi-enzyme
pathways.
Note that two of the amino acids in figure 2 have asterisks because they do not strictly fit the
definition of essential and nonessential amino acids. Arginine is listed as an essential amino acid
because humans require arginine in their diet to support rapid growth during childhood and
pregnancy. However, arginine is actually generated from argininosuccinate in the urea cycle,
which means that a small amount of this "essential" amino acid is made available for protein
synthesis through this route. Tyrosine is also highlighted because this conditional nonessential
amino acid is made in humans from the essential amino acid phenylalanine by the enzyme
phenylalanine hydroxylase. Therefore, as long as we have enough phenylalanine in our diets we
can generate tyrosine, although in fact, much of the tyrosine in our bodies actually comes directly
from dietary tyrosine.
The three dimensional structure of proteins, and their corresponding functions, are
determined by the primary amino acid sequence.
Figure 4.
Because amino acid side groups have different
Figure 3.

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chemical properties, the

Figure 5.
abundance of individual
amino acids in proteins is
not uniform. For example,
lysine, alanine, valine,
isoleucine, glycine and
glutamic acid are the most
common amino acids in
proteins, whereas,
cysteine, tyrosine,
histidine and methionine
are relatively rare.
Because of these
differences, it is critical
that metabolic flux through
various amino acid
biosynthetic pathways be
tightly regulated in plants
and bacteria to provide the required proportions of each amino acid in response to cellular needs.
As illustrated in figure 4, the general principle of feedback inhibition plays a pivotal role in
modulating flux through linked amino acid biosynthetic pathways.
As shown in figure 5, the biosynthesis of
Figure 6.
three nonessential amino acids (alanine,
aspartate and asparagine), and six essential
amino acids (methionine, threonine, lysine,
isoleucine, valine and leucine) in E. coli involves
two interconnected pathways utilizing pyruvate
and oxaloacetate as precursors. Note that the
metabolic intermediate -ketobutyrate links the
oxaloacetate and pyruvate pathways together
because the carbon skeleton of isoleucine is
derived from both -ketobutyrate and pyruvate.
While most all organisms contain orthologous
enzymes required for the synthesis of the
nonessential amino acids alanine, aspartate and
asparagine, the pathways leading to the synthesis
of the six essential amino acids shown in figure 5
vary considerably amongst plant and bacterial
species.
Aromatic amino acids are synthesized in
plants, fungi and bacteria by a pathway involving
the formation of a hydrocarbon ring following the
condensation of phosphoenolpyruvate and
erythrose-4-phosphate. In the first stage of this
pathway, sometimes called the shikimate
pathway, a C10 compound is formed called
chorismate which is the precursor to the three
aromatic amino acids tryptophan, tyrosine and
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phenylalanine. Since animal cells do not contain the enzymes required for chorismate
biosynthesis, enzyme inhibitors of this pathway have been developed for use as animal-safe
herbicides. One of the most widely used herbicides in this class is glyphosate, the active
ingredient in Roundup. As shown in figure 6, glyphosate is a competitive inhibitor of the enzyme
EPSP synthase which is required to convert shikimate 3-phosphate to EPSP. By spraying plants
with glyphosate, EPSP synthase activity is inhibited and the plants die because they are unable to
synthesize aromatic amino acids to support ongoing protein synthesis. Since glyphosate was
proven to be an animal-safe herbicide, and weed growth in crop fields reduces yields and
contaminants the agricultural product, plant scientists at the biotechnology company Monsanto
reasoned that by developing glyphosate-resistant crop plants it would be possible to spray
fields with the Roundup herbicide to eliminate weeds without killing the crop plants. The first
glyphosate-resistant crop plant developed was a strain of soybeans marketed as Roundup
Ready soybeans. Farmers planting fields with Roundup Ready soybean seeds can to spray
their crops with glyphosate throughout the growing season to kill weeds, resulting in higher crop
yields with improved quality (Figure 7).
The strategy used by the Monsanto scientists to develop Roundup Ready crop plants, which
now include wheat, corn and canola, was to isolate a mutated EPSP synthase gene from
glyphosate-resistant bacteria (the
Figure 7.
CP4 strain of Agrobacterium
tumefaciens). The CP4 EPSP
synthase enzyme does not bind
glyphosate, but importantly, is still
able to convert shikimate 3phosphate to EPSP and thereby
maintain flux through this amino acid
biosynthetic pathway. The plant
scientists used recombinant DNA
cloning techniques to create
transgenic plants by introducing
the mutated EPSP synthase gene
DNA into plant cells using a
technique called biolistics that
shoots DNA-coated gold particles
into plant meristematic tissue using
a high-pressure "gene gun" device.
In order to protect its investment in
the Roundup Ready crop plants, the
company sells seeds to the farmers
that are sterile so that the
transgenic plants cannot be
propagated from year to year.
Moreover, with well over 80% of the
soybeans planted in the United
States being the Roundup Ready
variety, the company has a robust market for its many Roundup herbicide products.
Amino acids are precursors of other biomolecules
The bulk of amino acids recovered from protein turnover, or obtained from the diet or de novo
synthesis, are used to support ongoing protein synthesis in cells. However, because of the
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nitrogen content of amino acids (the -amino group), they are also used as metabolic precursors
for numerous biomolecules, including heme groups
Figure 8.
(hemoglobin and cytochromes), nucleotide bases (purines
and pyrimidines) and a variety of signaling molecules
(neurotransmitters, hormones, nitric oxide). For example, the
prosthetic group of hemoglobin, myoglobin and cytochromes
is heme, a porphyrin ring containing iron. In 1945, David
Shemin and his colleagues discovered that glycine
contributed all four nitrogens to heme, with the carbon atoms
coming from both glycine and acetate. Shemin's approach
would not be considered good standard practice in modern
biochemistry, but at the time, it seemed like the best
experiment. Amazingly, he ingested gram quantities of 15Nlabeled glycine every hour over a three day period, and then
analyze 15N incorporation into hemoglobin by taking blood
samples at regular intervals. One of his findings was that the
glycine is the metabolic precursor to heme as shown in figure
8. In this pathway, which takes place in both the
mitochondrial matrix and the cytosol, the enzyme aminolevulinate synthase combines glycine and succinyl
CoA to form -aminolevulinate which is then exported to the
cytosol where two molecules condense to generate porphobilinogen. Following numerous other
reactions, the enzyme ferrochelatase incorporates Fe2+ into the heme ring. Most heme
biosynthesis takes place in erythrocyte precursors in the bone marrow to produce hemoglobin, and
the rest occurs in liver cells to provide heme for enzymes.
Tyrosine is the precursor to several important molecules in metabolic signaling and
neurotransmission, including epinephrine and dopamine. Figure 9 shows that tyrosine is oxidized
by the enzyme tyrosine hydroxylase in a reaction requiring the enzyme cofactor
tetrahydrobiopterin to form dihydroxyphenylalanine (L-DOPA), a metabolic precursor to dopamine
and dopaquinone. In the dopamine branch of the pathway, L-DOPA is converted to dopamine by
the enzyme aromatic
Figure 9.
amino acid
carboxylase.
Dopamine is a potent
neurotransmitter that is
required for numerous
brain functions. Patients
with Parkinson's
disease, a debilitating
neurological syndrome,
have fewer than 20% of
the normal dopamine
producing cells in the
substantia nigra region
of the brain. Dopamine
levels in the brain are
tightly-regulated as
over-production of
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dopamine is associated with the hallucinogenic

Figure 10.
symptoms found in schizophrenia. In fact,
Parkinson's patients who are treated with the drug
levodopa, a dopamine precursor that is metabolized
to dopamine once it enters the brain, can experience
schizophrenia symptoms if dopamine levels get too
high. It may be possible someday to treat
Parkinson's disease with stem cell therapy by
implanting dopamine producing cells into the effected
area of the brain, and thereby provide a better
treatment with fewer side effects.
Tyrosine is also the precursor to pigment
molecules called melanins that are produced from
dopaquinone. The two primary melanins are
eumelanins, which are dark pigments having a
brown or black color, and pheomelanins that have
red or yellow color (figure 10). The yellow color of
pheomelanin pigments comes from the sulfur in
cysteine that is combined with dopaquinone.
Melanocytes are cells that produce melanins, and
depending on the ratio of eumelanin and
pheomelanin pigments, one can have either dark hair
or light hair depending in the distribution of melaninfilled granules along the hair shaft. Natural loss of hair color occurs as Figure 11.
a result of aging when melanin production in human melanocytes
located near the base of hair follicles shuts down and these defective
cells are not replaced as they normally are in younger individuals
(figure 11). Gray hair can be colored by treating it with a mixture of
hydrogen peroxide and an ammonia based solution containing
artificial pigments. The ammonia causes the hair follicles to swell,
allowing the pigments to enter the hair shaft and undergo a chemical
reaction with the hydrogen peroxide to permanently stain the hair. However, as new hair grows
out from the hair follicle, it is easy to distinguish the difference between the unpigmented gray hair
and the artificially-colored hair.
Inborn errors of metabolism: Phenylketonuria,
albinism, and porphyria
The conversion of phenylalanine to tyrosine by the
enzyme phenylalanine hydroxylase serves as an
initiating step in phenylalanine degradation and is
also responsible for tyrosine production in animals.
Animals lack the enzymes needed to synthesize
phenylalanine de novo, but since they have the
enzyme phenylalanine hydroxylase, they can
convert dietary phenylalanine into tyrosine (figure
12). Tyrosine is used not only for protein synthesis,
but as described above, tyrosine is also the
precursor for neurotransmitter dopamine, as well
as, skin pigments (melanins) and epinephrine. A

Figure 12.

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genetic defect in the gene encoding

phenylalanine hydroxylase is responsible for the
Figure 13.
metabolic disease phenylketonuria (PKU).
The disease occurs about once in every 15,000
births, making it the most common genetic
metabolic disorder. Disease symptoms in
untreated individuals include severe mental
retardation, stunted growth and dental
problems. The clinical symptoms of PKU are
caused by the accumulation of phenylalanine in
the blood that is 30-50 times higher than normal.
This high level of phenylalanine leads to the
production of phenylalanine metabolites such as
phenylpyruvate, phenylacetate and
phenyllactate (figure 13), all of which are
associated with the observed neurological and
developmental problems.
Fortunately, PKU is treatable if diagnosed
shortly after birth using a simple blood test, and
in fact, this test is required by law in most
countries. Since the symptoms of PKU are
caused by excess phenylalanine and its metabolites, and humans require phenylalanine in their
diets (it is an essential amino acid), treatment regimens center around careful monitoring of
phenylalanine intake to provide just enough for protein synthesis without causing phenylalanine
accumulation. Phenylketonuriacs also have to be careful to avoid processed foods and beverages
containing the food additive aspartame (aspartylFigure 14.
phenylalanine methyl ester) because it is hydrolyzed to
aspartate and phenylalanine in the intestine (figure 14).
Aspartame is an artificial sweetener that was discovered in
1965 by James Schlatter, a chemist at the pharmaceutical
company G.D. Searle. Schlatter was synthesizing
compounds to be tested as a treatment for gastric ulcers,
and unknowingly, got one of the intermediate products on
his finger while working at the bench. When he licked his
finger later in the day to pick up a piece of paper, he
noticed an intense sweet flavor and initially thought it
came from his breakfast doughnut. He eventually figured
out it was the aspartyl-phenylalanine methyl ester
compound he had made, and twenty years later,
NutraSweet became a billion dollar a year product for
the company. Aspartame binds to G protein-coupled
receptors on taste cells in the tongue and is 150 times
sweeter than sucrose in human taste tests.
The phenylalanine hydroxylase gene is located on
chromosome 12 making it an autosomal recessive
genetic disease. An autosomal genetic disease is one in
which the mutation is located on one of the 22 autosomal chromosomes (all chromosomes except
the X or Y chromosome). As shown in figure 15, the probability that two PKU carriers will have a
child with the disease is 25% based on simple Mendelian genetics. Since the frequency of PKU
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carriers in the general population is ~2% (~1 in 50), then the probability that a baby will be born
with PKU by random chance is 0.02 x 0.02 x 0.25 = 0.0001, or 1 in 10,000, which is close to the
observed frequency of 1 in 15,000. Type 1 albinism is another autosomal recessive disease. In
this case, the defective gene encodes the enzyme tyrosinase. As shown in figure 16, a deficiency
in tyrosinase will result in loss of hair and skin pigments which explains the albino phenotype.
Interestingly, individuals with phenylketonuria can have
Figure 15.
light skin and hair at birth because of low levels of
tyrosine. However, phenylketonuriacs are not albinos
because they obtain sufficient amounts of tyrosine in their
diets to support melanin biosynthesis.
Numerous metabolic disease affecting heme
biosynthesis have been linked directly to enzymes in the
heme biosynthetic pathway. These diseases are
characterized by the accumulation of heme precursors in
the blood and liver and are collectively called porphyrias
because they inhibit porphyrin ring synthesis. As shown in
figure 17, defects in essentially every enzyme in the heme
biosynthetic pathway have been identified. Porphyrias are
caused by either autosomal recessive mutations similar to
phenylketonuria, or to autosomal dominant mutations.
Unlike recessive genetic diseases in which both copies of
the gene (maternal and paternal) are defective, dominant
genetic diseases cause symptoms when only one of the gene copies is mutated. In some cases
this is because the defective protein has "gained" a deleterious function through an inherited
mutation (e.g., unregulated enzyme activity), or it could be due to that fact that decreased levels of
protein are sufficient to cause the disease symptoms. As shown in figure 18, the inheritance
pattern of a dominant genetic disease is such that each child has a 50% chance of being afflicted if
one parent carries the mutation and the other parent is normal (100% of the children will get the
disease if both parents carry the dominant
Figure 16.
mutation).
One of the most common porphyrias is
called acute intermittent porphyria, which is due
to mutations in the gene encoding
porphobilinogen deaminase that lead to
decreased levels of the enzyme and a dominant
genetic phenotype. Under normal physiological
conditions, this disease is often asymptomatic
because the amount of enzyme made from the one
normal copy of the gene is usually sufficient to
maintain flux through the heme biosynthetic
pathway. However a variety of factors, including
hormones, drugs or dietary changes, can trigger
stomach pain and neurological problems stemming
from accumulation of the heme precursors aminolevulinate and porphobilinogen in the blood
and liver. It is thought that King George III of
England may have suffered from acute intermittent porphyria based on descriptions of his health
that were written at the time of the American Revolution (figure 19). Most notably, he suffered from
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excruciating stomach pain and episodes of delirium that may have been due to arsenic exposure,
one of the environmental factors known to aggravate porphyria symptoms in individuals carrying
the defective gene. A more rare form of porphyria is congenital erythropoietic porphyria that
results from recessive mutations in the gene encoding uroporphyrinogen III cosynthase. This
Figure 17.

Figure 18.

disease is characterized by the accumulation of uroporphyrinogen

metabolites that are excreted in the urine giving it a red color. One of the
metabolites is uroporphyrinogen I which builds up in the teeth causing
them to turn reddish brown and fluoresce under ultraviolet light. Moreover,
because uroporphyrinogen I also accumulates in the skin, these individuals
need to avoid excess exposure to sunlight which can result in the formation
of painful blisters. The striking symptoms of congenital erythropoietic
porphyria, combined with the common medieval practice of drinking animal
blood as a treatment for human ailments, led to an interesting proposal that
rare occurrences of porphyria might contribute to the legend of the
"vampires." According this legend, vampires must drink blood to survive
and can only come out at night to avoid the lethal affects of sunlight
(vampires are also usually depicted with strange looking teeth). Figure 20
shows the Bran castle in Romania which has come to symbolize the
folklore of Count Dracula, a fictional vampire created by
Figure 20.
Bram Stoker in 1897 when he published a story based
loosely on the life of Vlad the Impaler who lived in
Transylvania in the 15th century.

Figure 19.

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ANSWERS TO KEY CONCEPT QUESTIONS IN AMINO ACID METABOLISM:

What is the biochemical basis for the most common form of phenylketonuria?
Phenylketonuria (PKU) is a genetically-inherited metabolic disease caused by deficiencies in the
enzyme phenylalanine hydroxylase which is required to convert phenylalanine to tyrosine in animal
cells. Plants and bacteria contain all of the enzymes needed to synthesize the complete set of
twenty amino acids, whereas, animals must obtain ten amino acids in their diet (essential amino
acids) and synthesize the rest (nonessential amino acids) using intermediate metabolites obtained
from glycolysis, the citrate cycle and the pentose phosphate pathway. Newborn babies are tested
for PKU at birth by measuring the level phenylalanine in the blood, if they are found to be
phenylalanine hydroxylase deficient, they are immediately put on a phenylalanine-restricted diet.
Aspartame (aspartyl-phenylalanine methyl ester) is an artificial sweetener that is hydrolyzed to
aspartate and phenylalanine in the intestine, and therefore, cannot be included in the diet of an
individual with PKU. Interestingly, since tyrosine, the product of the phenylalanine hydroxylase
reaction, is required for the synthesis of hair and skin pigments (melanins), babies with PKU have
lightly colored hair and skin until they obtain sufficient amounts of tyrosine in their diets.

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