CHAPTER 25: Nitrogen acquisition and amino acid metabolism

The nitrogen (from Egyptian word nitre for salt) cycle

o Nitrogen in the environment exists primarily in an oxidized state, either as N 2 or nitrate

o
o
o
o
o

(NO3). Biological systems acquire nitrogen by reducing this nitrogen to ammonium ions
(NH4+, only N-containing compound that can be taken into organisms). Ammonium ions are
then incorporated into organic molecules. All of these processes can occur in bacteria, but
not in higher organisms. The oxidation of reduced nitrogen also occurs in nature. Hence
there is a large nitrogen cycle (Fig 25.1).
Nitrate assimilation is the conversion of nitrate to ammonia. It occurs in plants, some
fungi, and some bacteria.
Nitrogen fixation is the conversion of N2 to ammonia. It occurs only in bacteria. Sometimes
these bacteria associate with plants. This is an anaerobic process.
Animals can do neither, so they must acquire nitrogenous compounds from either plants or
bacteria.
Animals release nitrogen in a reduced form: ammonia, urea, or possibly uric acid. This
release occurs to dispose some waste products or as decomposition.
Nitrification. Bacteria then can take the reduced nitrogen, as ammonia, and oxidize it.
Nitrifying bacteria convert ammonia to nitrite and nitrate, and use the energy released for
growth. This process is called nitrification because it makes the nitrogen more readily
available to plants. Nitrate is more soluble than ammonia, and plants absorb it more quickly.
Denitrification is the conversion of nitrate to N2, which is released to the atmosphere. It is
done by denitrifying bacteria, which are very efficient. The function of this process is to
provide bacteria with an electron acceptor other than oxygen for energy generation. This is
an anaerobic process. Oxygen is a preferred electron acceptor, and shuts this off in E. coli.
Furthermore, oxygen interfers with this process.

o
o Note: the atmosphere is an oxidizing environment
o Note: fertilizer is NH4NO3
25.1 Biological N acquisition.
o There are two routes of such acquisition: nitrate assimilation (over 99% of N incorporation)
and nitrogen fixation (less than 1% of N incorporation, but most important). Both produce
ammonia.
o Nitrate assimilation occurs in two steps.
The first step is a two-electron reduction of nitrate to nitrite, catalyzed by nitrate
reductase.
NO3- + 2 H+ + 2 e- NO2- + H2O
Nitrate reductase transfers a pair of electrons via four protein-bound
intermediates to nitrate.
o NADH to SH to FAD to cyto b557 to MoCo and finally to nitrate.

MoCo is a molybdenum-containing cofactor (see Fig 25.2a) MoCo is a

cofactor for a variety of hydroxylase-type reactions: xanthine DH,
aldehyde oxidase, sulfite oxidase.
The second step is a six-electron reduction of nitrite to ammonia, catalyzed by nitrite
reductase.
NO2- + 6 H+ + 6 e- NH4+ + 2 H2O
Nitrite reductase (plants) obtains electrons from photosynthetically reduced
ferrodoxin. Electrons transferred to a 4Fe-4S cluster to siroheme (heme-like
group in plants and bacteria)(Fig 25.2b) and finally to nitrite, which binds the
siroheme and accepts electrons from it. Microbial nitrite reductases have more
complex electron transfer pathways.
Nitrogen fixation reduces N2. The enzyme nitrogenase is only found in bacterial cells. Eight
electrons are transferred, and one molecule of H2 is obligatorily produced.
N2 + 10 H+ + 8 e- 2 NH4+ + H2
It is the only mechanism that provides direct access to the vast reserves of N 2 gas in
the atmosphere.
N2-fixing bacteria can associate w/ higher plants - important b/c N availability often
limits plant growth.
All nitrogenases require the enzyme nitrogenase, a strong reductant (such as reduced
ferrodoxin strongest biological reductant), ATP, and O2-free conditions (O2 inhibits
nitrogenase; why bacteria can do this and plants dont).
enzyme contains 2 components: Fe-protein (nitrogenase reductase) & MoFe-protein
(nitrogenase)
The reductase hydrolyzes two ATP per electron transferred, or 16 ATP (rmr, 8
e- transfer) per N2. The ATP is used for overcoming the activation energy
required to break the N2 bonds (Fig. 25.4). This component has a single 4Fe4S cluster, and is very O2 sensitive (aka very readily oxidized).
The nitrogenase component is an 22 protein. Each dimer contains a Pcluster (8Fe-7S cluster see Fig 25.5a) and the FeMo-cofactor (a 7Fe-1Mo-9S
cluster see Fig 25.5b). This component is also O2 sensitive.
The reaction involves a complex electron transfer. It starts with reduced ferrodoxin
(see Table 20.1 on p 643 for its redox potential). The reductase is the electron donor.
ATP hydrolysis is coupled to electron transfer from the reductase to the nitrogenase
component. The process is slow, about 3 N2 per second, so that cells may contain
large amounts of the protein, about 5% of total.
o

Two factors commonly control nitrogenase

ADP inhibits activity (ATP is substrate; w/out energy, it cant proceed)

NH4+ inactivates a transcriptional activator (b/c NH4+ is product)
Some organisms have a third mechanism, ADP-ribosylation of the nitrogenase
reductase subunit (which is covalent modification of a subunit)
25.2 The fate of ammonia (ammonium ion): ammonia assimilation.
o Enzymes of ammonia assimilation
Once environmental nitrogen is reduced to ammonia, the ammonia must be
assimilated.
primarily done by bacteria, which have only two enzymes of ammonia
assimilation: glutamate dehydrogenase (GDH) and glutamine synthetase (GS).
(Carbamoyl phosphate synthetase does not assimilate ammonia in bacteria,
but it does in higher organisms, as part of the urea cycle, which is a
mechanism of nitrogen disposal.)
GDH catalyzes the reductive amination of -ketoglutarate, with NADPH as the
reducing agent

NADPH is in excess over NADP, so that the reaction goes in the synthetic
direction.

(Degradative GDHs will use NAD. NAD is in slight excess over NADH, and the
reaction with this cofactor degrades glutamate. In vertebrates, the catabolic
enzyme is activated by ADP and inhibited by GTP. This suggests that its
function is to provide citric acid cycle intermediates for energy.)
GS catalyzes the amidation of glutamate

This reaction requires ATP, which phosphorylates the carboxyl group

o This will be a general pattern for the acidic amino acid families
(glutamate and aspartate): the carboxyl group will be activated before
a modification.
Pathways of ammonia assimilation.
The functions of these pathways are two-fold
First, these pathways incorporate inorganic nitrogen into organic compounds
(assimilation).
Second, as will become apparent, these pathways also result in the synthesis
of the intracellular nitrogen donors, glutamate and glutamine. This will be
discussed shortly. It is the function of the pathways of ammonia assimilation
to make both of these compounds.
There are two pathways of ammonia assimilation. The primary differences are that
one requires more energy than the other, and
the former can assimilate a low concentration of ammonia.
The GDH pathway.
synthesizes glutamate and glutamine by the actions of GDH and GS
GDH has a high Km for ammonia (~2 mM in E. coli), whereas GS has a much
lower Km (~0.1 mM).

With high ammonia (which is not normal in nature), GDH can assimilate
ammonia into glutamate, and GS can assimilate ammonia into glutamine.
The GS-glutamate synthase pathway

When ammonia is low, which is normal in nature, this pathway first

synthesizes glutamine via GS from ammonia and glutamate.
Then, glutamate synthase, uses glutamine as a nitrogen donor for the
formation of glutamate
The difference in this pathway is energy consumption.
o GDH pathway does not need to consume ATP for glutamate synthesis.
o GS-glutamate synthase pathway does
o It can be calculated for E. coli that the difference in energy is 10% of
the cells total energy requirement.
o The intracellular nitrogen donors, and their relative quantitative importance.
In considering these pathways, they are not used as described in the book
With high ammonia, the cell needs 75% of its nitrogen as glutamate, and 25% as
glutamine.
GDH pathway assimilates 75% of the ammonia, and

GS 25%
With low ammonia, the GS-glutamate synthase pathway and GS assimilates 100% of
the ammonia, and
converts 75% of the glutamine made to glutamate.
25.3 Regulation of the pathways of ammonia assimilation: control of glutamine synthetase.
o Regulation of glutamine synthetase occurs at three levels:
Feedback inhibition (appears to be competitive inhibitors, but this is controversial)
Covalent modification
Gene expression, which determines how much is made
o GS is a dodecamer (12 subunits), 12 type protein. The enzyme is arranged as two stacks of
hexagons

active site is at the subunit interfaces within each hexagon

Two subunits contribute to the active site
Monomers are inactive.
Inhibition.
Nine compounds inhibit GS activity: Gly, Ser, Ala, His, Trp, CTP, AMP, carbamoyl~P,
and glucosamine-6-P


Gly and Ser will accumulate if the cell has enough purines, which require glutamine
for their synthesis. They are therefore indirect indicators of glutamine status. Why
alanine inhibits is not known. These amino acids bind to the glutamate site.
The other compounds require glutamine for their synthesis, and are endproducts.
Two of them also bind to substrate sites: AMP and carbamoyl~P
The others bind to allosteric sites.
Covalent modification

Each subunit can be adenylylated (add 1 AMP?) at a tyrosine residue. Adenylylation

inactivates GS.
ATase is an enzyme that adds and removes adenylyl groups depending on the
environment. The mechanism of this control is stated incorrectly in the text.
Glutamine allosterically (turns enzyme off) controls a bifunctional regulatory enzyme,
uridylyltransferase (UTase)/uridylyl-removing enzyme (UR) which controls the activity
of another regulatory protein, PII (which is the 2nd protein of a cascade), which in turn
controls ATase activity.

key: unmodified PII shuts the system off

when glutamine is highadd adenyl groups to glutamine
synthetase to turn off
when glutamine is lowregulatory enzyme adds modification
PIIisnt active system is active
With high glutamine, UR is active, which results in PII formation (instead of PIIUMP). PII interacts with ATase and adenylylates GS. This form of PII is called PIIA.
With low glutamine, UTase is active, and PII-UMP is formed. Its interaction with
ATase results in deadenylylation of GS. This form of PII is called PIID.
-ketoglutarate does have an effect, but only with unmodified P II. Therefore,
the story is complicated. Glutamine is the major effector. It is not as the book
describes.
expression is also controlled by UTase/UR and PII
o

Gene

o
High glutamine (nitrogen excess), which results in P IIA, results in interference
of phosphate transfer from NRII (nitrogen regulator II) to NRI (nitrogen
regulator I). NRI~P is a transcriptional activator, and the unphosphorylated
form is inactive. The gene for GS is not expressed.
Low glutamine (nitrogen limitation) has the opposite effect. It results in P IID
formation, and net transfer of phosphate from NRII which phosphorylates itself
and transfers phosphate to NRI. NRI~P is a transcriptional activator that turns
on the gene for GS.
Despite what the book says, it is the level of glutamine that is important, not
the ratio of glutamine to -ketoglutarate.
unmodified PII shuts down gene expression
25.4 Amino acid biosynthesis
o Plants and microorganisms can generally make all the amino acids from ammonia.
o The amino acids are classified by the precursors needed for their synthesis

For example, all members of the -ketoglutarate family require -ketoglutarate. The
precursors for all the amino acids are from intermediates in central metabolism:
glycolysis, the pentose and citric acid cycles.
Mammals can synthesize only about 10 of the amino acids, called the nonessential amino
acids (short pathways)
The ones that cant be synthesized are essential (long pathways), and mammals lack
the pathways

Excess amino acids are not stored (except for in skeletal muscles, but these are
unreachable). Instead, the nitrogen is removed, and the carbon skeleton is reutilized,
often by forming citric acid cycle intermediates.
Nitrogen donor for amino acids is glutamate

TRANSAMINATIONS (electrically and energetically aka reversible neutral)

Amino acid synthesis frequently involves addition of a nitrogen from glutamate

(common N donor) to an -keto acid (usually; the other type of recipient is an
aldehyde) by transamination

The process involves transfer of an -amino group from a donor, usually glutamate,
to the -keto position of an -keto acid.
Amino acid 1 (usually glutamate) + -keto acid 2 -keto acid 1 (usually ketoglutarate) + amino acid 2
The enzymes are named for their amino acid substrates, e.g., glutamate-aspartate
aminotransferases.
The reactions are usually readily reversible. Glutamate is virtually always in excess,
which determines the direction of the reaction.
The aminotransferases require pyridoxal phosphate as an essential cofactor (carries
e- in redox rxn; can be involved in C rearrangements, carboxylations, etc)
The -ketoglutarate family
This family includes glutamate, glutamine, proline, and arginine. In fungi, this family
also includes lysine.(not impt for us; for us, its in the aspartate family)
We already discussed glutamate synthesis as part of the mechanism of ammonia
assimilation.
The central fact about members of this family is that the modifications occur on the carboxyl group of glutamate.
major changes are often at this carbon
o This carboxyl group can have a nitrogen added (glutamine),
o can be replaced by an amino group (ornithine in arginine synthesis), or
o can be reduced to an aldehyde which is reactive (proline).
We already discussed glutamine synthesis. The -carboxyl group is activated by
phosphorylation, and ammonia is added to form an amide linkage.
Proline synthesis follows a similar pattern

The -carboxyl group is activated by phosphorylation.

The activated carboxyl is then reduced to an aldehyde. The resulting
compound is called glutamate-5-semialdehyde. A semialdehyde has the
properties of an aldehyde and a carboxyl group. It is an aldehyde.
The aldehyde spontaneously interacts with the -amino group and cyclizes.
The resulting compound is reduced to give proline.
Both reductions require NADPH.
Arginine synthesis initially follows a similar pattern, at least as far as ornithine
synthesis

The first part of the pathway is the synthesis of ornithine from glutamate.
The first step of arginine synthesis is N-acylation (addition of an acetyl group
to the -amino group of glutamate) to block the cyclization that occurs in
proline synthesis. After that, the steps are similar to those in proline synthesis.

10

The -carboxyl group is activated by phosphorylation, and the activated

carboxyl is reduced to an aldehyde. This would cyclize if the -amino group
had not been blocked.
Now the aldehyde is free to react with something else, in this case, an amino
group is added by an unusual aminotransferase. It should be noted that the
nitrogen is not added to an -keto group, but to a terminal aldehyde.
The next step is removal of the acetyl blocking group, which generates
ornithine.
The second part of the pathway is essentially addition of a urea (one C and 2
Ns) group to the nitrogen at the end of ornithine. This is relevant to the urea
cycle. The urea is added in pieces, starting with one carbon and one nitrogen
from carbamoyl-P (Fig 25.22).
o Carbamoyl-P is unusual since it consumes two ATPs. CPS-I is a
mitochondrial enzyme in higher organisms, probably because this is
where the ATP is. It uses ammonia as a nitrogen source. E. coli has only
one CPS, and it uses glutamine as a N source. The carbon source is
bicarbonate (HCO3).
Ornithine transcarbamoylase adds carbamoyl-P to ornithine to form citrulline.
This is also a mitochondrial enzyme.
The next steps replace an oxygen with a nitrogen. The oxygen of the ureido
group is activated by AMP addition, and aspartate displaces the AMP. The
resulting compound is cleaved, leaving behind the nitrogen of aspartate. Note
that aspartate is a nitrogen donor here. Aspartate is a nitrogen donor in only
three reactions: this one, and two in purine synthesis.

11

The urea cycle

This cycle was discovered by Hans Krebs before the citric acid cycle. Most of
the steps are part of arginine synthesis.

It is a mechanism to remove nitrogen. The nitrogen can be ammonia

(generated from a catabolic GDH, which will deaminate glutamate, which is
generated from aminotransferases) and aspartate. The ammonia and
bicarbonate are incorporated into carbamoyl-P.
The additional step that is not part of arginine synthesis is catalyzed by
arginase, which converts arginine to ornithine and urea.
Elevated amino acid degradation increases glutamate, ammonia, aspartate
(via an aminotransferase), and N-acetylglutamate. The latter compound
stimulates CPS-I activity.

12

It should be noted that this is an energy-intensive process.

The process is confined to the liver.

Lysine synthesis in fungi

To be honest, this is not important. However, the pathway is interesting.

The first five steps are essentially the citric acid cycle from oxaloacetate to ketoglutarate and then to glutamate, except that -ketoglutarate takes the
place of oxaloacetate. The carbon skeletons are one carbon longer. The logic
of the sequence is to add a carbon by adding acetyl-CoA followed by a
decarboxylation.

13

Acetyl-CoA is added to -ketoglutarate by a citrate synthase-like

reaction (forming homocitrate), followed by an aconitase-like reaction
(forming homoisocitrate), then an isocitrate DH-like reaction (forming
-ketoadipate), and finally an aminotransferase reaction (forming aminoadipate), which makes a compound with one more carbon than
glutamate (not quite a GDH-like reaction).
The next three steps are activation of the terminal carboxyl group (seen that
before), a reduction to a semialdehyde (seen that before), and an elaborate
mechanism to add a nitrogen.
o

The aspartate family.

The members are aspartate, asparagine, lysine (in bacteria), methionine, threonine,
and isoleucine.
Like glutamate family amino acids, almost all the chemistry is at the terminal
carboxyl group, and this requires activation of the carboxyl group by similar
mechanisms.
Aspartate itself is formed by transamination of oxaloacetate

Aspartate is a nitrogen donor in arginine and purine synthesis

required for pyrimidine synthesis
is withdrawn from the cycle which requires the aneuplerotic reactions.
Asparagine is synthesized in a reaction similar to the GS reaction

The carboxyl group is activated with ATP forming aspartyladenylate.

o All organisms have glutamine as the nitrogen donor.
o Bacteria can have a second enzyme that can directly use ammonia. In
this case, for example E. coli, the organism has two enzymes.

14

Threonine, methionine, and lysine are derived from a set of common

intermediates

For all of these amino acids, aspartate is first phosphorylated on the carboxyl
group, to form aspartyl phosphate.

15

Three aspartokinases catalyze formation of aspartyl-phosphate.

o Each is inhibited (enzyme activity) and repressed (gene control) by a
different endproduct

The result of this control is that one endproduct does not stop
synthesis of intermediates that are also required for other amino acids.
o Each amino acid also inhibits the first unique reaction for each amino
acid.
Aspartyl semialdehyde is formed from reduction of aspartophosphate by
NADPH. Note the similarity to the glutamate family members.
Lysine from aspartyl-semialdehyde
o In terms of carbon atoms, lysine has two more than aspartate
To get the number of carbons right, pyruvate (3 carbons) is
added to aspartyl semialdehyde
o The next reactions remove a carbon (a decarboxylation at the very
end), remove an hydroxyl group (via a dehydration and reduction), and
add a nitrogen
o The nitrogen addition is complex. There is an -keto group, but if an
aminotransferase were to act on the intermediate, it might remove
another -amino group instead. Therefore, the existing -amino group
is blocked, the nitrogen added by transamination, then the first amino group is unblocked.
Homoserine (serine with an extra CH2) is formed from NADPH-dependent
reduction of aspartyl semialdehyde. Homoserine is a precursor for both
methionine and threonine.
Threonine from homoserine
o Threonine and homoserine differ in the position of the OH. It is moved
by activation of the C4 OH
Methionine from homoserine
o Methionine differs from homoserine in that it has an SH instead of an
OH, and a methyl group is added to the SH.
o The first three steps of methionine synthesis are simply replacing the
OH with an SH.
First, the OH is activated by succinylation with succinyl-CoA.
Then cysteine, the universal sulfur donor in bacteria, replaces
the succinyl-CoA.
The cysteine is then removed, except for the SH which remains
behind. This produces a compound called homocysteine, this is
one carbon longer than cysteine.
o The last step is addition of a methyl group using a THF derivative, N5methyl-THF.
o Methionine has several important functions.
o The first amino acid for proteins.
o In proteins, the S group can be oxidized, and methionine residues
surround and protect active sites from oxidative damage.
o The third function is as a precursor for S-adenosylmethionine
(SAM), a methyl donor
SAM can also be a donor of propylamino groups
Note that the methyl group comes from a THF derivative.
o

16


Homocysteine and heart disease (p 834)
o Elevated homocysteine in the blood may be an independent risk factor
for heart disease.
o There are two forms of homocysteinuria:
o One results in elevated homocysteine and methionine. This one results
from a block in the conversion of homocysteine to cysteine. It is easily
cured by vitamin B6 therapy.
o A second results from high homocysteine, but normal methionine. This
is because of a block in converting homocysteine to methionine. It is
cured with vitamin B12 therapy.
o Folic acid (the precursor for THF) therapy could also help.
o Both forms of homocysteinuria result in extraordinary cardiovascular
damage. It is thought that homocysteine, S-adenosylhomocysteine, or
a compound derived from homocysteine is destroying the arteries.
The pyruvate family
Family members are alanine, valine, leucine. Isoleucine is a member of the aspartate
and pyruvate family.
Alanine is synthesized by a glutamate-alanine transaminase in all organisms.

17

Valine and isoleucine share the enzymes that catalyze the last four reactions of
their synthesis
o They differ by one methylene group.
o
pathways essentially begin with an -keto acid, pyruvate or ketobutyrate, respectively.
o

-Ketobutyrate is made from threonine by threonine deaminase. It is feedback

inhibited by isoleucine.
The next reaction for both isoleucine and valine synthesis is addition of a twocarbon fragment from hydroxyethyl-thiamine pyrophosphate (TPP), with
pyruvate providing the carbon. Other TPP-containing enzymes include
pyruvate DH, and transketolase.
The next step is a reduction and a carbon migration (this is difficult to see in
the book).
The third step is a dehydration to generate the -keto acid.
The last step is transamination.
The regulation is much more complicated than suggested by the book. We can
ignore it, except for the threonine deaminase regulation.

18

Leucine is at a branch point. Its synthesis requires the next to last intermediate of
valine synthesis

o
o
o

recapitulates TCA cycle

Leucine has one more carbon than valine, and it gets it by mimicking
the first reactions of the citric acid cycle.
The first step is addition of acetyl-CoA.
The second step is an aconitase-like reaction that results in migration of a
hydroxyl group.
The third step is an isocitrate DH-like decarboxylation that generates an keto acid.
Finally, the last step is a transamination.
The 3-phosphoglycerate family (from glycolysis): serine, glycine, cysteine, and
sulfur assimilation.
These amino acids are derived from 3-phosphoglycerate, a glycolytic intermediate.

19

Serine synthesis starts from oxidation (NAD as electron acceptor) of 3-PG to an keto acid. The -keto acid is transaminated to form 3-phosphoserine, and
dephosphorylated to generate serine. Serine inhibits the first enzyme of the pathway.

Glycine (two carbons) is made from serine (three carbons) in one step

The extra carbon from serine is donated to the C1 carrier, THF to form N5, N10methylene-THF. This reaction, serine hydroxylmethyltransferase, requires
pyridoxal phosphate.

20

Cysteine synthesis proceeds from serine, usually in two steps in bacteria

The reaction mechanism involves replacement of an OH with an SH group.

This is accomplished by first activating the hydroxyl group by acetylation with
acetyl-CoA.
o This reaction, the first committed step in cysteine synthesis, is
inhibited by cysteine.
The next step is sulfhydrylation by H2S, which replaces the acetyl group.
The sequence of reactions is the primary mechanism of sulfur assimilation.
Cysteine is the major donor of sulfur for reactions in bacteria (more
complicated in higher organisms, where methionine can provide the sulfur)
Sulfate assimilation

21

Sulfur in the environment is available as an oxidized compound, sulfate (b/c

envir. is oxidizing)
H2S is toxic (and reduced sulfur), and is rapidly consumed by bacteria, as a
reduced compound that can be used for energy generation.
Sulfate is reduced after activation of the sulfate by ATP to form APS and PAPS.
Thioredoxin is then used as a reductant (aka reducing agent) to reduce sulfate
to sulfite, and
then NADPH reduces sulfite to sulfide.
The aromatic amino acids
Chorismate (comes from Greek for branch), shikimate, and the functions of
the common pathway

22


All the aromatic amino acids are derived from chorimsate.
Chorismate is synthesized in seven steps from erythrose-4-P, an
intermediate of the pentose cycle, and 2 molecules of PEP.
o These seven steps can be considered a common pathway, which is
called the shikimate pathway.
o It is named after the first intermediate of the pathway that was
discovered.
The function of this pathway is to synthesize a compound that is nearly
aromatic.
The endproduct, chorismate, is also a precursor for several other aromatic
compounds; for example, folic acid, coenzyme Q, vitamin E. In some
organisms, such as plants, the carbon flow through this pathway is enormous,
resulting in the synthesis of lignin, which can be 35% of the dry weight of
higher plants. It is difficult to degrade because of the stability of the aromatic
ring.
o
o

23

The logic of the pathway is to provide an unsaturated ring structure, followed

by desaturation, and addition of a side chain from the second PEP.
The first step of the common pathway is regulated in a fashion similar to that
of the common pathway of the aspartate family.
o There are three enzymes that catalyze the first step in E. coli, and each
of the amino acid endproducts inhibits and represses one of the
enzymes. The inhibition is only partial because amino acids are not the
only compounds made from these pathways.
Phenylalanine and tyrosine (Fig 25.37). Both amino acids have prephenate as an
intermediate.

For both amino acids, CO2 is removed from chorismate, and for phenylalanine,
an OH is removed.
In both cases, the next step is a transamination with glutamate as the
nitrogen donor.
The two amino acids partially inhibit the common pathway at the first step,
and they also inhibit the first committed steps, i.e., the synthesis of
prephenate.
Some organisms can synthesize tyrosine from phenylalanine

24

o
o

This requires a hydroxylation with O2 as the oxygen donor,

tetrahydrobiopterin (a THF derivative), and NADPH to reduce the
oxygen to the OH level.
o Tyrosine is classified an a nonessential amino acid, as long as
phenylalanine is provided in the diet. Phenylalanine is essential.
Some individuals lack the monooxygenase that catalyzes phenylalanine
hydroxylation. Dietary phenylalanine is not converted to tyrosine, and is
instead deaminated. This results in accumulation of phenylpyruvate in the
urine, phenylketonuria. Mental retardation and other neurological disorders
develop without dietary intervention (a low phenylalanine diet).
Tryptophan

Synthesis requires phosphoribosyl-PP (PRPP) and serine, both of which provide

carbons. Tryptophan inhibits the first step
Otherwise the pathway is unique and complex, and that is all you need to
know about it.

25

Histidine

is in a family of its own.

26

unusual pathway which requires PRPP and ATP as carbon precursors.

fifth step is unusual in that it results in ring closure, which is required for formation of
the imidazole ring of histidine, and release of an intermediate in purine synthesis
from the partially altered ATP
ATP uses C and N from purine (6-membered ring) to from histidine
Histidine inhibits the first reaction of the pathway, and represses the synthesis of all
enzymes in E. coli.
Herbicides (weed killers) and amino acid analogs. Plants can synthesize all 20 amino acids.
Inhibitors that target precursors of our essential amino acids should not be toxic to us.
o Glyphosate (Roundup) is a PEP analog that targets the shikimate pathway of aromatic amino
acid synthesis.
o Sulmeturon (Oust) methyl inhibits an enzyme required for both valine and isoleucine
synthesis.
o Aminotriazole (Amitrole) blocks histidine synthesis.
o Phosphinothricin (PPT) inhibits glutamine synthetase. It is rapidly removed by the kidneys,
and does not cross the blood-brain barrier.

TAs review
Histidine
ATP provides
IN & IC
(~PRPP)

-ketoglutarate
Glutamate
Glutamine
Proline
Arginine
Features
See Notes

aspartate
Lysine (bact)
Aspartate
Asparagine
(Precursor to
aspartate)
Aspartokinase- 3
isozymes
havediff. prod.
that inhibits

pyruvate
Valine
Alanine
Isoleucine
leucine

3-P-glycerate
Serine
Glycine
Cysteine
(Reitzer
teaches this)
e.coli
cys = Sdonor of the
cell since
sulfate
sulfide in cys.

aroma
Chorismat
Derivat
Phenylalan
Tyrosine (n
essen
tryptopha

PKU can
conv. F toT
mental
retardatio

See Notes
AA precursors: TCA, glycolysis, pentose cycle
Common Steps Synthesis requires activation by phosphorylation, usually followed by
reduction (except glycine, etc.)
o THF = tetrahydrofolate single C carrier
o herbicides analogs to block aa synthesis; wont affect humans
o degradation
remove N ( amino) first, usually by transamination
inability to degrade Tyr alkaptonuria
Amino Acid Degradation: degradation of the carbon skeleton, N removal, inborn errors
o Degradation of the carbon skeleton
Higher organisms obtain their amino acids (many of them anyway) from their diet in
the form of peptides or proteins
These are degraded to amino acids, and either incorporated into protein, or
degraded.
o Amino acids can serve as energy sources
only provides about 10% of energy in humans
During starvation, skeletal muscle can be degraded to amino acids to provide energy.
o Synthesis of amino acids proceeds from a few compounds in central metabolism (glycolysis,
the pentose cycle, and the citric acid cycle).
Degradation of amino acids results in synthesis of a few compounds in central
metabolism: acetyl-CoA, succinyl-CoA, pyruvate, -KG, fumarate, OAA, and
acetoacetate
o
o

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degradation productions either directly eneter TCAcycle or become precursors

entering the cycle
responsible for knowing 2 types of aa degradation products/overall scheme
o dont need to know which aa goes where
All but acetate and acetoacetate can be converted to glucose.
2 types of C skeletons from aa degradation:
The glycogenic amino acids are those that can be converted to glucose.
Those degraded to acetate or acetoacetate are ketogenic, and can be used
to synthesize fatty acids or ketone bodies.
Nitrogen removal. The carbon skeletons of the amino acids can be degraded. An early
step in such catabolism is removal of the nitrogen.
The first step is often loss of the nitrogen by transamination (often), with -KG as the
acceptor, which generates glutamate.
The nitrogen of glutamate can be oxidatively deaminated by glutamate
dehydrogenase, which generates -KG and ammonia (Fig 25.4 from Zubay).
NAD is the cofactor.
The ammonia can enter the urea cycle in the formation of carbamoyl-P.
This GDH is mitochondrial, like carbamoyl-P synthetase. The synthetic GDH is
cytosolic.
An alternate fate of the glutamate nitrogen is donation to OAA, which forms
aspartate.
Aspartate provides a nitrogen for arginine synthesis during the urea cycle (Fig
25.9 from Zubay).
The aspartate forms fumarate, which can be reconverted to OAA.
Urea is just one of three different endproducts of nitrogen metabolism that are
excreted. The other two are ammonia (fish/bacteria) or uric acid (birds/reptiles) (Fig
from Zubay).
Catabolic pathways and diseases of defective amino acid degradation. (know well)
Phenylalanine and tyrosine (Fig. 25.47).
We have already considered one defect in phenylalanine catabolism, which
resulted in phenylketonuria.
failure to synthesize Tyr fro Phenylalanine causes a build up of phenylalanine
Another disease of amino acid metabolism is alkaptonuria.

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caused by a block in one enzyme of tyrosine catabolism.

results in darkening of the cartilaginous tissues, and urine that turns black on
exposure to air.
Feeding of phenylalanine results in increased secretions of homogentisic acid
A London pediatrician in 1908 proposed a defective enzyme, and that the
disease was inherited as a Mendelian recessive. In other words, he was
proposing that genes code for proteins (1st time Mendel and humans were
associated)
The 1958 Nobel Prize was awarded to Beadle and Tatum who proposed the
one gene-one enzyme hypothesis. They noted that their hypothesis was
implicit in Garrods work.
There are several inherited diseases in which amino acid catabolism is impaired
(Table from Zubay).
Extra
Beriberi
o Vitamin B1 deficiency from thiamine deficiency
o 1st seen in chickens eating polished rice
no disease associated w/ synthetic pathways we have b/c they would be lethal

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