Amino acid metabolism

Amino acid metabolism

(KEGG pathway MAP01150 common amino acids for protein biosynthesis)
(KEGG pathway MAP01160 non common amino acids)

Amino acid metabolism is complex because of the large number of metabolites involved. Amino acid metabolism
can be split into those 20 amino acids used for protein biosynthesis. They also function as precursors for the
synthesis of many signaling molecules (see neurotransmitter and nitric oxide chapter). They are distinct from the
unusual amino acids (e.g. ornithine) used for a large variety of intermediary pathways and activated one-carbon
units used for the synthesis of aromatic, nitrogen containing compounds such as nucleic acids and all its cofactor
derivatives like nicotinamides and coenzyme A, ubiquinone, heme and chlorophyll.

Degradation

Because of the multitude of pathways, only a selected few are presented here demonstrating basic principles found
in all other metabolic pathways of nitrogenous compounds. Amino acids serve as precursors for lipids,
carbohydrates, and nucleic acids including ribonucleotides used as cosubstrates and coenzymes in the production of
energy (ATP, NAD, FAD, CoA). Following the metabolic fate of carbon atoms of dietary amino acids, they can be
traced to all major metabolic intermediates because of the close interaction of amino acid metabolism with both the
citric acid cycle and glycolysis/gluconeogenesis. These intermediates containing carbons from dietary amino acids
include pyruvate, acetyl-CoA and acetoacetyl-CoA (ketone bodies), and the citric acid cycle intermediates
ketoglutarate, succinyl-CoA (heme synthesis), fumarate, malate, and oxaloacetate.

Amino acid metabolism is 'separated' into pathways according to the different length of carbon structures involved.
These are referred to as the C3, C4, and C5 families of amino acids. which produce common end products during
catabolism. The C3 family includes alanine, serine (glycine), and cysteine, all of which are degraded to pyruvate.
They are glucogenic amino acids because they can directly be utilized by the liver gluconeogensis (except their
amino groups which are excreted as urea). The C4 family of amino acids includes aspartate and asparagine, which
are degraded to oxaloacetate and are closely linked to glutamate and alpha-ketoglutarate interconversion by amino
transferases. The C4 amino acid threonine has a separate pathway leading to pyruvate and is a glucogenic amino
acid. The C5 family of amino acids includes glutamine, proline, arginine, and histidine, all of which are converted
ultimately to glutamate, which is deaminated to alpha-ketoglutarate.

The non-polar C4 amino acids methionine, isoleucine and valine are precursors for the synthesis of odd numbered
fatty acids via the intermediate propionyl-CoA (a C3 acyl-CoA). Propionyl-CoA can be reused for succinyl-CoA
(C4) synthesis (carboxylation) which in turn serves as heme precursor. The non polar amino acid leucine, however,
undergoes a more complex degradation pathway, including a decarboxylation-carboxylation detour leading to the
formation of acetyl-CoA and acetoacetyl-CoA (a ketone body).

Metabolism of aromatic amino acids

Aromatic amino acids include phenylalanine, tyrosine and tryptophan. Phe and Tyr are closely related. They contain
a benzene ring which is hydroxylated in tyrosine. Tyrosine is synthesized directly from the essential amino acid
phenylalanine. Tryptophan contains a conjugated indole ring and its metabolism is linked to that of vitamin B
(niacin C00253). These metabolic relations give rise to an intricate nutritional dependence. For example, a high level
of dietary tyrosine relieves the need for essential phenylalanine. Also, metabolic disorders like the impairment of
synthesizing tyrosine from phenylalanine makes the former an essential amino acid. This lack of amino acid
biosynthetic pathways in humans is the cause of many diseases associated to malnutrition. Pellagra is a vitamin
deficiency syndrome caused by an inadequate supply of niacin (vitamin B) because of problems in the pathway
leading from tryptophan to niacin synthesis. Vitamin C (C00072), which is a necessary coenzyme in tyrosine
metabolism, or vitamin B6 (C00250), which is required for tryptophan metabolism, cause deficiencies in the
metabolism of aromatic amino acids.

Degradation of aromatic ring structures is mostly performed in liver, also many specialized cells can use the benzene
and indole rings for the synthesis of more complex, biologically important molecules such as heme, pigments, and
hormones.

Phenylalanine
(KEGG pathway MAP00360)

Phenylalanine is first converted to tyrosine by the addition of a hydroxyl unit. This reaction is catalyzed by
phenylalanine hydroxylase (EC 1.14.16.1). This is a liver specific enzyme and belongs to the group of
monooxygenases. The reaction requires molecular oxygen, NADPH, and the coenzyme tetra-hydrobiopterine
(C00272). Note that the hydroxylation of the benzene ring is used to destabilize it preparing the ring structure for
breakup. The cofactor hydrobiopterine is oxidized during tyrosine formation. It is converted back to its reduced form
by dihydrobiopterin reductase (EC 1.6.99.7). The reaction of phenylalanine hydroxylase itself is irreversible.

Tryosine
(KEGG pathway MAP00350)

Tyrosine degradation is a liver resident process. It starts out with the transfer of its amino group to alpha-
ketoglutarate by tyrosine-glutamate aminotransferase (EC 2.6.1.5). This enzyme is specific for both tyrosine and
phenylalanine. Other L-tyrosine accepting transaminases are less substrate specific, including aspartate
transaminase (EC 2.6.1.1) which acts on aspartate, tyrosine, phenylalanine, and tryptophan. This latter enzyme
isoform is obtained from aromatic-amino-acid transaminase (EC 2.6.1.57) by controlled proteolysis. The
degradation intermediate of this transaminase reaction is 4-hydroxy-phenylpyruvate (C01179) which in turn is
oxidized in the presence of vitamin C to homogentisic acid (C00544). This reaction is catalyzed by 4-
Hydroxyphenylpyruvate dioxygenase (EC 1.13.11.27). The ring structure of homogentisate is subsequently broken
and the linear C8 unit degraded in two reaction steps to fumarate and acetoacetate, one citric acid cycle intermediate
and one ketone body.

Tyrosine transferase (EC 2.6.1.5) is under hormonal control. Glucocorticoids stimulate amino acid degradation in
liver which yields fumarate and acetoacetate. Fumarate is converted to oxaloacetate which stimulates
gluconeogenesis. Thus liver can synthesize and export glucose into the blood plasma. Acetoacetate is secreted and
used by neurons and muscle tissue for oxidative degradation. In enteric bacteria like E.coli, the major degradation
product of tyrosine (phenylalanine) is succinate. Also phenol, pyruvate, and free NH 3 can be derived by the action of
tyrosine-phenol-lyase.

Tryptophan
(KEGG pathway MAP00380)

Tryptophan degradation in human also yields precursors for glucose synthesis. The first step, catalyzed by
tryptophan oxygenase (EC 1.13.11.1), is controlled by cortisol (a glucocorticoid). Tryptophan stimulates its own
degradation by allosterically activating tryptophan oxygenase. This is an example of a feed-forward mechanism. 
The product of the oxygenase reaction is L-Formylkynurenine (C02700), which is further degraded by kynurenine
formidase (EC 3.5.1.9). The products are formate and kynurenine (C00328). The latter is degraded to 3-
hydroxyanthranilate (C00632) and alanine. This step requires the presence of the cofactor vitamin B6 (pyridoxal).
Hydroxyanthranilate can further be decarboxylated to form acetoacetate. Most of hydroxyanthranilate (95%) is used
for ketone body formation. The remaining 5 percent go into pyrimidine synthesis (nucleic acid synthesis).
Tryptophan is a minor source for the synthesis of NAD (nicotinamide dinucleotide). Most of the nicotinamide is
derived from niacin. A daily supply of NAD(P) can be obtained from ~1mg of niacin, while it takes 60mg of the
amino acid tryptophan to accomplish the same.

Synthesis
Amino acid synthesis includes the fixation of nitrogen in form of ammonia and the assimilation of the latter into
keto acids to form amino acids by means of glutamate dehydrogenase and glutamine synthetase. The keto acids are
provided by glycolysis and citric acid cycle. In total, there are six anabolic pathways for amino acids referred to as
biosynthetic families. Many non essential amino acids can directly be obtained by transamination from glutamate to
the respective keto acid (e.g. pyruvate to alanine; oxaloacetate to aspartate). Essential amino acids have to be
obtained from dietary proteins. Plants and micororganisms have all pathways for the net synthesis of amino acids
needed for protein biosynthesis. The synthesis of aromatic ring containing amino acids is discussed below.

Essential amino acids Non-essential amino acids

Histidine Alanine
Isoleucine Arginine
Leucine Aspartate
Lysine Asparagine
Methionine Cysteine
Phenylalanine Glutamate
Threonine Glutamine
Tryptophan Glycine
Valine Proline
  Serine
  Tyrosine

Synthesis aromatic amino acids in E.coli

(KEGG pathway MAP00400)

Only microorganisms and plants have the capacity to synthesize aromatic amino acids. Starting from intermediates
of glycolysis, the pentose phosphate pathway and/or photosynthesis (calvin cycle), these organisms form the
intermediate shikimate (C00493) in several steps. First, shikimate is converted in three steps to chorismate
(C00251), which serves as the committed precursor for the synthesis of all three aromatic amino acids, but also for
ubiquinone and folate synthesis. The enzyme chorismate mutase (EC 5.4.99.5) catalyzes an intramolecular
transferase reaction (isomerization) to form prephenate (C00254), a precursor of tyrosine and phenylalanine.
Alternately, chorismate is converted to anthranilate in the presence of glutamine by anthranilate synthase
(systematic name: chorismate pyruvate lyase; EC 4.1.3.27). This reaction commits chorismate towards the synthesis
of thryptophan.

Regulation

Amino acid biosynthesis is under allosteric feed back regulation. In general, the end product of a pathway, the amino
acid, inhibits the enzyme catalyzing the first (or committed step) of its own biosynthetic pathway. This ensures the
energy saving synthesis of building blocks for protein biosynthesis. Accumulating amino acids thus shuts down their
biosynthetic activity. A simple feed back mechanism is found in E.coli. Here, isoleucine, which is derived from
threonine, inhibits threonine deaminase (EC 4.2.1.16), the committed step in isoleucine biosynthesis which forms
alpha-ketobutyrate or oxo-butanoate.

For the biosynthetic pathways of more complex and branched aromatic amino acids, however, an analogous
sequential feedback mechanism has been elucidated, as found in the bacteria Bacillus subtilis. Simply put, the
endpoint of each branch inhibits the first enzymatic step of the immediately preceding branching point. Thus, the
aromatic amino acids do not only inhibit their early common pathways leading to shikimate, chorismate, or
prephenate intermediates. This assures flexibility for the cell by adjusting the levels of aromatic amino acids
according to actual needs. Protein synthesis may need different amounts for phenylalanine than tryptophan. Thus
tryptophan may shut down its own biosynthetic branch by inhibiting anthranilate synthase, leaving chorismate
mutase unaffected. Chorismate will still be used for phenylalanine or tyrosine formation before it accumulates to
shut down the entire pathway.
Although E.coli uses the same pathways for the synthesis of aromatic amino acids, it uses a different control
mechanism to ensure relative independence between the formation of phenylalanine, tyrosine, and tryptophan.
Instead of using this simple sequential feedback mechanism after branching points, E.coli relies on enzyme
multiplicity meaning that E.coli uses three different enzymes (2-Dehydro-3-deoxyphosphoheptonate aldolase) for
the early synthesis of shikimate. Each enzyme is under allosteric control of its 'own' amino acid end product. Thus
the level of enzymes in the cytoplasm determine the level of shikimate and chorismate.

For phenylalanine and tyrosine, an additional enzyme multiplicity control is used to allosterically suppress two
isoforms of chorismate mutase, one of which is specific for Phe, while the other binds only Tyr. Overall, enzyme
multiplicity, too, shows a sequential feedback mechanism because of the multiple branching points of the pathway.

Metabolism of sulfur containing amino acids

(KEGG pathway MAP00271 for methionine)
(KEGG pathway MAP00272 for cysteine)

Two sulfur containing amino acids are used in proteins   -   methionine and cysteine. Methionine is an essential
amino acids for humans.  It contributes to the hydrophobicity of a protein.  Its sulfur is non reactive. Cysteine in
contrast has a highly reactive sulfhydril group which is the cause for disulfide bridges (cysteine pairs) in
extracellular proteins  (cysteine pairs don't form in the cytoplasm, which has a reducing environment). Cysteines can
be part of catalytic sites of proteins or serve as metal ion binding sites, e.g. for Zn coordination in the DNA binding
protein domains called Zn-fingers.

The metabolism of both sulfur containing amino acids is closely related. Methionine is a precursor for cysteine
formation in human liver. Methionine, although it cannot be regenerated from cysteine, can be recycled through
methylation of homocysteine (C00155) using a single carbon (methyl-) donor such as betaine (from choline), folate,
or vitamin B12 (C05776; Cobalamin (III))  Homocysteine methylation is catalyzed by 5-methyltetrahydrofolate-
homocysteine methyltransferase (EC 2.1.1.13) tapping into the one carbon pool metabolism of folate and
tetrahydrofolates. Alternately, methionine is regenerated by betaine-homocysteine-S-methyltransferase (EC 2.1.1.5).
The methyl donor betaine (C00719) is a derivative of choline. Choline is directly obtained from phosphatidylcholine
by the hydrolytic activity of phospholipase C (PLC; EC 3.1.4.3). This reaction pathway is linked to glycine and
serine metabolism because the dimethylglycine product is reverted to glycine.

The direct recovery of methionine in liver is part of the activated methyl cycle transferring activated methyl groups
from a THF donor to S-adenosylmethionine (C00019). This major source for methyl compounds, in form of
activated S-adenosylmethionine, comes from dietary methionine. Methionine is linked with ATP to form S-
adenosylmethionine releasing both inorganic phosphate and pyrophosphate. This reaction is catalyzed by the
enzyme methionine adenosyltransferase EC 2.5.1.6.

ATP + L-Methionine + H2O = Orthophosphate + Pyrophosphate + S-Adenosyl-L-methionine

The phosphoester hydrolysis fuels the reaction forming an activated methyl group that can be donated to diverse
acceptor molecules such as nor-epinephrine (see Neurotransmitters) and ethanolamine (see Phospholipids) to form
epinephrine (adrenaline) and choline, respectively. The methyl group is transferred mostly to amino or hydroxyl
groups on the acceptor. The amino acid homocysteine cannot be used for protein synthesis and has to be shortened
by one carbon unit to cysteine in a process known as trans-sulfuration.

In this reaction, serine serves as acceptor. The short lived intermediate cystathionine (C02291; homocysteine -S-
serine) is split in a hydrolysis reaction to homoserine and cysteine. Both synthetase (4.2.1.22) and hydrolyse (EC
4.4.1.1) are vitamin B6 (pyridoxal) dependent. The reactions are irreversible.

In E.coli cysteine is synthesized from serine in two steps. First, serine is acetylated to acetyl-serine by serine
acetyltransferase (EC 2.3.1.30). Acetylserine is converted to L-cysteine by the addition of a sulfhydril group. The
enzyme cysteine synthase A (O-acetylserine sulfhydrylase A; EC 4.2.99.8) catalyzes this reaction.
Glycine and tetrahydrofolatemetabolism
(KEGG pathway MAP00790 for folate)
(KEGG pathway MAP00670 for one-carbon pool)
(KEGG pathway MAP00260 for glycine, serine, threonine)

Tetrahydrofolate (THF, C00101) as discussed above is an important carrier of activated one-carbon units. While S-
adenosylmethionine only transfers methyl groups, THF carries a great variety of activated one-carbon units of
different oxidation states:

most reduced -CH3 methyl

-CH2 methylene
-CH=O formyl
-CH=NH formimino
most oxidized -CH= methylene

Carboxylation (-COOH) cannot be mediated by THF, instead always requires biotin as coenzyme. One-carbon units
are interconvertibel (can be reduced or oxidized; see one carbon pool metabolism) and are activated on THF on the
nitrogens N5 or N10 or both (methylene).

The major one-carbon acceptor molecules are found in the biosynthetic pathways of methionine, glycine, purines,
and deoxythymine. The example for glycine is shown below. Glycine synthase catalyzes a reversible reaction and
thus produces either glycine or one carbon units and ammonium.

Glycine can be regenerated from serine. As the example shows, serine can donate a methylene (-CH2-) unit in form
of an activated N5,N10-methylene-THF, which in turn can be used to synthesize glycine from CO2 and   NH4. More
glycine can be used up to generate more N5,N10-methylene-THF. This is a de novo synthesis of one carbon units
from carbohydrates.

Captain's Log:

When I got ready for my adventure this morning, I had no idea how different this experience would be from the ones I had
already encountered. Unfortunately, I had to leave early in the Krebs Cycle so that I could make it on time for my photosynthesis
appointment. First, our acetyl CoA molecule traveled to find oxaloacetic acid which had four carbons of its own. We formed
citric acid, causing us all to be in a rather bitter mood since there were six carbons in such a small place. I guess one of the
carbons grew sick of the situation quickly because he left and joined oxygen to form carbon dioxide. I felt pretty uncomfortable
in ketoglutaric acid because I was the only carbon left from pyruvic acid. All of the other carbons seemed to know each other
pretty well since they travel in oxaloacetic acid in the Krebs Cycle most of the time. Even though I was interested in seeing what
would happen next, I got kicked out of the group to join some oxygen that was nearby. I really wasn't very upset about having to
leave the Krebs Cycle because I heard that those carbons do not know how to have any fun. They just go through the same
routine everyday for countless times. They are very good at producing energy, but they didn't need me to help them with the ATP
production. Instead, I got to travel through the bloodstream to the lungs, where I was exhaled. It was exciting to leave Tom's body
for the first time because I was all on my own. I've decided to just hang out in the air for a while until I reach my next destination.
I'm planning on going to a plant because I heard that carbon dioxide is always in high demand for something called
photosynthesis. It's kind of like a factory, they are going to take my carbon dioxide molecule and separate us. The oxygen will go
back into the air while I will become part of a sugar just like I was a couple of days ago!

An Overview:

The importance of the Krebs Cycle can not be overstated; almost all of our ATP is produced in this cycle. We can see two main
problems as a result of glycolysis:

1. not enough ATP produced

2. a lack of NAD+ molecules
Remember that the body needs NAD+ molecules to act as electron carriers receiving the extra hydrogen atom and electrons from
G3P to form NADH. Since the cell contains only a small supply of NAD +, there must be a way to recycle NAD+ from NADH to
continue the shuttling of electrons. Sometimes the body reacts by immediately passing the hydrogen atom from NADH to an
organic compound without the aid of an electron transport system. This process is called fermentation. However, fermentation
occurs in an anaerobic environment and will only support the muscles for a short period of time before invoking lactic acid
poisoning. Therefore, our bodies have evolved to "fix" the problems of our energy history. Unlike fermentation, the Krebs Cycle
is able to gather the energy trapped in the NADH molecules and reproduce more NAD +. Also the conversion of NADH to NAD+
is coupled with the production of 24 ATPs.

A Closer Look:

Step 1:

This step consists of the combination of pyruvic acid and coenzyme A to form acetyl CoA. (This is described fully on the
previous page)

Step 2:

Acetyl - CoA, consisting of two carbons, joins oxaloacetic acid, made up of four carbons, to form citric acid, a six-carbon
compound.

Step 3:

One of citric acid's two recently acquired carbons is oxidized to form CO 2. At the same time, a NADH is produced leaving
ketoglutaric acid.

Step 4:

The last carbon remaining from pyruvic acid is removed in the form of CO 2. Another NADH is formed, while the only ATP
directly generated in the Krebs Cycle is produced. Left behind is succinic acid, a four-carbon molecule lacking any of pyruvic's
original carbons

Step 5:

Two hydrogen atoms and their corresponding electrons are released from succinic acid and attach to FAD to form FADH 2. The
remaining molecule is malic acid.

Step 6:S

The continuing oxidation of succinic acid releases another hydrogen atom and two electrons forming NADH. This completes one
turn of the circle as oxaloacetic acid is reproduced and ready to pick up another Acetyl CoA and form citric acid.

Summary:

The Krebs Cycle begins with oxaloacetate and combines with Acetyl CoA to cycle through one complete turn. After Acetyl CoA
is oxidized to CO2 and H2O, the electrons drive proton pumps that generate ATP that is greatly needed by the cell. Remember
that the NADH molecules are important because they contain extracted electrons that ultimately reduce NAD +. However, when
the electrons do not have enough energy to reduce NAD+, they are stored temporarily in the FADH2 molecule, as in Step 5. Each
NADH molecule is responsible for the production of three ATP molecules while FADH 2 is responsible for the production of two
ATP molecules. In the table below, the products of glycolysis, oxidation of pyruvic acid, and the Krebs Cycle are summarized: