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• Amino acids are one of the first organic molecules to appear on Earth.
• The isolated amino acids are white crystalline solids. They have high melting and boiling
points due to their unique properties of ionic and dipolar in nature.
• Amino acids are the building blocks of the proteins.
Aside from their role in composing proteins, amino acids have many biologically important
(1) They are also energy metabolites, and many of them are essential nutrients.
(2) Amino acids can often function as chemical messengers in communication between cells.
For example, Arvid Carlsson discovered in 1957 that the amine 3-hydroxytyramine
(dopamine) was not only a precursor for the synthesis of adrenaline from tyrosine, but is
also a key neurotransmitter.
(3) Certain amino acids — such as citrulline and ornithine, which are intermediates in urea
biosynthesis — are important intermediaries in various pathways involving nitrogenous
(4) Although other amino acids are important in several pathways, S-adenosylmethionine
acts as a universal methylating agent.
Importance of Amino Acids
How many amino acids are possible theoretically?
Theoretically infinite amino acids
Plants have more than 250 non-protein amino acids involved in defenses as secondary
metabolites (Swain, 1977).
To date, scientists have discovered more than five hundred amino acids in nature, but only
twenty-two participate in translation (Gutiérrez-Preciado et al., 2010).
More than 700 amino acids occur naturally, but 20 of them are especially important.
How many amino acids are found in nature?
Citrulline Canaline Lanthionine
Non-standard amino acids are used as
(1) Amino acids for Non-Ribosomal Peptide Synthesis (NRPS) system
(2) Metabolic intermediates
(3) Defense mechanism in plants
(4) Others if any
What are the role of non-standard amino acids?
Leucine Aspartic acid
Methionine Glutamic acid
Selenocysteine (Bock 2000)
Pyrrolysine (Srinivasan et al. 2002)
Amino acids used for protein synthesis
20100=1.27 x 10130
Are 20 amino acids enough to code
for all proteins?
Amino acids are electrolytes i.e. they are electric conductive.
• Due to their dipolar in nature, they are also called zwitterions (or amphoteric behavior).
The acid-base properties of amino acids
At pH 7, the amino and carboxyl groups are charged. But, over a pH range from 1 to 14,
these groups exhibit a series of equilibrium involving binding and dissociation of a proton,
reflecting weak acids or bases.
Their acid-base properties are important as it influences the eventual properties of proteins,
permits methods of identification of different amino acids and dictates their reactivity.
The amino group, characterized by a basic pK value of ~9, is a weak base.
While the amino group ionizes around pH 9, the carboxyl group remains charged until a
pH of ~2 is reached. At this pH, a proton binds neutralizing the charge of the carboxyl
HA + H2O H3O+ + A-
Where HA, the proton donor, is either –COOH or –NH3
+ and A- the protein acceptor is
either –COO- or –NH2.
The extent of ionization depends on the equilibrium constant
K = [H+][A-]/[HA]
And it becomes straightforward to derive the relationship (Known as Henderson-Hasselbalch
pH = pK + log[A-]/[HA]
• One of the most important consequences of the asymmetric α-carbon is that it
gives rise to a chiral centre and the presence of two isomers (mirror image). The
two forms are known as L and D amino acids. All naturally occurring amino acids
found in proteins belong to the L absolute configuration. The Cα atom is
asymmetric or chiral or optically active molecule except for Glycine.
• The D and L stereoisomers of any amino acid have identical properties with two
exceptions: they rotate plane-polarized light in opposite directions and they
exhibit different reactivity with asymmetric reagents. This latter point is important
in protein synthesis where D amino acids are effective inhibitors.
Amino acids: Enantiomer (Stereoisomer)
The amino, carboxyl, hydrogen and R groups are arranged tetrahedrally around the central -
Amino acid classification
Non-polar (hydrophobic): Ala, Ile, Leu, Phe, Pro, Trp, Val
Positively charged: Arg, His, Lys
Polar, but uncharged: Asn, Gln, Ser, Thr
Negatively charged: Asp, Glu
Thiol group: Cys, Met
Aromatic: Phe, Trp, Tyr
Chemical and physical properties of amino acids
Glycine is the simplest amino acid. It is the only one in the
table that is achiral i.e. lacks an asymmetric centre and does not
occur as R/S isomers, possess little intrinsic chemical reactivity,
has conformational flexibility.
Glycine (Gly or G)
Amino acids having hydroxyl groups
Serine (Ser or S) Threonine (Thr or T)
Generally phosphorylated, strong nucleophile in
the presence of His, Asp.
Aliphatic amino acids
Alanine (Ala or A) Valine (Val or V) Leucine (Leu or L) Isoleucine (Ile or I)
Inactive side chain, hydrophobic
Acidic or Negatively charged amino acids
Aspartic acid (Asp or D) Glutamic acid (Glu or E)
Negative charge under physiological conditions,
exhibit chemical reactions including
esterification with alcohols or coupling with
amines, chelators of divalent metal ions.
Asparagine (Asn or N) Glutamine (Gln or Q)
Amino acids having amino groups
Unreactive group that is polar and acts as
hydrogen bond donor and acceptor, labile at
alkaline pH or extreme temperature being
deamidated to form the corresponding acidic
Basic or Positively charged amino acids
Lysine (Lys or K) Arginine (Arg or R)
Most basic side chain of Arg (pK=12). Lysine is strong basic and interacts with negatively
charged atoms. Lys can also go methylation, acetylation, arylation and acylation. One of the
most popular lysine modifications involves adding a nitrobenzene derivative (colored).
Methylation preserves the positive charge on the side chain.
In fungi, trimethylated lysine residues are found as natural components of proteins.
One of the most important reactions occurring with Lys is the reaction with aldehydes to form
a Schiff base. The reaction is important within the cell because pyridoxal phosphate reacts
with amino group of Lys and is found in the many enzyme's active site.
Amino acids containing sulfur
Methionine (Met or M)
The sulfur atom of methionine can be oxidized to form first a sulfoxide and finally a sulfone
derivative. This form of oxidative damage is known to occur in proteins and the reaction
scheme involves progressive addition of oxygen atoms.
The sulfur atom of Met is readily methylated using methyl
iodide in a reaction that is often used to put label on Met via
Sulfur of Met interacts with heavy metal complexes
particularly those involving mercury and platinum such as
K2PtCl4 or HgCl2 and these have proved extremely useful in
the formation of isomorphous heavy atom derivatives in
One of the most important reactions of Met involves cyanogen bromide – a reagent that
breaks the polypeptide chain on the C-terminal side of Met by sequestering the carbonyl
group of the next peptide bond in a reaction involving water and leading to formation of a
homoserine lactone. This reaction is used to split polypeptide chains into smaller fragments
for protein sequencing.
The thiol group ionizes at alkaline pH (~8.5) to form a reactive thioate anion (S-). This thiolate
anion reacts rapidly with many compounds, but the most importantly includes other thiols or
disulfides in exchange type reactions occuring at neutral to alkaline pH. A common reaction
of this type is between Cys and Ellman’s reagent (Dithionitrobenzoic acid DTNB). The
aromatic disulfide unergoes exchange with reactive thiolate anions forming a colored aromatic
thiol – nitrothiobenzoate. The benzoate anion absorbs intensely at 416 nm allowing the
concentration of free thiol groups to be accurately estimated in biological systems. Thiols are
oxidized by molecular oxygen in reactions catalyzed by trace amounts of transition metlas
including Cu and Fe. More potent oxidants such as performic acid oxidize the thiol groups to
a sulfonate (SO3
2-) and this reaction has been exploited as a method of irreversibly breaking
disulfide bridges to form two cysteic acid residues. More frequently, disulfide bonds are
broken by mercaptoethanol or dithiothreitol, dodium borohydride or molecular hydrogen.
Cysteine (Cys or C)
The sulfur group of Cys is more reactive than that of Met.
Functional group of Cys is called Thiol, sulfhydryl or mercapto. It is
the most reactive side chain found amongst the 20 naturally occuring
amino acids undergoing many chemical reactions with diverse
reagents. For example, some enzyme use Cys in their active site. It
can also form disulfide bonds.
Proline (Pro or P)
Unique side chain that covalently bonds with the backbone
nitrogen atom to form a cyclic pyrrolidine ring with groups
lacking reactivity. One of the few reactions involving prolyl side
chains is enzyme-catalyzed hydroxylation.
Hisdine (His or H)
pK of around 7.0. In its ionized state, it has positive charge whilst in
the unionized state the side chain remains neutral.
Experimental evidence suggests that the hydrogen atom is usually
located on the NE2 nitrogen but upon further protonation, it moves to
ND1 nitrogen. Thus, it exists in resonance between two nitrogen
atoms. The unprotonated nitrogen of the uncharged imidazole ring is a
potent nucleophile and has a capacity for the hydrogen bonding.
Aromatic amino acids
Phenylalanine (Phe or F) Tyrosine (Tyr or Y) Tryptophan (Trp or W)
They absorve UV light and are responsible for the absorbance and the fluorescence of proteins
frequently measure between 250 and 350 nm. In this region, the molar extinction coefficients
(an indication of how much light is absorbed at a given wavelength) of Phe, Tyr and Trp are
not equal. Trp exhibits a molar extinction coeff. ~four times that of Tyr and ~28 times greater
than Phe. Almost all spectrophotometric measurements of a protein’s absorbance at 280 nm
reflect the intrinsic Trp content of that protein. At equivalent molar concentrations, proteins
with a high number of tryptophan residues will give a much larger absorbance at 280 nm
when compared with proteins possessing a lower Trp content.
Amino acid Properties
Phenylalanine The aromatic ring of Phe is chemically inert and thus resistant to chemical
modification. However, it forms pi-pi interactions with other aromatic rings.
Tyrosine It is more reactive than Phe due to the presence of OH group. Nucleophiles such as
nitrating agents or activated forms of iodide react with tyrosine side chains in proteins
and change the acid-base properties of the ring.
Tryptophan The indole side chain is the largest side chain occurring in proteins and is responsible
for most of the intrinsic absorbace and fluorescence. As a crude approximation, the
molar extinction coeff. Of a protein at 280 nm may be estimated by adding up the
number of Tryp residues found in the sequence and multiplying by 5800.
Spectroscopic properties of the aromatic amino acids
Amino acid Absorbance Fluorescence
λmax (nm) ε (M-1 cm-1) λmax (nm) Quantum yield
Phe 257.4 197 282 0.04
Tyr 274.6 1420 303 0.21
Trp 279.8 5600 348 0.20
Detection, identification and quantification of amino acids and
The concentration of a protein solution is calculated using the data in Table and Beer-
A280 = ε280 x c x l (where A: absorbance, ε: molar absorptivity coeff, c: concentration in moles
dm-3 and l: light path length normally 1 cm).
The specific optical rotation of selected amino acids
L-amino acid [ ]D (H2O) L-Amino acid [ ]D (H2O)
Alanine 1.8 Isoleucine 12.4
Arginine 12.5 Leucine -11.0
Cysteine -16.5 Phenylalanine -34.5
Glutamic acid 12.0 Threonine -28.5
Histidine -38.5 Tryptophan -33.7
Amino acid composition in proteins
Based on the UnitProtKB/trEMBL database (release no.: 2013_06 on 29th May 2013) – It
contains 3,55,02,518 sequence entries and 11,38,44,40,438 amino acids.
Amino acid Frequency (%)
Amino acid Frequency (%)
Legend: gray = aliphatic, red = acidic, green = small hydroxy, blue = basic, black = aromatic,
white = amide, yellow = sulfur