Amino Acid Metabolism Lectures
Amino Acid Biosynthesis I
David Nelson Nov. 12, 1997
last revised Nov. 12, 1997 8:30 AM
You are now familiar with the central metabolic pathways of glycolysis, gluconeogenesis
and the TCA cycle. These pathways concern carbohydrate metabolism. You have also
been studying lipid metabolism and nucleotide metabolism. Now we will begin to look
at the metabolism of the last major cellular building blocks, the amino acids.
Mammals don't make all 20 amino acids, so it will be necessary to consider some of the
pathways found in microorganisms. In bacteria and other microorganisms, mutants that
are defective for growth without a specific supplement are called auxotrophs. If E. coli
is mutagenized and a large collection of auxotrophs for a particular amino acid are
isolated, these can be characterized genetically. Genetic crosses between different mutants
result in cells with a portion of the genome present in two copies (merodiploid). If the two
mutations are in different genes, then some crosses will have a mutant copy and a wild type
copy of the two affected genes and these will complement one another and the cells will
grow. If the mutations are in the same gene, then the cross will be unable to grow. The
mutants are then said to be in the same complementation group. These mutants are
considered to be in the same gene. In a biochemical pathway, 8 complementation groups
would suggest 8 genes in the pathway. Some enzymes are multisubunit, so 8
complementation groups may represent less than 8 enzymes.
In going over these pathways, I will add information about some metabolic diseases.
These have similar features. Usually there is a build up of precursors in front of a block in
a pathway (see Fig. 17.17 p. 563). After the block, intermediates are absent or depleted.
The total absence of an enzyme is not required to make people sick. Low activity of an
enzyme due to a point mutation or a premature stop codon can occur and this may lead to
disease. Since genes in eukaryotes are built from exons, alternative splicing or exon
skipping can cause disease. We will see that there are many more diseases caused by
defects in amino acid breakdown, than in amino acid biosynthesis. That is due to
accumulation of intermediates in the breakdown pathways that can act in deleterious ways.
Many amino acids are neurotransmitters and high concentrations of them can lead to mental
retardation. Some intermediates are inhibitors of other enzymes and they can shut down
needed metabolic activity. These types of problems are not as likely in the biosynthetic
pathways, because missing amino acids can be obtained in the diet, and feedback inhibition
of pathways can shut down a pathway if intermediates accumulate to high levels. The loss
of a biosynthetic pathway can be harmful if an intermediate is required as a precursor in
another pathway.
Treatment of metabolic diseases is frequently dietary. The accumulation of amino acids that
are toxic can be controlled by a low protein diet supplemented with essential amino acids.
Other possible treatments are transplants of cells, that can provide the missing enzyme.
There have been several trials of treating Parkinson's disease (defect in L-dopa synthesis,
in the tyrosine biosynthetic pathway) by surgical implants of fetal brain tissue into the
brains of patients. This has helped some patients. There was one dramatic case of a man
who had taken a street drug that was contaminated with a toxic chemical. This resulted in
killing the cells of his substantia nigra where dopamine is made and he became frozen,
completely unable to move. A transplant of fetal brain cells, done in Sweden, helped him
to regain some movement.
Not all defects in amino acid pathways result in disease. Failure to breakdown proline for
example causes proline to build up in the blood. It is excreted and no ill effects are noted.
GETTING STARTED IN AMINO ACID BIOSYNTHESIS
In biosynthesis, all 20 amino acids are made from only 7 precursors from the central
metabolic pathways.
1. fructose 6 phosphate precursor to aromatic amino acids
2. glyceraldehyde 3 phosphate precursor to aromatic amino acids
3. 3-phosphoglycerate precursor to 3-phosphogycerate family of amino acids
4. phosphoenolpyruvate precursor to aromatic amino acids
5. pyruvate precursor to pyruvate family of amino acids
6. alpha ketoglutarate precursor to alpha ketoglutarate family of amino acids
7. oxaloacetate precursor to aspartate family of amino acids
The Kyoto Encyclopedia of Genes and Genomes has 16 maps on amino acid metabolism.
Some of them are more complex than the pathways we saw already for purines and
pyrimidines. There is probably enough material for more than a dozen lectures, but we
have only three, so you will be getting just the highlights of amino acid metabolism. To
give you an idea of what we are not going to cover, here is the map for
phenylalanine metabolism.This does not include phenylalanine biosynthesis
that is shown as a small cirlce near the top of the figure.
SOURCES OF NITROGEN FOR SYNTHESIS, GLUTAMATE
AND GLUTAMINE
In plants and microorganisms, the 20 amino acids all receive their amino groups from
glutamate in a transamination reaction. In some pathways the transamination is the first
step (methionine, threonine). In other pathways, transamination is the last step (valine,
leucine and isoleucine). In mammals, only 10 amino acids are synthesized. The rest are
acquired in the diet.
The source of nitrogen is clearly important. The nitrogen in glutamate is incorporated
from ammonia. Ammonia is a reduced form of nitrogen that is created by reduction of N2
gas (nitrogen fixation) or by reduction of nitrate (nitrate assimilation). We will talk about
these processes after we consider amino acid biosynthesis.
Glutamate can be synthesized in two ways, by glutamate dehydrogenase in a reductive
amination that couples ammonia to alpha ketoglutarate(fig. 26.7, p. 832), or by glutamate
synthase that transfers the amide group from glutamine to alpha ketoglutarate(fig. 26.10,
p. 834). This last reaction makes two glutamates, one from glutamine and one from alpha
ketoglutarate. The glutamine used in this reaction is made by glutamine synthase.
The two methods of forming glutamate are both present in bacteria. However, glutamate
dehydrogenase has a higher Km for ammonia than glutamate synthase. In real life
situations, where ammonia concentration is low, glutamate synthase is the preferred
pathway. Since glutamate synthase only transfers a nitrogen from glutamine, glutamine
synthase is the key enzyme for introducing ammonia into carbon compounds.
Glutamine synthase is a highly regulated enzyme in E. coli, and we will spend some time
discussing it (see text, pp. 835-838).
GLUTAMINE SYNTHASE
Glutamine synthase (GS) requires activation of the gamma carboxyl group by ATP. The
activated unit is then transferred to ammonia to make the amide (fig. 26.8, p. 833). The
enzyme is a double hexamer made from 12 identical subunits, like two donuts sitting on top
of one another. Each active site has two Mn2+ ions. The active sites are at the interfaces
between the subunits, so monomers of the enzyme are not active. Nine different
nitrogenous compounds feedback inhibit GS. Six of these are made directly from
glutamine (carbamoyl phosphate, glucosamine 6 phosphate, tryptophan, histidine, CTP
and AMP). The other three, alanine, serine and glycine are indicators of the cells nitrogen
state (see fig. 26.13, p. 836). Each inhibitor can only inhibit to a maximum of 11%. All
nine must be present to inhibit the enzyme 100%. This means that there are binding sites
on each subunit (or subunit interface) for 9 inhibitors, 3 substrates and two metal ions
(14 sites per subunit).
In addition to feedback inhibition, the 12 subunits are identical and each may be covalently
modified by adenylylation of a tyrosine residue to inhibit activity. Adenylylation is
addition of an AMP group. This reaction is reversible. Levels of adenylylation are
dependent on alpha ketoglutarate/glutamine ratios. When glutamine is high the enzyme is
adenylylated (inhibited) when alpha ketoglutarate is high it is deadenylylated (activated).
A cascade mechanism is involved to increase sensitivity (see fig 26.15, p. 837). There are
two enzymes in the cascade, an adenylyl transferase (AT), that acts to add or remove AMP
groups from GS, and a uridylyl transferase (UT), that acts to add or remove UMP groups
to or from a regulatory protein PII. The addition and removal of AMP groups by AT is
catalyzed by two different active sites on this enzyme, so AT is a multifunctional enzyme.
Which of the two active sites is operating depends on the regulatory protein PII.
PII is not an enzyme. It is a tetramer of identical 11 kDa subunits. Each subunit has a
tyrosine that can also be modified by covalent attachment of a UMP group. The enzyme
uridylyl transferase (UT) is responsible for the addition or removal of UMPs to or from
the four subunits of PII. The direction that UT operates is dependent on the glutamine or
alpha ketoglutarate concentration. High glutamine signals that the GS has made enough
glutamine and should be shut down. High glutamine causes the UT enzyme to remove the
four UMPs from PII. This stimulates the adenylyl transferase to Adenylylate GS and thus
inactivate it. PII without the four UMPs is called PIIA, because it is in the Adenylylation
mode.
High alpha ketoglutarate does the opposite. It causes UT to add UMPs to PII, and this
leads the AT enzyme to Deadenylylate GS. PII with the four UMPs is called PIID,
because it puts the AT enzyme in Deadenylylation mode. When GS is deadenylylated it is
active and makes glutamine.
The steady state ratio of glutamine to alpha ketoglutarate adjusts the enzymatic activity of
GS to match the needs of the cell. It is not an all or none system.
Besides feedback inhibition and covalent modification, there is regulation at the level of
gene expression. (see fig 26.16, p. 838) The gene in E. coli for GS can only be
transcribed if the transcriptional enhancer NRI is phosphorylated. NRI is converted to
NRI-P by a kinase called NRII. This kinase becomes a phosphatase when complexed with
PIIA (remember this inhibits GS). The phosphatase dephosphorylates NRI-P to NRI and
gene transcription stops.
AMINO ACID BIOSYNTHESIS
There are five families of amino acids. Four are based on the precursors they are made
from. The fifth, the aromatic amino acids, all share the first part of their pathway up to
chorismate. Histidine is in a group all by itself.
THE ALPHA KETOGLUTARATE FAMILY OF AMINO ACIDS
Glutamate, glutamine, proline, arginine and lysine(in fungi, and lower eukaryotes) are all
derived from alpha KG. We have already seen how glutamate and glutamine are made.
Proline is made starting from glutamate(see fig 26.18, p. 841). The gamma carboxyl group
is activated by phosphorylation. This compound is reduced and dephosphorylated to form
glutamate gamma semialdehyde, a compound we will see again in amino acid degradation.
The aldehyde spontaneously cyclizes by forming an internal Schiffs base with the alpha
amino group. This is then reduced to form proline.
Hydroxyproline is made in collagen after the protein is synthesized. The enzyme prolyl
hydroxylase is an ascorbate dependent enzyme. Lack of ascorbate causes scurvy. Polar
explorers traveling with Robert Scott to the South Pole in 1911/1912 developed scurvy.
This weakens collagen tissue. One man who had suffered a bullet wound during a war
began to have his wound reopen. Ascorbate, or vitamin C is required to maintain scar
tissue and to keep blood vessels from becoming fragile. None of Scott's polar expedition
survived. They traveled 1400 miles and died within 15 miles of their home base.
Arginine is also made from glutamate via an activated gamma carboxyl group. However,
the alpha amino group is acetylated first, so the gamma aldehyde cannot react with the
alpha amino group to form a cyclic product(fig. 26.19, p. 842). After this is done, the
aldehyde is produced and then glutamate donates the terminal amino group. The acetyl
group is removed, leaving ornithine. Ornithine is converted to arginine in the urea cycle.
We will talk about the urea cycle when we cover amino acid degradation, so we will stop
at ornithine for now. N-acetylglutamate is an activator of an important enzyme,
carbamoyl phosphate synthase I. This enzyme catalyzes a major route of ammonia
removal, so a defect in arginine biosynthesis leads to toxic levels of ammonia that result in
death a few days after birth.
Ornithine is a precursor to putrescine, spermine and spermidine. These are polyamines that
interact with DNA. Putrescine is formed by decarboxylation of ornithine. It has a simple
structure of a four carbon chain with amino groups on each end. The conversion to
spermine and spermidine involves transfer of propylamine groups from an S-adenosyl
methionine derivative. We will talk about that when we cover methionine biosynthesis.
Experiments with yeast have shown that polyamines are essential, but the cell makes a huge
supply that takes several generations of yeast cell division, after the gene is turned off, to
dilute stores of polyamines enough to have any effects on growth.
Lysine can be made starting from alpha ketoglutarate in lower eukaryotes, but in higher
eukaryotes, it is made from aspartate. The fungal pathway of lysine synthesis is shown in
fig. 26.22, p. 846. This pathway has several steps similar to the TCA cycle. Lysine has
one more carbon than alpha ketoglutarate, so the starting compound must be lengthened.
This is done by reacting acetyl CoA with alphaKG in a reaction that is similar to the citrate
synthase reaction. The product has an hydroxyl on the beta carbon, just like citrate. This
has to be moved to the alpha carbon in order to decarboxylate the homoisocitrate. The
product alpha ketoadipate is similar to alpha ketoglutarate in the TCA cycle. The
compound is transaminated from glutamate to make a new amino acid alpha aminoadipate.
It is an amino acid that is next in the series aspartate (4 carbons), glutamate (5 carbons),
alpha aminoadipate (6 carbons). This compound is activated by phosphorylation and
reduced to an aldehyde as was seen in proline and ornithine biosynthesis. The aldehyde
reacts with a glutamate amino group to form an adduct that is hydrolyzed on the other side
of the nitrogen to form lysine and release alpha ketoglutarate. Remember in purine
biosynthesis that aspartate was an amino donor that was cleaved to release fumarate
The main difference here is the hydrolytic cleavage adds an oxygen back to the product.
THE ASPARTATE FAMILY OF AMINO ACIDS
The amino acids in this family include aspartate, asparagine, methionine, threonine,
isoleucine and lysine by a different pathway from the one seen in fungi.
Aspartate is formed by transamination of oxaloacetate from the TCA cycle. Both alpha
ketoglutarate and oxaloacetate are sources of carbon skeletons for amino acid synthesis.
These amino acids are sources of nitrogen and carbon for building other amino acids and
nucleotides. These are exit points from the TCA cycle and these compounds have to be
replenished or the cycle will run out of oxaloacetate to react with acetyl CoA. (See
section 19.13, pp. 616-617) The cycle is refilled by anaplerotic reactions that produce
TCA cycle intermediates. (see section 19.14, p. 618) The most important is pyruvate
carboxylase that makes oxaloacetate from pyruvate and CO2. When there is a deficit of
oxaloacetate, acetyl CoA builds up, because it cannot enter the TCA cycle. Acetyl CoA
activates pyruvate carboxylase. Malic enzyme is another anaplerotic enzyme that converts
pyruvate and CO2 to malate. We will see later that all amino acids are broken down to
TCA cycle intermediates, so amino acids can serve as sources of these intermediates to
keep the TCA cycle going, even if carbohydrates are not available.
Asparagine is made in two ways. In bacteria, the amide nitrogen comes from ammonia, as
in the glutamine synthase reaction. In other organisms glutamine is the amino group
donor to aspartate. This reaction requires ATP hydrolysis to AMP and PPi. (see fig.
26.24, p. 848) The AMP group activates the carboxyl. This is a recurring theme that you
should be aware of.
Threonine, methionine and lysine are all made starting from aspartate(fig. 26.25, p. 849).
Since it is important to make the appropriate amount of each amino acid, the first enzyme
in the pathway, aspartokinase, is subject to regulation. In fact in E. coli, there are three
aspartokinase enzymes. Aspartokinase I is feedback inhibited by threonine .
Aspartokinase III is feedback inhibited by lysine. Aspartokinase II is not feedback
inhibited, but its gene expression is repressed by methionine. Methionine is called a co-
repressor(see fig. 17.16, p. 562). Lysine is a co-repressor of aspartokinase III and
threonine and isoleucine (isoleucine is made from threonine) are co-repressors of
aspartokinase I.
The pathways for these 4 amino acids all start the same way. Aspartate is phosphorylated
and reduced to form the aldehyde, just as we saw with proline, ornithine and fungal lysine
biosynthesis. This requires an ATP and NADPH. At this point lysine biosynthesis
branches off, through an 8 step pathway that we won't cover in detail. The main features
are addition of three carbons as pyruvate to make a 7 carbon skeleton. An extra amino
group is added from glutamate and one carbon is removed in a decarboxylation reaction at
the end of the pathway.
The three remaining amino acids have one more common step before they branch off.
This is the reduction of the aspartyl semialdehyde to an alcohol by NADPH. The alcohol
of aspartic acid is called homoserine. This is very similar to threonine except the hydroxyl
is on the gamma carbon instead of the beta carbon. A two step pathway phosphorylates
the gamma hydroxyl and shifts it to the beta carbon to make threonine.
Homoserine is also the precursor for methionine. Again the hydroxyl is activated, but this
time it is by succinyl CoA that transfers a succinyl group and releases CoA. Methionine
has a sulfur, and this is donated from cysteine. The whole cysteine molecule is covalently
attached to the activated carbon through the sulfur. This is cleaved off as pyruvate and
ammonia to leave the SH group on the product homocysteine. The terminal methyl is
donated by N5 methyl tetrahydrofolate.
Methionine can react with ATP to form S-adenosylmethionine (SAM). (see fig. 26.26, p.
851) The ATP is hydrolyzed to release PPi and Pi, so SAM costs three high energy
phosphate bonds to make. The methyl group on SAM is readily donated to methyl
acceptors in synthetic reactions producing S-adenosyl homocysteine. This product is
cleaved to form adenosine and homocysteine that are recycled. Another route SAM can
enter is the decarboxylation of the methionine part of the molecule to make decarboxylated
SAM. This structure is now a propylamine donor rather than a methyl donor. This group
is transferred to a putrescine amino group (made by decarboxylation of ornithine) to make
spermidine. One more propylamine can be added to the other amino group of spermidine
to make spermine.
Isoleucine is made from threonine after it is oxidatively deaminated to form alpha
ketobutyrate. This compound is similar to pyruvate, except it has one more methylene
group. (see fig. 26.27, p. 852) Pyruvate and alpha ketobutyrate are both processed by the
same four enzymes to make valine and isoleucine. We will go over these in the pyruvate
family of amino acids. Since the deamination of threonine is unique to the isoleucine
pathway, this step is feedback inhibited by isoleucine. The next step is feedback inhibited
by valine, so valine inhibits its own synthesis and isoleucine's as well, but isoleucine only
inhibits its own pathway, not valine's. (Remember isoleucine also inhibits aspartokinase I
at the first step in its pathway.)
THE PYRUVATE FAMILY OF AMINO ACIDS
Alanine valine and leucine are the three members of this family. Alanine is formed by
transamination of pyruvate from glutamate. Valine is made by the same pathway as
isoleucine starting from the alpha keto acids pyruvate and alpha ketobutyrate. In the first
common step, pyruvate donates an hydroxyethyl group to the alpha carbon of the keto
acid. In the next step, the newly added acetyl group inserts itself between the alpha and
beta carbons, forming a dihydroxy acid. This is then dehydrated to make the alpha keto
acids that are directly transaminated by glutamate to make valine and isoleucine.
Leucine biosynthesis branches from alpha ketoisovalerate, the immediate precursor to
valine. This compound undergoes a reaction similar to oxaloacetate in the citrate
synthase reaction. It reacts with acetyl CoA to make a product that is both
hydroxylated and carboxylated at the beta carbon. As in the TCA cycle conversion of
citrate, the hydroxyl is shifted to the alpha carbon and the carboxyl is removed as CO2.
This forms a new alpha keto acid that is transaminated to form leucine.
THE 3-PHOSPHOGLYCERATE FAMILY OF AMINO ACIDS
Serine, glycine and cysteine belong in this group. 3-phosphoglycerate is oxidized to
convert the alpha hydroxyl to a keto group. This is then transaminated and
dephosphorylated to make serine. (see fig. 26.29, p. 854)
Glycine is made from serine in two different ways. In the first, an hydroxymethyl group is
transferred from serine to tetrahydrofolate to make N5 N10 methylenetetrahydrofolate. The
second pathway is the glycine oxidase reaction acting in reverse. Here CO2, NH4+ and
N5 N10 methylenetetrahydrofolate condense to form glycine. Serine is the source for one
carbon units used in biosynthesis of many products like lipids, purines, thymine,
tryptophan, cysteine and methionine. Glycine is used in purine, glutathione and heme
synthesis.
There are several ways to make cysteine. In bacteria, H2S can react with serine to make
cysteine directly. This requires a pyridoxal phosphate dependent enzyme. In other
organisms the hydroxyl is first activated by adding an acetyl group from acetyl CoA. The
acetyl group is then displaced by H2S to form cysteine.
An analog of acetyl serine called acetyl homoserine can be made from aspartate by doing
reductions and acetylation of the gamma carboxyl group. This can be modified by H2S to
form homocysteine. Once this is formed, serine is added to form cystathionine and the
structure is cleaved on the opposite side of the sulfur to form cysteine and alpha
ketobutyrate. The carbons and nitrogen come from the serine, not from homocysteine.
H2S is highly toxic, because it can react with heme A3 in cytochrome oxidase and poison
electron transport. Therefore, it must be made in small quantities when it is needed. H2S
comes from sulfate SO4 (2-), at +6 oxidation state. Eight electrons are required to get it
to H2S (-2 oxidation state). This is done by a series of reactions (fig. 26.32, p. 856).
sulfate is added to the beta position of ATP liberating PPi. The ribose ring of this
compound is then phosphorylated to activate the sulfonyl group further. This compound
called PAPS reacts with thioredoxin to form a disulfide bond and release sulfite SO3 (2-)
at +4 oxidation state. Sulfite is converted in a six electron reduction to sulfide by the
enzyme sulfite reductase, using 3 NADPH as electron donors. A heme derivative called
siroheme is in this protein.
Through this series of reactions, you should be noticing that there are common principles
that are used in the pathways again and again. Often, carboxyl groups are activated by
phosphorylation and then reduced to make an aldehyde before some other reaction occurs.
The series of reactions seen in the TCA cycle that react acetyl CoA with an alpha keto acid
and then proceed to shift an hydroxyl group and decarboxylate the product, are used in the
fungal lysine pathway and the leucine pathway. There are a variety of activating groups
that can be added to carboxyls and hydroxyls. These include phosphates, CoA, succinate,
acetyl groups or AMP. You should be aware of similarities in the different pathways.