Amino Acids Biochemistry 2022

BIOMOLECULES BIOMOLECULES
CHEMISTRY OF AMINO ACIDS AMINO ACIDS
6-Dec-22 HNS103 EGK UON-2021 6-Dec-22 HNS103 EGK UON-2021

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Summary Nature of Amino Acids (AAs)

¡ About 300 of them found in nature of
¡ Amino acids Classification which 20 are known to be building blocks
¡ Chemistry of Amino acids for proteins
¡ Functional Significance of R-groups
¡ Monomers of all peptides and
¡ Non-standard Amino Acids polypeptides
¡ Acid-Base Properties of AAs
¡ non-protein associated AAs perform
¡ Other chemical properties
specialized functions
¡ The peptide Bond and peptide formation
¡ Several of the AAs found in proteins also
serve other functions
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Properties of Amino Acids Structure

¡ capacity to polymerize ¡ The AAs in peptides and proteins consist of a
¡ novel acid-base properties l A Hydrogen atom (H)
l carboxylic acid (-COOH)
¡ varied structure and chemical functionality
l an amino (-NH2)
¡ chirality l A distinct side chain or R group
¡ All are attached to the same tetrahedral
carbon atom, the alpha (α)-carbon.

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1
Basic Amino Acid Structure
General Structure of an Amino Acid
§ α-carbon is chiral (except for
glycine)
§ at pH 7.0 uncharged amino

acids are zwitterions
Chiral center
§amino acids have a

tetrahedral structure
Major
determinant of
their properties
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7 8
Nonsuperimposable Mirror Images of Each Other

General structure of AAs
¡ All the 20 standard AAs have a common

general structure
¡ The α-carbon is a chiral centre with 4 different

substituent gps AAs occur as sterioisomers (enantiomers) in two different
arrangements
AAs occur in two configurations, Levorotatory and
Dextrorotatory
Naturally occurring amino acids are exclusively of L-form,
Most amino acids have
HNS103 EGKmore than one carbon atom
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D- sterioisomers have only EGK
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been found in bacteria
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Classification of Amino Acids AA Classifications cont’d

¡ AAs differ from each other in their ¡ Classes based polarity or tendency to interact
1. Side chain structure with water at biological pH (pH 7.0)
2. Size
¡ Polarity varies from nonpolar (Hydrophobic)
3. Electric Charge to highly polar (hydrophilic)
¡ Classification based on: ¡ Hydrophobic amino acids tend to repel the
¡ Chemical Nature of Side chain (R group) aqueous environment
¡ Polarity, charge ¡ hydrophilic amino acids tend to interact with the
¡ Type of R group aqueous environment and are involved in
formation of H-bonds
¡ Hydroxyl, carboxyl, sulfuryl
¡ There are five major classes and here AAs
differ by polarity, size and shape
¡ The R chains influence the stability of the AAs
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11 12
2
Smallest of
all side chains
Nonpolar, but
does not make
Classification of any real
Amino Acids contribution to
hdrophpbic
interaction
Adds to the
flexibility of
polypeptides
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13 14
Nonpolar and
hydrophobic
Aliphatic side
chain
Tend to cluster
within proteins
Nonpolar
thioester group
Stabilize protein
structure
through
hydrophobic
interactions

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15 16
Aromatic side
chains, relatively
nonpolar Site for
phosphorylation
Participate in
hydrophobic Can form H-
interaction bond, & has
some polar
characteristics
A phenyl A phenol An indole

group group group Present in
linked to Enzyme OH O
- + H+
linked to linked to
alanine alanine alanine active sites
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17 18
3
Absorbs UV light
Makes them
significantly
more polar than
phelnylalanine
Accounts for the characteristic strong absorbance of light

by most proteins (at 280nm)
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19 20
Soluble in
water & more
hydrophilic
Present in active
sites of many
Side chains contain protein digesting
functional groups that
enzymes (Serine
can H- bond with proteases)
water

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Cystine
Has a polar
sulfhydryl group
Could dissociate to Oxidized state can

a nonprotonated form disulfide
form linkage
SH S
- + H+ SH S
- + H+
Present in active
sites of some
enzymes (cysteine
protease)
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23 24
4
Sulfhydryl groups
are polar
(hydrophilic)
Disulfide linkages
are highly nonpolar
(hydrophobic)
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Basic Amino Acids-Positively charged

AAs
The a-amino group
is held in a rigid ¡ Hydrophillic nitrogenous bases
ring structure ¡ Positively charged at physiological pH
involving the side ¡ Histidine – imidazole ring protonated/ionized,
chain -CH2 groups
only amino acid that functions as buffer in physiol
range.
Reduces structural ¡ Lysine - diamino acid, protonated at pH 7.0
flexibility of the ¡ Arginine - guianidinium ion always protonated,
polypeptides when most basic amino acid
present

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27 28
Has a 2nd primary

amine group at
the e position
Contributes to its
hydrophilicity
Have significant positive charge at pH 7.0 (neutral pH)

Rest of the side
Very hydrophilic chain is aliphatic
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5
Aliphatic part
is smaller than
in Lysine
Contributes to its
Has a positively
hydrophilicity Has an
charged guanidino
HNS103 EGK group
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imidazole group
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Present in many Acidic Amino Acids

enzyme active
sites • Contain carboxyl groups (weaker acids than a-
carboxyl-group)
Serves as a
• Negatively charged at physiological pH, present as
proton donor or
proton acceptor conjugate bases (therefore –ate not –ic acids)
• Carboxyl groups function as nucleophiles in some

enzymatic reactions
+ H +
H

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Both have second

carboxyl group
Have R groups with net negative charge at pH 7.0

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Essential/Non-Essential Amino Acids
¡ Essential –histidine, isoleucine, leucine, lysine,

methionine, phenylalanine, threonine,
tryptophan, valine
¡ Non-essential – alanine, arginine, aspartate,

asparagine, cysteine, glutamate, glutamine,
Present in active glycine, proline, serine, tyrosine
sites of enzymes

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Nonstandard Amino Acids

¡ 4-hydroxyproline derived
from Proline through
Hydroxylation
¡ δ-hydroxylysine derived
from Lysine through
hydroxylation d
¡ Both found in Collagen.

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39 40

¡ A constituent of myosin, a
contractile protein of muscle. ¡ Derived from glutamate.
¡ Found in certain Ca 2+ binding proteins

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¡ Introduced during protein synthesis.

¡ Contains Selenium instead of sulfur.
¡ Derived from 4 molecules of Lysine.

¡ Found in Elastin, a fibrous protein.

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ASSIGNMENT
1. Classify AAs based on the type of R-

group

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Acid-Base Properties of AAs Acid-Base Properties of AAs

¡ The a-COOH and a-NH2 groups in amino
¡ Amino acids in aqueous solution are
acids are capable of ionizing when placed in an
ionized and can act as an acid or a base
aqueous medium
¡ These acid-base properties gives proteins
¡ as are the acidic and basic R-groups of the
their physical and biological properties
amino acids).
¡ Separation, identification and
¡ The following ionic equilibrium reactions may
quantification of AA composition and
be written:
sequence is based on these acid-base
l R-COOH <--------> R-COO- + H+
behavior
l R-NH3 + <---------> R-NH2 + H +

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Acid dissociation constants of
Ionization of AAs weak acids
¡ AAs act like weak organic acids ¡ That tendency is defined by the equilibrium
¡ R-COOH- protonated Form (proton donor) constant for a reversible rxn
¡ R-NH3- “ HA(aq) H+(aq) + A−(aq)
Ka = [H+] [A-]
¡ R-COO- deprotonated Form (Conjugate
[HA]
base)
Ka is the acid dissociation constant
¡ R-NH2- “
¡ The stronger the acid, the higher the dissociation
¡ Acidic and Basic side gps also ionize
constant and vice versa

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Dissociation constant of amino acids

Amino acids as buffers
¡ The acidic strength of the COOH, NH3 and
ionizable R-groups in amino acids can be defined
¡ AAs can act as buffers which resist pH changes
l Dissociation constant, Ka or the pKa just like in weak on addition or removal of protons
acids
¡ When the concentration of the proton donor
exactly equals that of the proton acceptor
¡ The COOH, NH3 as well as the ionizable R-Gp of each
AA has a distinct pKa value
(base), the buffering power is maximal
¡ At this point pH changes least on addition or
removal of H+ and OH-
¡ In solution, they can protonate and deprotonate and thus
behave like weak acids ¡ The pH at this point in the titration curve is
equal to the pKa
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Titration Curves of Amino Acids Titration of Glycine Cont’d
¡ a -COOH, a-NH2 and Ionizable R-Gps in ¡ At low pH, the COOH gp will lose its
solution consist of a proton donor and its
conjugate base (proton acceptor) proton and at a certain midpoint, there
¡ A titration curve indicates the changes in pH as are equal concentrations of the proton
the protons are removed or added e.g Glycine donor and acceptor
+NH -CH -COOH
3 2
¡ +NH3-CH2-COOH +NH -CH -COO- +
3 2 H+
¡ At low pH, the predominant form is
¡ +NH3-CH2-COOH fully protonated form ¡ The pH at this midpoint = pKa = 2.34,
¡ After titration begins, the COOH gp is very thus the COOH gp has a pKa of 2.34
acidic and ionizes first

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Titration of Glycine Cont’d Titration of Gly Cont’d
¡ As the titration continues, all the Gly is in the
form +NH3-CH2-COO- called zwitterion (fully ¡ The a-NH2 has a pKa of 9.6
ionized form). An AA has a characteristic pH ¡ The titration is complete at pH 12 at
called Isoelectric pH (pI) where it exists as a
zwitterion. which point, the predominant form of
¡ At pI, the AA has no net charge, is electrically Gly is NH2-CH2-COO-
neutral and remains stationary in an electric ¡ So Gly has two pKa, one for the COOH
field. pI of Gly = 5.97
and the other for the NH3 gp
¡ Removal of the 2nd proton from NH3 begins
and at the midpoint, it exists as
+
¡ NH3-CH2-COO- NH2-CH2-COO-
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55 56
Isoelectric pH or point Titration of AAs

¡ An AA has a characteristic pH called isoelectric ¡ The net charge (the sum of all the charged
point (pI). The AA exists as a dipolar form groups present) of any amino acid, peptide or
with no net charge (zwitterion) protein, will depend upon the pH of the
¡ When the net charge of an amino acid or surrounding aqueous environment.
protein is zero the pH will be equivalent to the
isoelectric point: pI. ¡ As the pH of a solution of an amino acid or
protein changes so too does the net charge.
¡ In an electric field, it remains stationary
¡ This phenomenon can be observed during the
¡ For AA with no ionizable R gp, the pI is the
average of the two pKa values titration of any amino acid or protein.
¡ pI = pKa1 + pKa2/2= 5.97
¡ At any pH above the pI, Gly has a net –ve charge and at
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any pHHNS103
below the pI,
EGK
it has a net +ve charge
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Titration Curve for Alanine Titration Curve

for Glutamic Acid
pK1 carboxylic acid = 2.34
pK2 amino group = 9.69 pK1 carboxylic acid = 2.2
pI = (pK1+ pK2)/2 pK2 R group = 4.3
pK3 amino group = 9.7
pI = (pK1+ pK2)/2
pI = (2.2+4.3)/2
pI = 3.25
pI (isoelectric point) = the pH at which the number of positive and

negative charges on a population of molecules is equal (i.e. no net charge).
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Titration Curve for pKa’s of charged amino acids R-groups
Lysine
¡ Aspartate/Glutamate = 4.0
pK1 carboxylic acid = 2.2 ¡ Histidine = 6.0
pK2 amino group = 9.0
pK3 R group = 10.5 ¡ Cysteine = 8.4
pI = (pK2+ pK3)/2 ¡ Tyrosine = 10.5
pI = (9+10.5)/2
pI = 9.75 ¡ Lysine = 10.5
¡ Arginine = 12.5

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Assignment The Peptide Bond

¡ Amino acids combine to form proteins.
¡ What is the structure of Ser at a pH of 2?
¡ They combine by the reaction of the amine part
¡ What is the structure of Asp at pH 13? of one amino acid with the carboxylic acid part
of the second to form an amide bond.
¡ In biochemistry, these amide linkages are called
peptide bonds. The acid on the end with the
amine still on it is called the N terminal and the
acid with the carboxylic acid group still attached
is called the C terminal.

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63 64
Standard Amino Acids Formation of a Peptide Bond

A V L I G
M S T P M
F Y W R K
H E D N Q M V V N
G
MV VN
A L WF
A G M
P
K P MG T R
M P
M Q R R L
G S H L W S G I
L L Y WS G I
V
Polypeptide 1 A
Polypeptide 2
65 66
65 66
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A Pentapeptide Protein Nomenclature
¡ Peptides 2 – 50 amino acids

¡ Proteins >50 amino acids
¡ Amino acid with free a-amino group is the amino-
terminal or N-terminal residue
¡ Amino acid with free a-carboxyl group is the carboxyl-
terminal or C-terminal residue
¡ Three letter code – Met-Gly-Glu-Thr-Arg-His
¡ Single letter code – M-G-E-T-R-H

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67 68
Peptides Example of a tripeptide

¡ These small (relatively few amino acid) proteins ¡ So, for example, this molecule would be called
are called peptides. Glutamyl-methionyl-lysine
¡ When naming a peptide, the C terminal amino acid O O O
is treated as the parent and all the other amino H2N CH C N CH C

H
N CH C OH
acids are named as substituents. H
CH2 CH2 CH2

¡ Written in the order from the N terminal end to the
C terminal end. CH2 CH2 CH2
¡ Amino acids are named as substituents by C O S CH2

changing the ine at the end of the name to yl.
NH2 CH3 CH2
NH2
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69 70
Reactions of Amino Acids

More examples
¡ Free amino acids (excluding proline)
¡ So what is the name of this peptide? share similar chemical reactivities due to
the common amino and carboxyl groups.
O O
¡ Different amino acid side chains have
H
H2N CH C N CH C OH different chemical reactivities.
¡ Therefore, reactivities of different
CH2 CH2
proteins reflects the composition of the
CH CH3 SH
unique sequence of amino acids in their
structure.
CH3
71 72
71 72
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Reactions of AAs
Ninhydrin reaction
¡ The Ninhydrin reaction is used to detect and quantify AAs
¡ Ninhydrin degrades amino acids into aldehydes, ¡ Proline yields a yellow colour
ammonia, and CO2 through a series of reactions ¡ The intensity of the colour is proportional to the AA
¡ The net result is ninhydrin in a partially reduced form concentration
hydrindantin: Ninhydrin then condenses with ammonia ¡ Comparing the absorbance to a standard solution gives
and hydrindantin to produce an intensely blue or purple the AA concentration
pigment, sometimes called Ruhemann's purple ¡ A ninhydrin solution in ethanol or other volatile solvents
is often used as a developer for amino acids in paper
chromatography or thin layer chromatography.
¡ Ninhydrin spray is also used on crime scenes to
visualize fingerprints, which contain trace amounts of
amino acids
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N-terminal Labeling Reagents

¡ The first amino acid residue at the N-terminus of a protein is identified by
N-terminal sequence determination reaction of the protein with one of the reagents such as DNF (1-fluoro-2,4-
¡ There are three major chemical techniques for sequencing dinitrobenzene).
peptides and proteins from the N-terminus. ¡ Following labeling the protein is hydrolyzed and the modified N-terminal
¡ These are the Sanger, Dansyl chloride and Edman techniques. residue is identified by chromatography. FDNB is also known as Sanger’s
¡ Sanger's Reagent: This technique utilizes 2,4- reagent. Other more sensitive fluorescent reagents (e.g., dansyl chloride
dinitrofluorobenzene (DNF) which reacts with the N-terminal and dabsyl chloride) now are used when working with small quantities of a
residue under alkaline conditions. purified protein. N-terminal labeling also identifies which sequenced
¡ The derivatized amino acid can be hydrolyzed and will be labeled polypeptide occurs first in the complete sequence of the protein
with a dinitrobenzene group that imparts a yellow color to the
amino acid.
¡ Separation of the modified amino acids (DNP-derivative) by
electrophoresis and comparison with the migration of DNP-
derivative standards allows for the identification of the N-terminal
amino acid.
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75 76
Edman degradation:
¡ Edman degradation allows for additional amino acid sequence to be
Dansyl chloride obtained from the N-terminus inward.
¡ Like DNF, dansyl chloride reacts with the N-terminal ¡ This method utilizes phenylisothiocyanate to react with the N-terminal
residue under alkaline conditions. residue under alkaline conditions.
¡ The resultant phenylthiocarbamyl derivatized amino acid is hydrolyzed
¡ Analysis of the modified amino acids is carried out in anhydrous acid. The hydrolysis reaction results in a rearrangement of
similarly to the Sanger method except that the dansylated the released N-terminal residue to a phenylthiohydantoin derivative.
amino acids are detected by fluorescence.
¡ As in the Sanger and Dansyl chloride methods, the N-terminal residue is
¡ This imparts a higher sensitivity into this technique over tagged with an identifiable marker, however, the added advantage of the
that of the Sanger method. Edman process is that the remainder of the peptide is intact.
¡ The entire sequence of reactions can be repeated over and over to obtain
the sequences of the peptide. Using this method it is possible to obtain
the entire sequence of peptides.
¡ This process has subsequently been automated to allow rapid and
EGK UON HBC205/210 Biochemistry of
efficient sequencing of even extremely small quantities of peptide.
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Edman Degradation Sequencing
The chemical sequencing is based on a two-step procedure developed by Paper chromatography
Pehr Edman. The reagent phenylisothiocyanate is used to label the N-
terminal residue of the polypeptide, which then is released and identified ¡ Paper chromatography is a technique that involves placing
without damaging the remainder of the polypeptide.
a small dot or line of sample solution onto a strip of
Then the process is repeated. About 40 amino acids can be sequenced for
each polypeptide. Polypeptides themselves are generated by enzymatic or chromatography paper.
chemical fragmentation of the starting protein . ¡ The paper is placed in a jar containing a shallow layer of
solvent and sealed.
¡ As the solvent rises through the paper, it meets the sample
mixture which starts to travel up the paper with the solvent.
¡ This paper is made of cellulose, a polar substance, and the
compounds within the mixture travel farther if they are
non-polar.
¡ More polar substances bond with the cellulose paper more
quickly, and therefore do not travel as far.
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Ion Exchange Chromatography
¡ Each individual AAs exhibits a distinct overall

net charge at a given pH.
¡ Some will be negatively charged and some will
be positively charged at the same pH. This
property of Aas is the basis for ion exchange
chromatography.
¡ Resins are used that are either negatively
(cation exchanger) or positively (anion
exchanger) charged
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14

Amino Acids Biochemistry 2022

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BIOMOLECULES BIOMOLECULES

CHEMISTRY OF AMINO ACIDS AMINO ACIDS

6-Dec-22 HNS103 EGK UON-2021 6-Dec-22 HNS103 EGK UON-2021

Summary Nature of Amino Acids (AAs)

Properties of Amino Acids Structure

6-Dec-22 HNS103 EGK UON-2021 6-Dec-22 HNS103 EGK UON-2021

§ at pH 7.0 uncharged amino

§amino acids have a

Nonsuperimposable Mirror Images of Each Other

¡ All the 20 standard AAs have a common

¡ The α-carbon is a chiral centre with 4 different

Classification of Amino Acids AA Classifications cont’d

6-Dec-22 HNS103 EGK UON-2021 6-Dec-22 HNS103 EGK UON-2021

A phenyl A phenol An indole

Accounts for the characteristic strong absorbance of light

6-Dec-22 HNS103 EGK UON-2021 6-Dec-22 HNS103 EGK UON-2021

Could dissociate to Oxidized state can

Basic Amino Acids-Positively charged

6-Dec-22 HNS103 EGK UON-2021 6-Dec-22 HNS103 EGK UON-2021

Has a 2nd primary

Have significant positive charge at pH 7.0 (neutral pH)

Present in many Acidic Amino Acids

• Carboxyl groups function as nucleophiles in some

6-Dec-22 HNS103 EGK UON-2021 6-Dec-22 HNS103 EGK UON-2021

Both have second

Have R groups with net negative charge at pH 7.0

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¡ Essential –histidine, isoleucine, leucine, lysine,

¡ Non-essential – alanine, arginine, aspartate,

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Nonstandard Amino Acids

¡ Both found in Collagen.

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Nonstandard Amino Acids

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¡ Introduced during protein synthesis.

¡ Derived from 4 molecules of Lysine.

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Nonstandard Amino Acids

1. Classify AAs based on the type of R-

6-Dec-22 HNS103 EGK UON-2021 6-Dec-22 HNS103 EGK UON-2021

Acid-Base Properties of AAs Acid-Base Properties of AAs

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Dissociation constant of amino acids

Titration Curves of Amino Acids Titration of Glycine Cont’d

6-Dec-22 HNS103 EGK UON-2021 6-Dec-22 HNS103 EGK UON-2021

Isoelectric pH or point Titration of AAs

Titration Curve for Alanine Titration Curve

pI (isoelectric point) = the pH at which the number of positive and

6-Dec-22 HNS103 EGK UON-2021 6-Dec-22 HNS103 EGK UON-2021

Assignment The Peptide Bond

6-Dec-22 HNS103 EGK UON-2021 6-Dec-22 HNS103 EGK UON-2021

Standard Amino Acids Formation of a Peptide Bond

¡ Peptides 2 – 50 amino acids

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Peptides Example of a tripeptide

is treated as the parent and all the other amino H2N CH C N CH C

CH2 CH2 CH2

¡ Amino acids are named as substituents by C O S CH2

Reactions of Amino Acids

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