Amino Acid Metabolism
Amino Acid Biosynthesis II
For Nov. 14, 1997
Last modified Nov. 14, 1997 8:45 AM
Note on an error in the first amino acid biosynthesis notes: There was an error in the notes
in the section on the pyruvate family of amino acids. The paragraph on leucine
biosynthesis says that alpha ketobutyrate undergoes a reaction similar to "pyruvate in the
pyruvate dehydrogenase reaction". This has been corrected to "oxaloacetate in the citrate
synthase reaction". The lecture was correct, the notes were in error until Wed. morning.
AROMATIC AMINO ACIDS
The aromatic amino acids do not derive from a single metabolic intermediate starting point,
like the other families of amino acids we already discussed. It takes three intermediates
from the glycolysis pathway to start aromatic amino acid biosynthesis. Fructose-6-
phosphate and glyceraldehyde-3-phosphate are reconfigured by transketolase (see Fig
21.42 page 691). If you remember from the pentose phosphate pathway, transketolase
moves a two carbon ketol group from a keto sugar to an aldose. So the 6 carbon fructose-
6-P becomes the four carbon erythrose-4-P and glyceraldehyde-3P picks up two carbons to
become the 5 carbon xylulose-5-P (precursor to PRPP). The erythrose-4-P reacts with
phosphoenolpyruvate to start biosynthesis of the aromatic amino acids.
Phenylalanine, tyrosine and tryptophan are synthesized from the intermediate chorismate.
This is also a precursor of many compounds in biology that contain a benzene ring.
Vitamins E, K, folic acid, ubiquinone, plastoquinone and lignin are all made from
chorismate(see fig. 26.33, p. 857). Two carbohydrate skeletons phosphoenol pyruvate
and erythrose 4 phosphate are condensed to form a seven carbon compound called 2-keto-
3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP). (see fig. 26.34, p. 858) This
compound is cyclized with loss of the phosphate to form a six membered ring that
undergoes dehydration and reduction to form shikimate. This pathway is referred to as
the shikimate pathway. Shikimate is phosphorylated and then reacts with PEP, with the
loss of phosphate from PEP to make the intermediate 3-enolpyruvyl-shikimate-5-
phosphate. This loses its phosphate to become chorismate.
Here the pathway branches in two directions to make trp on one branch and tyr and phe on
the other branch.
Tyrosine and phenylalanine are similar amino acids and their synthesis is similar(fig.
26.35, p. 859). The enolpyruvyl group on chorismate is shifted to the para position of the
ring to make prephenate. At this point the pathway diverges again. If the carboxyl group
is oxidized to CO2, the resulting alpha keto acid is 4-hydroxyphenylpyruvate, which is
transaminated from glutamate to form tyrosine. If the carboxyl and the hydroxyl are both
removed, the resulting alpha keto acid is phenylpyruvate. This is also transaminated to
form phenylalanine.
Tryptophan is also formed from chorismate, in a five enzyme pathway. In the first step of
this pathway, the enolpyruvyl group and the hydroxyl are removed and an amino group
from glutamine is added to the ring, which is now aromatic. This compound, anthranilate
is condensed with PRPP, which you saw in purine biosynthesis. The phosphoribosyl
group is added to the amino group. In step three, the ribose sugar is isomerized to a
ribulose sugar. The enzyme that catalyzes this step is one of the common genes in yeast
that is used as a selectable marker TRP1. Yeast that have a mutation in TRP1 cannot grow
on media lacking tryptophan. A functional copy of TRP1 on a plasmid complements this
defect and can be selected for in a yeast transformation, on tryptophan minus plates. If
your favorite gene is on this plasmid, you can select for your gene at the same time, by
selecting for trp plus colonies from a trp1 auxotrophic yeast.
In step 4, the intermediate made by TRP1 is decarboxylated and dehydrated to form the
indole ring of tryptophan, attached to glycerol three phosphate. The last enzyme,
tryptophan synthase actually has two active sites and two different steps are carried out on
the alpha and beta subunits. The glycerol three phosphate is removed as glyceraldehyde 3
phosphate, on the alpha subunit and serine is added to replace it on the beta subunit. The
intermediate is transferred from the alpha to the beta subunit active sites through a tunnel
on the enzyme. This is called channeling. (see fig. 26.37, p. 861).
The five enzymes in this pathway have been studied in many different organisms, including
bacteria, fungi and yeast. (see handout) Some of the genes are fused together, so two
enzymes are on one polypeptide chain. Some of the enzymes have alpha and beta
subunits, that are coded in separate proteins, but they have no activity unless they are
associated with each other. Anthranilate synthase is like this in all species, tryptophan
synthase is like this in some species, in other species the alpha and beta subunit are fused
as one protein. Some of the proteins that are not sequential in the pathway are covalently
linked by disulfide bonds. All this points to the existence of a super complex of all the
tryptophan biosynthetic enzymes. However, this complex is pretty loose, since it cannot
be isolated intact.
REGULATION OF AROMATIC AMINO ACID BIOSYNTHESIS
In E. coli, there are three different DAHP synthase enzymes (first step in chorismate
synthesis). This is similar to aspartokinase in the thr, ile, met and lys pathways. Each
product, trp, tyr and phe inhibits one of the three different DAHP synthases. The gene
expression of these three enzymes is also repressed by the end products. Trp represses the
five enzymes unique to the trp pathway, since they are in a single operon.
In Bacillus subtilis, there is just one enzyme and it is not inhibited by the three amino acid
products of the pathway. Instead it is inhibited by chorismate and prephenate, the
products that occur at the two branch points of the pathway. The branches that lead to tyr
and phe are inhibited by these amino acids, at the first step after prephenate is formed.
HISTIDINE BIOSYNTHESIS
The first step of histidine biosynthesis couples PRPP and ATP to make an unusual
compound that has two ribose rings attached to the same adenine ring. The compound
has four phosphates. (see fig. 26.38, p. 862) Two of these are cleaved off and then the
adenine ring is broken. The ribose from PRPP is isomerized to the open chain
conformation then, in reaction 5, glutamine donates its amide and displaces the remainder
of the ATP molecule, leaving behind one nitrogen and one carbon from the adenine ring
that was broken. This cyclizes to make the imidazole ring. Here we see imidazole
coupled to glycerol phosphate. This is like the tryptophan pathway, where the indole ring
was coupled to glycerol phosphate. Rather than swapping the glycerol phosphate for
serine like we saw in the tryptophan pathway, histidine is made by dehydrating to make
an alpha keto group that is transaminated by glutamate. The product is dephosphorylated
and oxidized twice by the same enzyme histidinol dehydrogenase to make the carboxyl.
The genes of the histidine biosynthetic pathway are in a single operon and they are
repressed by histidine.
The histidine biosynthetic pathway is the basis for the Ames mutagenesis test. Mutants in
the histidine pathway are plated on a his minus medium and a test substance is included.
The number of his+ colonies that appear compared to a control shows that a compound is
mutagenic. It changes the his minus mutants back to wild type and restores growth.
EXPLOITING INHIBITORS OF MISSING PATHWAYS
Plants make all twenty amino acids, but humans make only 10. Therefore, inhibitors of
the pathways that are present in plants but not humans should not affect humans (and
other mammals). These are useful as herbicides. (see p. 863) The aromatic amino acids
and histidine as well as the branched chain amino acids are all essential in humans,
meaning we cannot make them. (We can convert phenylalanine to tyrosine by a
phenylalanine breakdown pathway, but we are missing the shikimate pathway.) There are
three inhibitors listed in your book that block three of these pathways: the shikimate
pathway, the histidine pathway, and an enzyme used in common to make Ile, Leu and Val.
NITROGEN FIXATION AND NITRATE ASSIMILATION
Early on in amino acid biosynthesis we talked briefly about how nitrogen is reduced to
ammonia so it can be incorporated into biological molecules. There are two routes.
Either nitrogen gas is converted to ammonia in a process called nitrogen fixation, or
nitrate is converted to ammonia by nitrate assimilation. Nitrogen fixation represents only
1% of all the nitrogen that is converted to ammonia in the biosphere, but it is very
important commercially since it occurs in bacteria associated with leguminous plants, like
soybeans. Nitrate assimilation accounts for the other 99% of natural ammonia production.
The amount of nitrogen fixed by man in the chemical production of fertilizer is not
insignificant and amounts to 30% of nitrogen in the biological nitrogen cycle. Before the
Haber process was invented this was 0%. A Nobel prize was given for this invention. It
is suitable that Nobel would award a prize to someone who invented a better way to fix
nitrogen, an essential ingredient in explosives. Nobel made his fortune from inventing
dynamite.
NITRATE ASSIMILATION
Nitrate assimilation is done in two reactions. First, nitrate NO3- is reduced to nitrite
NO2-. Nitrogen changes from a +5 oxidation state to +3, which takes two electrons. The
enzyme is called nitrate reductase. The second enzyme is nitrite reductase that converts
NO2- to NH4+, moving nitrogen from a +3 to -3 oxidation state. This takes six electrons.
Nitrate reductase is a fairly big protein (a homodimer of 210-270 kDa) that contains a
series of redox active centers to pass electrons from NADH on to nitrate. These include
redox active sulfhydryls, an FAD, a b-type heme and molybdenum cofactor (see fig. 26.1,
p. 828).
The second enzyme, nitrite reductase gets its electrons from reduced ferredoxin in
chloroplasts of plants. The electrons came from light driven activation of P700 in
photosystem I. (remember, only one electron/ferredoxin) The protein is fairly small, and
it has a 4Fe-4S center and siroheme, an unusual heme derivative (see fig. 26.1). NO2-
binds to the siroheme as an iron ligand. In plants, the nitrate reductase is in the cytosol
and the nitrite reductase is in the chloroplasts, so nitrite must be transported into the
chloroplasts.
In microorganisms, the nitrite reductase is more like the nitrate reductase, and it gets its
electrons from NADPH.
NITROGEN FIXATION
Nitrogen fixation is only done by bacteria. Frequently, these bacteria are associated with
plants in an endosymbiotic relationship. Legumes are particularly valuable examples. The
process requires an enzyme called nitrogenase in an oxygen free environment. If ATP
concentrations are low the enzyme is shut down, because it is expensive to reduce N2. It
takes 16 ATP to reduce one N2 molecule. Also, if ammonium ion is abundant the genes
for nitrogen fixation are repressed. There are at least 20 genes involved. A source of
strong reductant like reduced ferredoxin is needed as a source of electrons.
Nitrogenase is composed of two proteins, nitrogenase reductase and nitrogenase.
Nitrogenase reductase is a small (60kDa) dimer with only one 4Fe-4S center. It receives
electrons from ferredoxin and passes them on to nitrogenase, one electron at a time. The
reductase is very sensitive to oxygen, which inactivates it. Nitrogenase is much larger,
(220kDa alpha 2 beta 2 heterotetramer) with many redox active centers. There are four
4Fe-4S centers and two molybdenum iron cofactors shown in fig. 26.5, p. 831. So there
are 32 metal ions/molecule (30 Fe and 2 Mo) and 32 sulfides (your text has this
backwards). The nitrogenase is also sensitive to oxygen poisoning. Of course the easiest
way to keep O2 low is to live in an anaerobic environment. That option is not available to
leguminous plants, where the oxygen concentration is kept low by leghemoglobin (leg for
legume) that binds O2 and transfers it to an electron transport chain. In other organisms,
the O2 concentration is kept low by very rapid electron transport chains that consume O2.
During each N2 reduction cycle, H2 is formed and it must be released from the active site
so that N2 can bind there. This means that 2 electrons from the 8 needed to complete one
cycle are lost as H2. The active site is on the nitrogenase enzyme, presumably between the
two molybdenum cofactors, so each can contribute electrons to the N2 molecule.
Ferredoxin, or sometimes flavodoxin, donates one electron to nitrogenase reductase. This
binds two ATP and associates with nitrogenase. (see handout) The two ATP are
hydrolyzed and the electron is transferred to the nitrogenase redox centers, while the
reductase is released. For each electron transferred, a proton accompanies it. After three
cycles, two electrons are used to reduce two protons to H2, which is released from the
active site, allowing N2 to bind. Five more cycles bring the total number of electrons
added to the nitrogen up to 6. The nitrogen is then released as two NH3 molecules. Every
electron transfer takes two ATP.
When the H2 gas is released, the oxidation state of the nitrogenase may slip back by losing
an electron, which forces the process to start over. This means the average number of ATP
used per N2 reduced can be more than 16.
HEME BIOSYNTHESIS
Some amino acids are precursors to cellular components that are not proteins or
nucleotides. These include heme, glutathione, the peptidoglycan of bacterial cell walls,
antibiotics and physiologically active amines like dopamine, serotonin, epinephrine,
norepinephrine, histamine, gamma amino butyric acid(GABA) and others. We will discuss
heme and glutathione biosynthesis.
Porphyrin is needed for hemes found in hemoglobin, myoglobin and cytochromes. The
cytochromes are found in the electron transport chain, and in numerous other enzymes
including cytochrome P450. These are metabolic enzymes involved in steroid
biosynthesis, lipid metabolism and drug oxidations. There are more than 50
different cytochrome P450 enzymes in humans.
Porphyrin is made in different pathways in mammals and plants. (see handout) In
mammals, the carbon and nitrogen skeleton required for making porphobilinogen (the four
ring structures in heme) comes from glycine and succinyl CoA. These react with the
release of CoA to form an enzyme bound intermediate that undergoes decarboxylation to
form delta aminolevulinate. Two delta aminolevulinate molecules condense to form
porphobilinogen.
In plants, glutamate is the source of the delta aminolevulinate synthesis. As we have seen
often in amino acid biosynthesis, the glutamate has to be activated before it can be
modified. This is done in an unusual way. The glutamate is transferred to a tRNA
molecule where a specific reductase reduces the alpha carboxyl (not the gamma carboxyl) to
form an aldehyde. This compound is rearranged in a rare intramolecular transamination
where the alpha amino group migrates to react with the terminal aldehyde. The product is
delta aminolevulinate.
Once the porphobilinogen is formed, 4 of these condense with each other to form a linear
tetrapyrrole called hydroxymethylbilane. This is rearranged and cyclized to form the first
cyclic tetrapyrrole uroporphrinogen III. This is the precursor to all the porphyrin rings we
have seen, and will see, including cobalamin (in methyl malonyl CoA mutase, of beta
oxidation), siroheme (in nitrite reductase and sulfite reductase), chlorophyll and the more
common hemes.
Heme is synthesized in cytosolic and mitochondrial compartments. The first steps are done
in the mitochondria to make delta aminolevulinate, which is exported to the cytosol to form
the tetrapyrrole ring. Once the ring is decarboxylated it is returned to the mitochondrion to
be finished and have iron inserted by ferrochelatase. Heme is a very insoluble structure, so
it may be able to pass through a lipid bilayer without a transport protein. It probably exists
in association with lipid membranes until it can be inserted into proteins.
PORPHYRIAS
Defects in heme biosynthesis lead to a build up of precursors. A block in uroporphrinogen
III cosynthase results in accumulation of a cyclic form of hydroxymethylbilane that is not
the enzymatically made form, and a decarboxylated version of this. These products are
excreted in the urine and turn it red. They are also deposited in the skin and teeth, making
the skin highly light sensitive and making the teeth a fluorescent reddish brown color. The
skin can easily form ulcers and scars due to light exposure and hair growth on the face
arms and legs is increased. This disease may be the basis for the werewolf legends.
Since there are many steps in the synthesis of heme, the severity and character of the
porphyrias vary. King George the III, ruler of England during the American revolution
may have suffered from a porphyria. His urine was the color of port wine, and he had
other symptoms, including intermittent neurological symptoms. These are alluded to in the
movie The Madness of King George. There is also speculation that Vincent van
Gogh may have had a porphyria.
DEGRADATION OF HEME
Heme is broken down by heme oxygenase to open the ring and form biliverdin, a green
linear tetrapyrrole. This has a conjugated double bond system that causes it to be colored.
Reduction of the central double bond shifts the spectrum of light it will absorb to make it a
red orange colored compound called bilirubin. The shift in a bruise from purple to greenish
to yellow orange is due to heme degradation under the skin.
The first step of ring cleavage that forms biliverdin releases a CO as one of the products.
This reacts to bind tightly with hemoglobin, so that at any given time, about 1% of a
persons hemoglobin is bound to CO. In a mutated form of hemoglobin(Hb Zurich), the
affinity of CO for heme is about 100 fold higher because a histidine ligand to oxygen is
changed to Arg. This his residue normally distorts CO binding to make it bind less tightly.
In patients with Hb Zurich, about 10% of their hemoglobin is bound to CO.
Bilirubin is very insoluble and has to be transported on serum albumin through the blood.
Excessive production of bilirubin, or failure to eliminate it causes deposition of the
yellowish pigment in tissues. The eyes and skin turn yellow and this is called jaundice,
from the Latin word for yellow. The eventual fate of bilirubin is to end up in bile, as
bilirubin diglucuronide, in the urine as urobilin (that makes urine yellow), and in the feces
as stercobilin (a deep red brown).
GLUTATHIONE AND THE GAMMA GLUTAMYL CYCLE
The tripeptide glutathione is composed of glutamate cysteine and glycine. It is not made on
ribosomes, and the linkages are not standard peptide linkages. Glutamate and cysteine are
coupled with hydrolysis of ATP. The gamma carboxyl of glutamate is the site of peptide
bond formation, not the alpha carboxyl. Thus the name of the product is gamma glutamyl
cysteine. This dipeptide then forms a conventional peptide linkage with glycine at the alpha
carboxyl of cysteine. The resulting tripepetide is gamma-glutamylcysteinylglycine.
Glutathione is important for multiple reasons. In E. coli, it is a source of reducing
equivalents for glutaredoxin, a small protein that donates electrons to ribonucleotide
reductase. Ribonucleotide reductase converts nucleotides to deoxynucleotides, by
removing the 2' hydroxyl group on the ribose ring. The oxidized glutathione is reduced
again by NADPH and glutathione reductase. Glutathione is an electron source for
reduction of sulfate to sulfite. Glutathione is also important for maintaining iron in
myoglobin as Fe2+, and for maintaining the cells proteins in a reduced state (free of
disulfide bonds).
The gamma glutamyl cycle is a means of importing amino acids into cells. This is
important in kidney and liver tissues . (see handout) In this cycle, glutathione is exported
and it serves as a donor of the gamma glutamyl group in a transpeptidase reaction catalyzed
by gamma glutamyl transpeptidase. This is an ecto-enzyme, an enzyme located outside the
cell. The gamma glutamyl group is transferred from cys-gly to the amino acid that will be
imported. A gamma glutamyl amino acid is formed and this is imported into the cell. Once
inside the amino acid is cleaved off in a reaction that cyclizes the glutamate to 5-oxoproline.
This is hydrolyzed to reform glutamate.
On the outside of the cell, the cys-gly dipeptide is also imported back into the cell, where it
is cleaved into cys and gly. All the components of glutathione are now back inside, and the
glutathione is resynthesized, for another round of the cycle.
Glutathione is also involved in detoxification of hydrophobic compounds, by glutathione-
S-transferase. In drug metabolism, there are two phases called phase I, where poorly
soluble compounds are modified (often by cytochrome P450s) to introduce a reactive
group, like a hydroxyl. In phase II, some soluble adduct is made by attaching a compound
like glutathione to the reactive group added in phase I. Glutathione-S-transferase does this
reaction and adds glutathione to activated drugs to make them more water soluble so they
can be excreted.
COMMERCIAL USE OF GLUTATHIONE-S-TRANSFERASE
Glutathione-S-transferase has a high affinity for glutathione, that has been exploited in
protein purification schemes. Pharmacia sells a kit for making GST-fusion proteins with
high expression promoters in E. coli. Your gene is fused to the gene for GST and both are
expressed together with a specific protease site built into the fusion joint. (see handout)
Lysis of the cells releases the protein into a soluble fraction. The mixture is either batch
adsorbed to glutathione Sepharose beads, or it is run over a column of the same material.
The protein fusion binds to the glutathione where it can be washed and then eluted with free
glutathione or it can be released from the beads by proteolysis with the specific protease,
(thrombin or factor Xa). If it is released as the fusion protein, this can be cleaved with the
protease and the GST part can be adsorbed back onto the glutathione Sepharose. Either
way gives you pure protein in just a few easy steps and at high concentrations. Further
enhancements of this technology have added a protein kinase recognition site into the
fusion, so the protein can be specifically labeled with P32.