STRUCTURE OF THE AMINO ACIDS GPH 2021.pdf

Notes on Amino acids

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PROTEIN STRUCTURE AND FUNCTION
Learning objectives
• Define and understand the general structure an amino acid and the concept of optical activity
• Know the 20 common amino acids including their three letter notation and their classification
• Be able to draw the structures of the 20 common amino acids
• Understand the role played by side chains of amino acids
• Understand the concept of ionization of an amino acid (Zwitterion, isoelectric point)
• Formation of peptide bond and nomenclature of peptides
• Be able to explain the four levels of protein structure
• Understand the biosynthesis and catabolism of amino acids including transamination and urea
cycle

STRUCTURE OF THE AMINO ACIDS
Although more than 300 different amino acids have been described in nature, only 20 are commonly found
as constituents of mammalian proteins.

Each amino acid (except for proline, which has a secondary amino group) has a carboxyl group, a primary
amino group, and a distinctive side chain (“R-group”) bonded to the α-carbon atom (as in the figure below)

General structure of an amino acid

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Optical properties of amino acids
The α-carbon of an amino acid is attached to four different chemical groups and is, therefore, a chiral or
optically active carbon atom. Glycine is the exception because its α-carbon has two hydrogen substituents
and, therefore, is optically inactive. Amino acids that have an asymmetric center at the α-carbon can exist
in two forms, designated D and L, that are mirror images of each other (see figure below). The two forms in
each pair are termed stereoisomers, optical isomers, or enantiomers. Enantiomers are physically and
chemically indistinguishable by most techniques, but can be distinguished on the basis of their different
optical rotation of plane-polarized light. Molecules are classified as dextrorotatory (D) or levorotatory (L)
depending on whether they rotate the plane of plane-polarized light to the right or to the left. All amino
acids found in proteins a protein are L-amino acids, that is, they rotate plane-polarized to the left. However,
D-amino acids are found in some antibiotics and in plant and bacterial cell walls.




D and L forms of alanine are mirror images

At physiologic pH (approximately pH 7.4), the carboxyl group is dissociated, forming the negatively charged
carboxylate ion (–COO
–
), and the amino group is protonated (–NH3
+
). In proteins, almost all of these
carboxyl and amino groups are combined through peptide linkage and, in general, are not available for
chemical reaction except for hydrogen bond formation



Thus, it is the nature of the side chains that ultimately dictates the role an amino acid plays in a protein. It
is, therefore, useful to classify the amino acids according to the properties of their side chains, that is,
whether they are nonpolar (hydrophobic) or polar (hydrophilic).

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Names of the 20 common amino acids including their corresponding three letter
abbreviations
Name
3 letter
abbreviation Name
3 letter
abbreviation
Cysteine Cys Threonine Thr
Histidine His Arginine Arg
Isoleucine Ile Asparagine Asn
Methionine Met Aspartate Asp
Serine Ser Glutamate Glu
Valine Val Glutamine Gln
Alanine Ala Phenylalanine Phe
Glycine Gly Tyrosine Tyr
Leucine Leu Tryptophan Trp
Proline Pro Lysine Lys


CLASSIFICATION OF AMINO ACIDS

The standard 20 amino acids differ only in the structure of the side-chain or ‘R’ group. They can be
subdivided into smaller groupings on the basis of similarities in the properties of their side-chains.
They display different physicochemical properties depending on the nature of their side-chain.
Some are acidic, others are basic. Some have small side-chains, others large, bulky side-chains.
Some have aromatic side-chains, others are polar. Some (like proline) present conformational
inflexibility; others can participate either in hydrogen bonding or covalent bonding. Some are
chemically reactive.
I. Hydrophobic, aliphatic amino acids
The aliphatic side-chains of alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile) and
methionine (Met) are chemically unreactive, but hydrophobic in nature.
• Proline (Pro) is also hydrophobic but, with its aliphatic side-chain bonded back on to the
amino group, it is conformationally rigid.
• The sulfur-containing side-chain of cysteine (Cys) is also hydrophobic and is highly
reactive, capable of reacting with another cysteine to form a disulfide bond

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II. Hydrophobic, aromatic amino acids

Phenylalanine (Phe), tyrosine (Tyr) and tryptophan (Trp) are hydrophobic by virtue of their aromatic rings.
All can participate in hydrophobic interactions. The hydroxyl group of tyrosine can form hydrogen bonds,
and it is an important functional group in some enzymes.
Tyr and Trp to a lesser Phe extent absorb UV light at 280nm. This accounts for the characteristic strong
absorbance of light by most proteins at a wavelength of 280 nm, a property exploited by researchers in the
characterizationof proteins

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III. Hydrophilic Amino acids

The remaining amino acids all have polar, hydrophilic side-chains, some of which are charged at
neutral pH
a. Polar, charged amino acids
The amino groups on the side-chains of the basic amino acids arginine (Arg or R) and lysine (Lys or K)
are protonated and thus positively charged at neutral pH.
The side-chain of histidine (His) can be either positively charged or uncharged at neutral pH.
In contrast, at neutral pH the carboxyl groups on the side-chains of the acidic amino acids aspartic acid
(aspartate; Asp) and glutamic acid (glutamate; Glu) are de-protonated and possess a negative charge.



b. Polar, uncharged amino acids
The side-chains of asparagine (Asn) and glutamine (Gln), the amide derivatives of Asp and Glu,
respectively, are uncharged but can participate in hydrogen bonding.
Serine (Ser) and threonine (Thr) are polar amino acids due to the reactive hydroxyl group in the
side-chain, and can also participate in hydrogen bonding (as can the hydroxyl group of the
aromatic amino acid Tyr)

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LOCATION OF NONPOLAR AMINO ACIDS IN PROTEINS
In proteins found in aqueous solutions––a polar environment––the side chains of the nonpolar amino acids
tend to cluster together in the interior of the protein. This phenomenon, known as the hydrophobic effect, is
the result of the hydrophobicity of the nonpolar R-groups, which act much like droplets of oil that coalesce
in an aqueous environment. The nonpolar R-groups thus fill up the interior of the folded protein and help
give it its three-dimensional shape. However, for proteins that are located in a hydrophobic environment
such as a membrane, the nonpolar R-groups are found on the outside surface of the protein, interacting
with the lipid environment

NOTE: Sickle cell anemia, a sickling disease of red blood cells, results from the substitution of polar
glutamate by nonpolar valine at the sixth position in the β subunit of hemoglobin

Proline: Proline differs from other amino acids in that proline’s side chain and α-amino N form a rigid, five-
membered ring structure. Proline, then, has a secondary (rather than a primary) amino group. It is
frequently referred to as an imino acid. The unique geometry of proline contributes to the formation of the
fibrous structure of collagen and often interrupts the α-helices found in globular proteins

Disulfide bond: The side chain of cysteine contains a sulf hydryl group (–SH), which is an important
component of the active site of many enzymes. In proteins, the –SH groups of two cysteines can become
oxidized to form a dimer, cystine, which contains a covalent cross-link called a disulfide bond (–S–S–).
NOTE: Many extracellular proteins are stabilized by disulfide bonds. Albumin, a blood protein that functions
as a transporter for a variety of molecules, is an example.

Polar Side chains as sites of attachment for other compounds: The polar hydroxyl group of serine,
threonine, and, rarely, tyrosine, can serve as a site of attachment for structures such as a phosphate group.
In addition, the amide group of asparagine, as well as the hydroxyl group of serine or threonine, can serve
as a site of attachment for oligosaccharide chains in glycoproteins


ACIDIC AND BASIC PROPERTIES OF AMINO ACIDS
Amino acids in aqueous solution contain weakly acidic α-carboxyl groups and weakly basic α-amino
groups. In addition, each of the acidic and basic amino acids contains an ionizable group in its side chain.
Thus, both free amino acids and some amino acids combined in peptide linkages can act as buffers. Recall
that acids may be defined as proton donors and bases as proton acceptors. Acids (or bases) described as
“weak” ionize to only a limited extent

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Isoelectric point: At neutral pH, alanine exists predominantly as the dipolar Form II in which the amino and
carboxyl groups are ionized, but the net charge is zero. The isoelectric point (pI) is the pH at which an
amino acid is electrically neutral, that is, in which the sum of the positive charges equals the sum of the
negative charges. Amino acids in solution at neutral pH exist predominantly as dipolar ions (also called
zwitterions). In the dipolar form, the amino group is protonated (–NH3
+
) and the carboxyl group is
deprotonated (–COO
-
). The ionization state of an amino acid varies with pH. For an amino acid, such as
alanine, that has only two dissociable hydrogens (one from the α-carboxyl and one from the α-amino
group), the pI is the average of pK1 and pK2 (pI = [2.3 + 9.1]/2 = 5.7). The pI is thus midway between pK1
(2.3) and pK2 (9.1). pI corresponds to the pH at which the Form II (with a net charge of zero) predominates,
and at which there are also equal amounts of Forms I (net charge of
+
1) and III (net charge of
–
1).

FORMATION OF THE PEPTIDE BOND

In proteins, amino acids are joined covalently by peptide bonds, which are amide linkages between the -
carboxyl group of one amino acid and the α-amino group of another. For example, valine and alanine can
form the dipeptide valylalanine through the formation of a peptide bond (as shown below). Peptide bonds
are not broken by conditions that denature proteins, such as heating or high concentrations of urea



Naming the peptide
By convention, the free amino end (N-terminal) of the peptide chain is written to the left and the free
carboxyl end (C-terminal) to the right. Therefore, all amino acid sequences are read from the N- to the C-
terminal end of the peptide. For example, in Figure above, the order of the amino acids is “valine, alanine.”
Linkage of many amino acids through peptide bonds results in an unbranched chain called a polypeptide.
Each component amino acid in a polypeptide is called a “residue” because it is the portion of the amino
acid remaining after the atoms of water are lost in the formation of the peptide bond. When a polypeptide is
named, all amino acid residues have their suffixes (-ine, -an, -ic, or -ate) changed to -yl, with the exception
of the C-terminal amino acid. For example, a tripeptide composed of an N-terminal valine, a glycine, and a
C-terminal leucine is called valyl glycyl leucine.

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Polarity of the peptide bond
Like all amide linkages, the – C=O and –NH groups of the peptide bond are uncharged, and neither accept
nor release protons over the pH range of 2–12. Thus, the charged groups present in polypeptides consist
solely of the N-terminal (α-amino) group, the C-terminal (α-carboxyl) group, and any ionized groups present
in the side chains of the constituent amino acids. The – C=O and – NH groups of the peptide bond are
polar, and are involved in hydrogen bonds, for example, in α-helices and β-sheet structures

Levels of protein structure
The 20 amino acids commonly found in proteins are joined together by peptide bonds. The linear sequence
of the linked amino acids contains the information necessary to generate a protein molecule with a unique
three-dimensional shape. The complexity of protein structure is best analyzed by considering the molecule
in terms of four organizational levels, namely, primary, secondary, tertiary, and quaternary

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Primary structure of proteins
The primary structure is the sequence of amino acids along the polypeptide chain. This sequence is
determined by the sequence of nucleotide bases in the gene encoding the protein.

i. By convention, the sequence is written from left to right, starting with the N-terminal amino acid and
ending with its C-terminal amino acid.
ii. Because there are no dissociable protons in peptide bonds, the charges on a polypeptide chain are
due only to the N-terminal amino group, the C-terminal carboxyl group, and the side chains on amino
acid residues.
iii. A protein will migrate in an electric field, depending on the sum of its charges at a given pH (the net
charge).
– Positively charged proteins are cations and migrate toward the cathode (–).
– Negatively charged proteins are anions and migrate toward the anode (+).
– At the isoelectric pH (the pI), the net charge is zero, and the protein does not migrate.

Understanding the primary structure of proteins is important because many genetic diseases result in
proteins with abnormal amino acid sequences, which cause improper folding and loss or impairment of
normal function. If the primary structures of the normal and the mutated proteins are known, this
information may be used to diagnose or study the disease.

Secondary level of Protein Structure
The secondary level of structure in a protein is the regular folding of regions of the polypeptide chain in
which the atoms of the side chains are not involved.
The two most common types of protein fold are
– The α-helix
– The β-pleated sheet.
In the rod-like α-helix, the amino acids arrange themselves in a regular helical conformation. The carbonyl
oxygen of each peptide bond is hydrogen bonded to the hydrogen on the amino group of the fourth
amino acid away, with the hydrogen bonds running nearly parallel to the axis of the helix. In an α-helix
there are 3.6 amino acids per turn of the helix covering a distance of 0.54 nm
– The side chains of the amino acid residues in an α-helix extend outward from the central axis of the
rodlike structure. This allows the formation of high tensile strength fibrillary proteins.
– A very diverse group of proteins contains α-helices. For example, the keratins are a family of
closely related, fibrous proteins whose structure is nearly entirely α-helical. They are a major
component of tissues such as hair and skin, and their rigidity is determined by the number of
disulfide bonds between the constituent polypeptide chains.
– The a-helix is disrupted by proline residues, in which the ring imposes geometric constraints, and
by regions in which numerous amino acid residues have charged groups or large, bulky side
chains.

β-pleated sheets are formed by hydrogen bonds between two extended polypeptide chains or between
two regions of a single chain that folds back on itself. The hydrogen bonds in β-pleated sheet can be
intrachain or interchain whereas α-helix has only intrachain hydrogen bonds
– These interactions are between the carbonyl of one peptide bond and the –NH of another.
– The chains may run in the same direction (parallel) or in opposite directions (antiparallel).

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An α-helix. The dotted lines represent the hydrogen bonding that occurs between the carbonyl (C=O) of one peptide bond and
the -NH of another peptide bond that is four amino acid residues further along the chain.

Tertiary structure of a protein
The tertiary structure of a protein refers to its overall three-dimensional conformation. It is produced by
interactions between disparate amino acid residues that may be located at a considerable distance from
each other in the primary sequence of the polypeptide chain.
i. Hydrophobic amino acid residues tend to reside and cluster in the interior of globular proteins,
where they exclude water, whereas hydrophilic residues are usually found on the surface, where they
interact with water.
ii. The types of noncovalent interactions between amino acid residues that produce the three
dimensional shape of a protein include;
– hydrophobic interactions,
– electrostatic (ionic) interactions,
– hydrogen bonds, and
– van der Waals interactions.
– Covalent disulfide bonds also occur in tertiary structure.
iii. All the information required for proteins to correctly assume their tertiary structure is defined by their
primary sequence. Sometimes molecules known as ‘‘chaperones’’ interact with the polypeptide to
help find the correct tertiary structure. Such proteins either catalyze the rate of folding or protect the
protein from forming ‘‘nonproductive’’ intramolecular tangles during the folding process.

The structure of an antiparallel β-sheet. The orientation is indicated by arrows, and the hydrogen
bonds by dotted lines.

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Quaternary structure of a protein

Quaternary structure refers to the spatial arrangement of subunits in a protein containing more than one
polypeptide chain.
The subunits are joined together by the same types of noncovalent interactions within a single polypeptide
to form its tertiary structure. In some cases, covalent disulfide bonds are also found in quaternary structure.

Denaturation and renaturation
– Proteins can be denatured by agents such as heat and urea that unfold polypeptide chains without
causing hydrolysis of peptide bonds. Such agents only break the noncovalent interactions.
– If a denatured protein returns to its native state after the denaturing agent is removed, the process
is called renaturation.

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BIOSYNTHESIS OF AMINO ACIDS
The carbon skeletons of all twenty amino acids are derived from just seven metabolic intermediates, that
together, are found in three metabolic pathways.
– glycolytic pathway
– pentose phosphate pathway
– citrate cycle
These include
1. three glycolytic pathway intermediates;
 3-phosphoglycerate,
 phosphoenolypyruvate,
 and pyruvate,
2. Two citrate cycle intermediates;
 α-ketoglutarate
 oxaloacetate.
3. Two pentose phosphate pathway intermediates;
 ribose 5-phosphate and
 erythrose 4-phosphate

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Based on the principles of nutritional biochemistry, it was determined that humans (and most animals)
require ten of the twenty amino acids in their diet in order to thrive.
As shown in below, these ten amino acids are called essential amino acids, whereas, the other ten amino
acids which humans can synthesize on their own, are called nonessential amino acids.

Nonessential Essential
Alanine Arginine*
Asparagine Histidine
Aspartate Isoleucine
Cysteine Leucine
Glutamate Lysine
Glutamine Methionine*
Glycine Phenylalanine*
Proline Threonine
Serine Tyrptophan
Tyrosine Valine

Note*: The amino acids arginine, methionine and phenylalanine are considered essential for reasons not
directly related to lack of synthesis. Arginine is synthesized by mammalian cells but at a rate that is
insufficient to meet the growth needs of the body and the majority that is synthesized is cleaved to form
urea. Methionine is required in large amounts to produce cysteine if the latter amino acid is not adequately
supplied in the diet. Similarly, phenyalanine is needed in large amounts to form tyrosine if the latter is not
adequately supplied in the diet.

Source of Nitrogen
In order to synthesize amino acids, a source of nitrogen is needed. In animals glutamate and glutamine
play the pivotal roles. The α−amino group of most of the amino acids comes from the transamination
reaction transferring the amino group from glutamate to an α-ketoacid acceptor. Glutamate is synthesized
from ammonia and α-ketoglutarate by the action of glutamate dehydrogenase
Transamination is the reaction between an amino acid and an α-keto acid. The amino group is removed
from the amino acid and donated to the α-keto acid converting that into an amino acid.

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AMINO ACID CATABOLISM
The strategy of amino acid degradation is to transform the carbon skeletons into major metabolic
intermediates that can be converted into glucose, or oxidized by the citric acid cycle. The carbon skeletons
of a diverse set of 20 amino acids are funneled into only seven molecules: the seven major metabolic
intermediates are
 Pyruvate
 acetyl CoA
 acetoacetyl CoA,
 α- ketoglutarate,
 succinyl CoA,
 fumarate
 oxaloacetate.

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Ketogenic Amino acids
The seven amino acids that are degraded entirely or in part to acetoacetyl-CoA and/or acetyl-CoA—Phe,
Tyr, Ile, Leu, Try, Thr, and Lys—can yield ketone bodies in the liver, where acetoacetyl-CoA is converted to
acetoacetate and then to acetone and β-hydroxybutyrate (Ketone bodies). These are the ketogenic amino
acids
Glucogenic Amino acids
The amino acids that are degraded to pyruvate, α-ketoglutarate, succinyl-CoA, fumarate, and/or
oxaloacetate can be converted to glucose and glycogen. They are the glucogenic amino acids. Glucogenic
AAs: AAs that are converted to metabolites that can be converted glucose. TCA cycle intermediates and
pyruvate can be converted PEP and then glucose.

The division between ketogenic and glucogenic amino acids is not sharp; Five amino acids—tryptophan,
phenylalanine, tyrosine, threonine, and isoleucine—are both ketogenic and glucogenic. Only Leu and Lys
are solely ketogenic.

Catabolism of amino acids is particularly critical to the survival of animals with high-protein diets or during
starvation. Leu is an exclusively ketogenic amino acid that is very common in proteins. Leu degradation
makes a substantial contribution to ketosis under starvation conditions

REMOVAL OF NITROGEN FROM AMINO ACIDS
The presence of the α-amino group keeps amino acids safely locked away from oxidative breakdown.
Removing the α-amino group is essential for producing energy from any amino acid, and is an obligatory
step in the catabolism of all amino acids. Once removed, this nitrogen can be incorporated into other
compounds or excreted, with the carbon skeletons being metabolized.

Transamination: the funneling of amino groups to glutamate
The first step in the catabolism of most amino acids is the transfer of their α-amino group to α-ketoglutarate
(Figure below).

Aminotransferase reaction using α-ketoglutarate as the aminogroup acceptor.

The products are an α-keto acid (derived from the original amino acid) and glutamate. α-Ketoglutarate
plays a pivotal role in amino acid metabolism by accepting the amino groups from most amino acids, thus
becoming glutamate. Glutamate produced by transamination can be oxidatively

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deaminated (see below), or used as an amino group donor in the synthesis of nonessential amino acids.
This transfer of amino groups from one carbon skeleton to another is catalyzed by a family of enzymes
called aminotransferases (formerly called transaminases). These enzymes are found in the cytosol and
mitochondria of cells throughout the body—especially those of the liver, kidney, intestine, and muscle. All
amino acids, with the exception of lysine and threonine, participate in transamination at some point in their
catabolism.

Alanine and the Glucose-Alanine Cycle
Aside from its role in protein synthesis, alanine is second only to glutamine in prominence as a circulating
amino acid. In this capacity it serves a unique role in the transfer of nitrogen from peripheral tissue to the
liver. Alanine is transferred to the circulation by many tissues, but mainly by muscle, in which alanine is
formed from pyruvate at a rate proportional to intracellular pyruvate levels. Liver accumulates plasma
alanine, reverses the transamination that occurs in muscle, and proportionately increases urea production.
The pyruvate is either oxidized or converted to glucose via gluconeogenesis. When alanine transfer from
muscle to liver is coupled with glucose transport from liver back to muscle, the process is known as
the glucose-alanine cycle. The key feature of the cycle is that in 1 molecule, alanine, peripheral tissue
exports pyruvate and ammonia (which are potentially rate-limiting for metabolism) to the liver, where the
carbon skeleton is recycled and most nitrogen eliminated.
There are 2 main pathways to production of muscle alanine: directly from protein degradation, and via the
transamination of pyruvate by alanine transaminase (ALT).

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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
Glutamate dehydrogenase: the oxidative deamination of amino acids
In contrast to transamination reactions that transfer amino groups, oxidative deamination by glutamate
dehydrogenase results in the liberation of the amino group as free ammonia (NH3) (Figure below). These
reactions occur primarily in the liver and kidney. They provide α-keto acids that can enter the central
pathway of energy metabolism, and ammonia, which is a source of nitrogen in urea synthesis.
Glutamate dehydrogenase: As described above, the amino groups of most amino acids are ultimately
funneled to glutamate by means of transamination with α-ketoglutarate. Glutamate is unique in that it is the
only amino acid that undergoes rapid oxidative deamination—a reaction catalyzed by glutamate
dehydrogenase (see Figure below). Therefore, the sequential action of transamination (resulting in the
collection of amino groups from most amino acids onto α-ketoglutarate to produce glutamate) and the
Alanine plays a special role in transporting amino
groups to liver.
Ala is the carrier of ammonia and of the carbon
skeleton of pyruvate from muscle to liver.
The ammonia is excreted and the pyruvate is used
to produce glucose, which is returned to the muscle.

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oxidative deamination of that glutamate (regenerating α-ketoglutarate) provide a pathway whereby the amino
groups of most amino acids can be released as ammonia.
Coenzymes: Glutamate dehydrogenase is unusual in that it can use either NAD
+
or NADP
+
as a coenzyme
(see Figure below). NAD
+
is used primarily in oxidative deamination (the simultaneous loss of ammonia
coupled with the oxidation of the carbon skeleton and NADPH is used in reductive amination (the
simultaneous gain of ammonia coupled with the reduction of the carbon skeleton.


Oxidative deamination by glutamate dehydrogenase


Direction of reactions: The direction of the reaction depends on the relative concentrations of glutamate,
α-keto glutarate, and ammonia, and the ratio of oxidized to reduced coenzymes. For example, after
ingestion of a meal containing protein, glutamate levels in the liver are elevated, and the reaction proceeds
in the direction of amino acid degradation and the formation of ammonia. [Note: the reaction can also be
used to synthesize amino acids from the corresponding α-keto acids]

Allosteric regulators: Guanosine triphosphate (GTP) is an allosteric inhibitor of glutamate
dehydrogenase, whereas adenosine diphosphate (ADP) is an activator. Thus, when energy levels are low
in the cell, amino acid degradation by glutamate dehydrogenase is high, facilitating energy production from
the carbon skeletons derived from amino acids.

UREA CYCLE
Urea is the major disposal form of amino groups derived from amino acids, and accounts for about 90% of
the nitrogen-containing components of urine. One nitrogen of the urea molecule is supplied by free
ammonia, and the other nitrogen by aspartate. [Note: Glutamate is the immediate precursor of both
ammonia (through oxidative deamination by glutamate dehydrogenase) and aspartate nitrogen (through
transamination of oxaloacetate by AST).] The carbon and oxygen of urea are derived from CO2. Urea is
produced by the liver, and then is transported in the blood to the kidneys for excretion in the urine.

Reactions of the cycle
The first two reactions leading to the synthesis of urea occur in the mitochondria, whereas the remaining
cycle enzymes are located in the cytosol.

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1. Formation of carbamoyl phosphate: Formation of carbamoyl phosphate by carbamoyl phosphate
synthetase I is driven by cleavage of two molecules of ATP. Ammonia incorporated into carbamoyl
phosphate is provided primarily by the oxidative deamination of glutamate by mitochondrial glutamate
dehydrogenase. Ultimately, the nitrogen atom derived from this ammonia becomes one of the nitrogens of
urea. Carbamoyl phosphate synthetase I requires N-acetylglutamate as a positive allosteric activator.
[Note: Carbamoyl phosphatesynthetase II participates in the biosynthesis of pyrimidines. It does not require
N-acetylglutamate, uses glutamine as the nitrogen source, and occurs in the cytosol.]
2. Formation of citrulline: The carbamoyl portion of carbamoyl phosphate is transferred to ornithine by
ornithine transcarbamoylase (OTC) as the high-energy phosphate is released as Pi. The reaction
product, citrulline, is transported to the cytosol. [Note: Ornithine and citrulline are basic amino acids that
participate in the urea cycle, moving across the inner mitochondrial membrane via a cotransporter. They
are not incorporated into cellular proteins because there are no codons for these amino acids] Ornithine is
regenerated with each turn of the urea cycle, much in the same way that oxaloacetate is regenerated by
the reactions of the citric acid cycle.

3. Synthesis of argininosuccinate: Argininosuccinate synthetase combines citrulline with aspartate to
form argininosuccinate. The α-amino group of aspartate provides the second nitrogen that is ultimately
incorporated into urea. The formation of argininosuccinate is driven by the cleavage of ATP to adenosine
monophosphate (AMP) and pyrophosphate. This is the third and final molecule of ATP consumed in the
formation of urea.

4. Cleavage of argininosuccinate: Argininosuccinate is cleaved by argininosuccinate lyase to yield
arginine and fumarate. The arginine formed by this reaction serves as the immediate precursor of urea.
Fumarate produced in the urea cycle is hydrated to malate, providing a link with several metabolic
pathways. For example, the malate can be transported into the mitochondria via the malate shuttle, reenter
the tricarboxylic acid cycle, and get oxidized to oxaloacetate (OAA), which can be used for
gluconeogenesis . Alternatively, the OAA can be converted to aspartate via transamination and can enter
the urea cycle
5. Cleavage of arginine to ornithine and urea: Arginase cleaves arginine to ornithine and urea, and
occurs almost exclusively in the liver. Thus, whereas other tissues, such as the kidney, can synthesize
arginine by these reactions, only the liver can cleave arginine and, thereby, synthesize urea.
Fate of urea: Urea diffuses from the liver, and is transported in the blood to the kidneys, where it is filtered
and excreted in the urine. A portion of the urea diffuses from the blood into the intestine, and is cleaved to
CO2 and NH3 by bacterial urease. This ammonia is partly lost in the feces, and is partly reabsorbed into
the blood. In patients with kidney failure, plasma urea levels are elevated, promoting a greater transfer of
urea from blood into the gut.