AMINO ACID SYNTHESIS:

Amino acids are biologically important organic compounds containing amine and carboxyl
functional groups, along with a side-chain.

- Building blocks of proteins

- Carry out important body functions

About half of the 20 amino acids are biosynthesized from intermediates in the TCA.

By Transamination:

a-ketoglutarate  glutamate

oxaloacetate  aspartate

private  alanine

By direct conversion:

Glutamate  glutamine

Aspartate  asparagines

Glutamate  proline and arginine

Synthesis of Glutamine

Transamination – an amino acid transfers its amino group to a keto acid and is converted itself to
a keto acid and vice versa.

Enzyme: Glutamate dehydrogenase

or

a-ketoglutarate + Gln + NADPH  Glutamate

Enzyme: Glutamate synthase

Synthesis of Glutamate

Glutamate – the most active of all the amino acids in terms of its number of metabolic roles.

Enzyme: Glutamine synthetase

Decarboxylation of glutamate yields ƴ-aminobutyric acid (GABA)

Glutamate is a neurotransmitter.

2 major metabolic fates:

1. Synthesis of glutathione, along with cysteine and glycine

2. Synthesis, via an ATP-dependent conjugating system, of polyglutamate forms of folic

acid and its coenzymes.

Synthesis of Asparagine

Transamination

Asparagine has few metabolic functions aside from serving as a protein constituent.
Or

Enzyme: Asparagine synthetase

Synthesis of Alanine

Alanine synthesisis is a bit of a mystery. Several reactions have been identified, but it has been
impossible to generate an alanine auxotroph and therefore positively identify a required pathway.
There are several pathways and the most likely is formation of alanine by transamination from
glutamate onto pyruvate. A transamination using valine instead of glutamate is also possible.
The glucose-alanine cycle is used primarily as a mechanism for skeletal muscle to eliminate
nitrogen while replenishing its energy supply. Glucose oxidation produces pyruvate which
can undergo transamination to alanine. This reaction is catalyzed by alanine transaminase,
ALT (ALT used to be called serum glutamate-pyruvate transaminase, SGPT). Additionally,
during periods of fasting, skeletal muscle protein is degraded for the energy value of the
amino acid carbons and alanine is a major amino acid in protein. The alanine then enters the
blood stream and is transported to the liver. Within the liver alanine is converted back to
pyruvate which is then a source of carbon atoms for gluconeogenesis. The newly formed
glucose can then enter the blood for delivery back to the muscle. The amino group
transported from the muscle to the liver in the form of alanine is converted to urea in the urea
cycle and excreted.

Synthesis of Lysine, Methionine, Theronine, and Isoleucine

In plants and bacteria, aspartate is a precursor to three amino acids (Met, Thr, Ile) via its
conversion to homoserine.

Aspartic B-semialdehyde is also a precursor to lysine.

 Enzymes involved:

Aspartokinase – major site for regulation of each biosynthetic pathway

- bacteria contain 3 distinct forms of this enzyme
1) One form is inhibited by threonine
2) One from is inhibited by lysine
3) The synthesis of the enzyme is repressed by methionine

Homoserine also has a role in microbial metabolism, unrelated to its function in amino acid
synthesis.

Phenomenon called “quorumsensing”

N-Acylhomoserine lactone (a signalling molecule)

Biosynthesis of Ornithine and Creatine Phosphate

An important reaction of glutamate is the energy-requiring reduction of the ƴ-carboxyl

group, to give glutamic ƴ-semialdehyde.
 Leads toward both ornithine and proline (by transamination).
In plants and bacteria, glumate is acetylated to N-acetyglutamate before reduction
 Acetylation prevents cyclization of the molecule after reduction, by condensation
between the resulting aldeyde and the a-amino group.
 In muscle, arginine is the precursor to the energy storage compound creatine
phosphate.

 The guanidino group is transferred to glycine, with regeneration ornithine.

 Ornithine is a participant in the Urea Cycle

 The other product, guanidinoacetic acid, is methylated by S-adenosylmethionine

to give creatinine, followed by phosphorylation to creatine phosphate.

 Enzyme: creatine kinase

Ornithine undergoes decarboxylation to give 1,4-diaminobutane (putrescine).

 Putrescine is the precursor to a class of important and ubiquitous compounds, the

polyamines.

Polyamines include putrescine, spermidine, spermine.

Ornithine is a major precursor for synthesis of polyamines.

Conversion of ornithine to putrescine is catalyzed by Ornithine Decarboxylase.

Biosynthesis of Arginine

Synthesis of arginine is an eight step process starting with the amino acid glutamate. Two
ATP and one NADPH + H+ are utilized to synthesize each arginine.

Arginine is a precursor to NO·

NO· - signal-transducing agent in vasodilation of endothelial vascular cells and

underlying smooth muscle
Enzyme: NO· synthase – contains bound FMN, FAD, nonheme iron, and tetrahydrobiopterin

- in endothelial vascular cells is sensitive to Ca2+

- activation of this enzyme by Ca2+ causes accumulation.

Biosynthesis of Proline, Hydroxyproline, and Collagen

Proline is a precursor to hydroxyproline

 Hydroyproline residues are generated by posttranslational modification
modification, following completion of the polypeptide chain.
 Procollagen – nonhydroxylated collagen precursor
 A proline residue two positions to the carboxyl side of a glycine residue is
the preferred substrate for the action of procollagen proline hydroxylase.
Decarboxylation of glutamate yields ƴ-aminobutyric acid (GABA)
Glutamate is a neurotransmitter.

2 major fates metabolic fates:

1. Synthesis of glutathione, along with cysteine and glycine

 2. Synthesis, via an ATP-dependent conjugating system, of polyglutamate forms of folic
acid and its coenzymes.

Reduction of Inorganic Sulfur

S + H  H2S
3’-phosphoadenosine-5’-phosphosulfate (PAPS) – activated sulfate compound used as an
active agent for sulfate esterification (in all organisms)
 Serves as substrate for sulfate reduction (in bacteria)
 SO42- + ATP + PPi  adenosine-5’-phosphosulfate + ATP -> PAPS + ADP

Synthesis of Cysteine and Methionine in Plants and Bacteria

Serine + H2S  Cysteine

In plants and most microorganisms:

 Serine+ acetyl-CoA  O-acetylserine + CoA-SH

 O-Acetylserine + H2S -> cysteine + acetate + H2O

Cysteine provides the sulfur for methionine synthesis, with the carbon coming fro homoserine.
Biosynthesis of Aromatic Rings: The Shikimic Acid Pathway

Synthesis of the aromatic amino acids begins with the synthesis of chorismate - an important
intermediate for many biosynthetic pathways. Phosphoenol pyruvate and erythrose 4-phosphate
serve as beginning substrates for the pathway. A price of one NADPH + H+ and one ATP is
exacted for every chorismate formed. In the sixth step of the synthesis another phosphoenol
pyruvate molecule is added to the growing molecule.
Synthesis of Phenylalanine
Chorismate is converted to phenylpyruvate in two steps and phenylalanine is synthesized by a
transamination reaction with glutamate. No energy is required to run these reactions.

Synthesis of Tyrosine

It is synthesized from Phenylalanine

Synthesis of Tryptophan

Trytophan synthesis is complex and involves 5 steps from chorismate. Glutamate donates an
amine group in the first step of the pathway and pyruvate is lost from chorismate. In the next
threes steps a ribose sugar is added, this eventually contributes to the 5 membered ring of
tryptophan. Energy is contributed to the process in the form of hydrolysis of pyrophosphate. This
hydrolysis helps drive the addition of the ribose sugar in the second step of the reaction. In the
last step of the pathway serine serves as the donor of the a carbon amino group of tryptophan.

Synthesis of Histidine

The synthesis of histidine is long and complex and its pathway is intertwined with nucleic acid
biosynthesis (specifically purine). The pathway seems to be universal in all organisms able to
synthesize histidine. The first five steps of the pathway take ribose from phosphoribosyl
pyrophosphate (PRPP) and transform it into Imadiazoleglycerol phosphate. Once the imadiazole
ring is formed, glutamate donates the a-amino group and the newly formed amine is oxidized to
histidine in the last step of the pathway. Energy is required in the form of ATP (in this case
elements of the ATP molecule actually becomes part of the amino acid) and pyrophosphate
which is lost from phosphoribosyl pyrophosphate and ATP help drive the reaction.

Synthesis of Serine, Lycine and Threonine

- Simple pathways
- Serine is involved in glycine, phospholipid, and cystein synthesis. Glycine
is active in biosynthesis of purine nucleotides and porphyrins.
- Serine and glycine are both major contributors to the pool of activated
one-carbon groups.

Synthesis of Serine

3-phosphoglycerate + NAD+  3-Phosphopyruvate

3-Phosphopyruvate (transamination)  3-Phosphoserine

Phosphoserine + H2O  Serine

Synthesis of Threonine

Threonine – essential amino acid

- its synthesis is limited to plants and prokaryotes

Homoserine + ATP  O-Phosphohomoserine + ADP

O-Phosphohomoserine  Pi H20  Threonine

Synthesis of Valine, Leucine and Isoleucine

-Essential amino acids

-Synthesized primarily in plant and bacterial cells

-No significant metabolic roles

Synthesis of Lysine

Diaminopimelic acid pathway – operates in bacteria, some lower fungi, algae, and higher plants

Condensation of pyruvate with aspartate B-aldehyde  decarboxylation of diaminopimelate 

Lysine

Diaminopimelate – constituent of bacterial cell walls

PROTEIN DEGRADATION:

Review: Proteins

Proteins are complex organic compounds. They are polymers consisting of amino acids (nitrogen
containing bases) linked by covalent peptide bonds.

Formation from Amino Acids:

There are 20 different amino acids that join together to make all types of protein.

- essential amino acids – not synthesized by the body

- can be obtained from digestion of dietary proteins and degradation of pre-existing
proteins in the body
- non-essential amino acids – synthesized by the body
  Arginine and histidine, although not required in the diets of adults, are required for
growth (children and adolescents), because the amounts that can be synthesized are
not sufficient to maintain normal growth rates.

Proteins in the body are constantly synthesized and degraded, partially draining and refilling the
cellular amino acid pools.

Amino Acid Metabolism:

Two major pathways for Protein Degradation:

1) Lysosome Proteolysis
2) Ubiquitin/Proteasome Pathway
Lysosome Proteolysis

It is a major pathway for protein degradation in eukaryotic cells which involves the uptake of
proteins in the lysosomes. Lysosomes are membrane-enclosed organelles that contain an array of
digestive enzymes, including several proteases. Proteolytic enzymes (proteases) degrade dietary
proteins into their constituent amino acids in the stomach and intestine. They are produced by the
stomach (pepsin), pancreas (trypsin, chymotrypsin, elastase, carboxypeptidases), and the
intestine (enteropepdidase, aminopeptidases). The lysosomal proteolysis degrades proteins
usually by hydrolysis at one or more of its peptide bonds.

The containment of proteases and other digestive enzymes within lysosomes prevents
uncontrolled degradation of the contents of the cell. Therefore, in order to be degraded by
lysosomal proteolysis, cellular proteins must first be taken up by lysosomes.

Autophagy - The principal pathway for this uptake of cellular proteins. - It is generally
activated under conditions of nutrient starvation, allowing cells to degrade nonessential proteins
and organelles so that their components can be reutilized.

Steps:
1) Formation of vesicles (autophagosomes) in which small areas of cytoplasm or cytoplasmic
organelles are enclosed in membranes derived from the endoplasmic reticulum.
2) Autophagosomes fuse with lysosomes
3) Degradative lysosomal enzymes digest their contents.

Proteolytic enzymes exhibit the preference for particular types of peptide bonds

Proteinases preferentially attacks the bond after:

Pepsin aromatic (Phe, Tyr) and acidic AA (Glu, Asp)

Trypsin basic AA (Arg, Lys)

Chymotrypsin hydrophobic (Phe, Tyr, Trp, Leu) and acidic AA (Glu, Asp)

Elastase AA with a small side chain (Gly, Ala, Ser)

Peptidases:

Carboxypeptidase A nearly all AA (not Arg and Lys)

Carboxypeptidase B basic AA (Arg, Lys)

aminopeptidase nearly all AA

Prolidase proline

Dipeptidase only dipeptides

Most proteins are completely digested to free amino acids. Amino acids and sometimes short
oligopeptides are absorbed by the secondary active transport and are transported via the blood to
the cells of the body.
Ubiquitin/Proteasome Pathway

The major pathway of selective protein degradation in eukaryotic cells uses ubiquitin as a marker
that targets cytosolic and nuclear proteins for rapid proteolysis

Ubiquitin:

Ubiquitin is a 76-amino-acid polypeptide that is highly conserved in all eukaryotes (yeasts,

animals, and plants). Proteins are marked for degradation by the attachment of ubiquitin to the
amino group of the side chain of a lysine residue.
Steps:

1) Ubiquitin is first activated by the enzyme El.

2) Activated ubiquitin is then transferred to one of several different ubiquitinconjugating
enzymes (E2).
3) A ubiquitin ligase (E3) then associates with E2 and directs the transfer of ubiquitin to a
specific target protein.
4) Multiple ubiquitins are then added, and the polyubiquinated proteins are degraded by a
protease complex (the proteasome).

Enzymes of the Ubiquitination

E1: ubiquitin-activating enzyme.

- exists as two isoforms of 110- and 117-kDa, which derive from a single gene and are
found in both the nucleus and cytosol. Inactivation of this gene is lethal.
- In mammals there is a single E1.

E2: ubiquitin-conjugating enzymes.

- superfamily of related proteins.

 - There are eleven E2s in yeast, and 20-30 E2s in mammals.

E3s: ubiquitin-protein ligases.

- play a key role in the ubiquitin pathway, as they are responsible for the selective
recognition of protein substrates.
- can be subdivided into at least six subtypes.

E4:

- catalyzes the efficient polymerization of very long polyubiquitin chains, it has been
characterized in yeast.

How is ubiquitin activated?

- C-terminus of ubiquitin gets adenylated

- Rearrangement to intermolecular thioester with a E1 (activation enzyme)
- Transfer of activierted ubiquitin from E1 to E2 (ubiquitin-conjugating enzyme) (thioester
bond)
- Transfer form E2 via E3(ubiquitin ligase) to target enzyme

A number of proteins that control fundamental cellular processes, such as gene expression and
cell proliferatio~ are targets for regulated ubiquitination and proteolysis. An interesting example
of such controlled degradation is provided by proteins (known as cyclins) that regulate
progression through the division cycle of eukaryotic cells.

DEAMINATION OF AMINO ACIDS:

What happens next after protein degradation to amino acids?

 Biosynthesis of structural proteins

Tissue proteins

 Biosynthesis of nucleotide bases

 Biosynthesis of structural proteins

Hemoglobin, protein hormones, enzymes

 Biosynthesis of small peptides with biological importance

Gluthatione, Endorphines
Transamination

•Transamination means transfer of amino group from α-amino acid to α-keto acid with formation
of a new α-amino acid and a new α-keto acid.

•The liver is the main site of transamination

•All amino acids can be transaminated except lysine, threonine, proline and hydroxyl proline.

•All transamination reactions are reversible

•It is catalyzed by aminotransferases (transaminases).

•It needs pyridoxal phosphate as coenzyme.

Role of Pyridoxal phosphate

• It acts as an intermediate carrier for amino group

• It accepts the amino group from amino acid to form pyridoxamine phosphate, which in turn
gives the amino group to α-keto acid.

Example of Transaminases

Alanine transaminase

Aspartate transaminase

Glutamate transaminase

Deamination

Deamination means the removal of amino group from α-amino acid in the form of ammonia with
formation of α-keto acid. The liver and kidney are the main sites for deamination.

Oxidative deamination

•It is catalyzed by one of the following enzymes

•L-amino acid oxidases

•D-amino acid oxidases

•Glutamate dehydrogenase

Non-oxidative deamination
•It is catalyzed by one of the following enzymes:

•Dehydrates

•Desulfhydrases

Oxidative Deamination: Catalyzed by Lamino acid oxidase

 This enzyme is present in the liver and kidney. Its activity is low.
 It is an aerobic dehydrogenase that needs FMN as a coenzyme.
 It deaminates most of the naturally occurring L-amino acids.

Oxidative Deamination: Catalyzed by D amino acid oxidase

 D-amino acids are present in plants and bacterial cell wall.

 They are not used in protein biosynthesis in humans and animals.
 D-amino acids are deaminated by D-amino acid oxidase resulting in ammonia and α-keto
acids.
 D-amino acid oxidase is present in the liver.
 It is an aerobic dehydrogenase.
 It needs FAD as a coenzyme.

Oxidative Deamination: Catalyzed by Glutamate dehydrogenase

 This enzyme is present in most tissues

 It is present both in cytoplasm and mitochondria
 Its activity is high.
 It is an anaerobic dehydrogenase
 It needs NAD or NADP as a coenzyme
 It deaminates glutamic acid resulting in α-ketoglutaric acid and ammonia

UREA CYCLE:

Urea is an organic compound having a chemical formula of CO(NH2)2.It is also called

carbamide; it is a waste product by the body after the metabolism of proteins. High intake of
protein would result to an excess amino acid catabolism which will lead to elevated glutamate
levels and N-acetylglutamate which will raise the urea cycle activity. It is known to be more
toxic than ammonia.
 Structure of Urea

Urea cycle is the process where excess nitrogen is excreted through the breakdown of arginine.
In occurs mainly in the liver but it can also occur in the kidney. The two nitrogen atoms of urea
in the Urea cycle are from ammonia and the nitrogen group of aspartate. The two products of
urea cycle are ornithine and urea, thus the cycle is closed after the regeneration of ornithine. For
each cycle, citrulline must leave the mitochondria, and ornithine must enter the mitochondrial
matrix. Urea cycle is related with the citric acid cycle through fumarate which is an intermediate
in TCA.

The urea cycle requires five reactions (of which four are part of the actual cycle). The first
reaction is the primary regulated step. Carbamoyl phosphate synthetase I5 is the mitochondrial
enzyme that catalyzes the formation of carbamoyl phosphate from inorganic ammonium and
carbonate. This enzyme is thus another enzyme capable of fixing ammonium. The usual fate of
the ammonium fixed by carbamoyl phosphate synthetase I is excretion in the form of urea, and
therefore this enzyme is usually considered separately from glutamine synthetase and glutamate
dehydrogenase, which fix ammonium for use in metabolism. In eukaryotic organisms, a different
carbamoyl phosphate synthetase forms carbamoyl phosphate in the cytoplasm as the first step in
pyrimidine biosynthesis. Unlike carbamoyl phosphate synthetase I, however, carbamoyl
phosphate synthetase II uses glutamine as the ammonium donor instead of free ammonium.
Carbamoyl phosphate synthetase I requires the presence of the allosteric activator N-
acetylglutamate (the product of the first step in ornithine biosynthesis) for activity. This
regulation means that carbamoyl phosphate synthetase I is the rate-limiting enzyme of the urea
cycle. The other four enzymes are part of the actual cycle. The cycle begins with the addition of
carbamoyl phosphate to ornithine by ornithine transcarbamoylase to produce citrulline. Citrulline
then leaves the mitochondria using a specific transporter, because the remaining reactions occur
in the cytoplasm. Once in the cytoplasm, citrulline is combined with aspartate by
argininosuccinate synthetase to form argininosuccinate, in a reaction that requires ATP, and
produces AMP and pyrophosphate. The next enzyme, argininosuccinase, performs a cleavage
reaction that releases the TCA cycle intermediate fumarate and the amino acid arginine. Note
that the arginine contains nitrogens derived from ornithine, from the free ammonium, and from
the aspartate. Arginine is then cleaved by arginase to release urea and to regenerate ornithine.
Ornithine also has a specific transporter that allows the ornithine to re-enter the mitochondria,
completing the cycle. As with the TCA cycle, the urea cycle is controlled by two factors:
regulated enzymes and substrate availability. For the urea cycle the regulated enzyme is
carbamoyl phosphate synthetase I. For the urea cycle, the availability of cycle intermediates and
free ammonium also control the cycle. Thus, high levels of ornithine allow the cycle to proceed
more rapidly. 5 Some textbooks call this enzyme carbamoyl phosphate “synthase” rather than
“synthetase”. The strict nomenclature rule states that a “synthetase” is an enzyme that combines
two molecules using ATP to provide the driving force, while a “synthase” combines two
molecules without using ATP. For the purpose of this course, “synthase” and “synthetase” are
effectively used interchangeably, although I am attempting to eliminate inconsistent usage. In
principle, the urea cycle can be used to synthesize or degrade arginine. Note, however that net
synthesis of arginine requires input of one of the other urea cycle intermediates; net degradation
of arginine requires net removal of one of these intermediates. As described about, the urea cycle
does not result in an alteration in the amount of arginine.

REFERENCES:

Matthews, C. K., van Holde, K., & Ahern, K. G. (2002). Biochemistry (Third ed.). Singapore:
Pearson Education Asia Pte Ltd.

https://www.rose-hulman.edu/~brandt/Chem330/Urea_cycle.pdf

http://lecturer.ukdw.ac.id/dhira/Metabolism/aminoacids.html

http://themedicalbiochemistrypage.org/amino-acid-metabolism.php#phenylalanine