The document discusses amino acids and peptides. It provides information on: 1. The structure and properties of the 20 common amino acids found in proteins, including their ionization states and isoelectric points. 2. How amino acids can act as acids or bases depending on pH due to ionization of their carboxyl and amino groups. 3. The formation of peptide bonds between amino acids and how this leads to the creation of polypeptides and proteins of physiological significance.

1. AMINO ACIDS
AND
PEPTIDES
by
Ramon S. del Fierro, Ph.D. (Tokyo)
Professor of Biochemistry

2. TOPIC OUTLINE
 Nature of Amino Acids
 Amino Acid Structure and Properties
 Types of Amino acids
 Ionization/Titration of Amino Acids
 Isoelectric pH
 Nature of Peptide Bonds
 Formation of Peptides
 Peptides of Physiological Significance

3. Fundamentals
• While their name implies that amino acids are
compounds that contain an amino (—NH2)
group and a carboxyl (—COOH) group, these
groups are actually present as —NH3
+ and
—COO–, respectively.
• They are classified as a, b, g, etc. amino acids
according to the carbon that bears the
nitrogen.

4. Amino Acids
NH3
+
COO
–
an a-amino acid that is cyclic in
structure; an intermediate in the
biosynthesis of ethylene
+
H3NCH2CH2COO– a b-amino acid that is one of
the structural units present in
Coenzyme A
+
H3NCH2CH2CH2COO–
a g-amino acid involved in
the transmission of nerve
impulses
a
b
g
1-amino-cyclopropane
β-alanine
γ-aminobutyric acid
a
a
b

5. Amino Acids
• Amino acid: a compound that contains
both an amino group and a carboxyl
group
• a-Amino acid has an amino group
attached to the carbon adjacent to
the carboxyl group
• a-carbon is also bound to side
chain group, R
• R gives identity to amino acid
• Two stereoisomers of amino acids
are designated L- or D- based on
similarity to glyceraldehdye

6. Amino acid
• Chiral center
• Optically active: rotate
plane-polarized light
• Amino acid in protein:
L - stereoisomer
α
Amino group
Carboxyl group
R (Alkyl or Aryl) group

7. Absolute configuration: D,L system
Not all L-amino acids are levorotatory (rotating polarized light to the left)

8. Specifying configuration: R S system
S configuration R configuration

9. Amino Acid Structure and Properties
• With the exception of glycine, all protein-derived
amino acids have at least one stereocenter
(the a-carbon) and are chiral (stereoisomers)
• the vast majority of a-amino acids have the
L-configuration at the a-carbon (Proline is usually D)
• Side-chain carbons in other amino acids designated
with Greek symbols, starting at carbon (bg…etc)
• Amino acids can be referred to by three-letter or
one-letter codes

10. The 20 Key Amino Acids
• More than 700 amino acids occur
naturally, but 20 of them are especially
important.
• These 20 amino acids are the building
blocks of proteins. All are a-amino
acids.
• They differ in respect to the group
attached to the a carbon.

11. Properties and Conventions Associated with the
Common Amino Acids found in Proteins
-COO- -NH2
-OH→-O-

12. Properties and Conventions Associated with the
Common Amino Acids found in Proteins

14. Achiral Carbon
α-carbon

15. Individual Amino Acids
Group A: Nonpolar side chains- Ala (A), Val (V),
Leu (L), Ile (I), Pro (P). Phe (F), Trp (W), Met (M).
• Ala, Val, Leu, Ile, Pro- contain aliphatic hydrocarbon
group. Pro has cyclic structure.
• Phe- hydrocarbon aromatic ring.
• Trp- Indole ring side chain, aromatic.
• Met- Sulfur atom in side chain.

16. Aliphatic Amino Acid Structures
Branched-Chain Amino Acids
(All are Essential Amino Acids)
α
β
a
β
- defective metabolism results in
Maple Syrup Urine
b
(I)
(Ile)

17. Proline, a cyclic amino acid
• Proline (Pro, P)
- has the a-amino nitrogen in the
aliphatic ring system
• The heterocyclic pyrrolidine ring
restricts the geometry of
polypeptides
α
not free

18. Aromatic Amino Acid Structures
Indole group
Phenyl group
β
Both are Essential Amino Acids

19. Phenylalanine
hydroxylase
Phenylalanine

20. 5-Hydroxytryptophan
(5-HTP)
Hydroxylation
Decarboxylation
Serotonin
(5-HT)
Monoamine Oxidase
(MAO)
5-HydroxyIndoleacetic Acid
(5-HIAA)

22. Methionine
Sulfur –containing
Essential Amino Acid

23. Amino Acids (cont’d)
Group B: Neutral Polar side chains- Ser (S), Thr (T),
Tyr (Y), Cys (C), Gln (Q), Asn (N)
• Ser, Thr- Side chain is polar hydroxyl group (-OH)
• Tyr- hydroxyl group (-OH) bonded to aromatic
hydrocarbon group
• Cys- Side chain contains thiol (sulfhdryl) group (-SH)
• Gln, Asn- contain amide bonds in side chain
(-CONH2)

24. Side Chains with Alcohol Groups
• Serine (Ser, S) and Threonine (Thr, T) have
uncharged polar side chains
Catalytic role, phosphorylation,
o-linked glycosylation,
side chain hydrogen bonding
β
Essential
Amino Acid

25. Aromatic amino acid structure
phosphorylation

26. Cysteine
Sulfhydryl group
b

27. Formation of Cystine
Disulfide bond

28. Structures of Asparagine and Glutamine
Amide
β
γ

29. Amino Acids (cont’d)
Group C: Acidic Side Chains: Glu (D), Asp (E)
• Both have a carboxyl group (-COOH) in side chain
• Can lose a proton, forming a carboxylate ion (-COO-)
• These amino acids are negatively charged at
neutral pH

30. Structures of Aspartate, Glutamate
Monoamino Dicarboxylic acids
γ
β

31. Amino Acids (cont’d)
Group D: Basic side chains: His (H), Lys (K), Arg (R)
• Side chains are positively charged at pH 7
• Arg-side chain is a guanido (guanidinium) group
• His-side chain is an imidazole group
• Lys-side chain ε-NH2 group is attached to an aliphatic
hydrocarbon chain

32. Structures of Histidine, Lysine and Arginine
-Guanido group
imidazole group
ε-amino group
All Essential Amino acids

33. Lysine

34. Class Activity :
1. Write the General Molecular Formula of an α-Amino
Acid. Explain the nature of the attached group.
2. Classify amino acids as :
a. Essential and Non-Essential Amino Acids in Man.
b. To its R-group composition :
i. Polar amino acids
ii. Non-Polar amino acids
iii. Acidic amino acids
iv. Basic amino acids
v. Aromatic amino acids
vi. Positively-charged amino acids

35. Class Activity
3. What amino acid is responsible for the formation of
disulfide linkages?
4. Explain the difference between D- and L- forms of
amino acids. Which form is predominantly present in
proteins?
5. In protein enzymes involved in phosphorylation
reactions, which amino acid/s is/are present on its
active site/s?
6. What amino acid is the precursor of :
a. serotonin
b. dopamine

41. Amino Acid summary
Important structural features:
1. All 20 are a-amino acids
2. For 19 of the 20, the a-amino group is primary; for proline,
it is secondary
3. With the exception of glycine, the a-carbon of each is a
stereocenter
4. Isoleucine and threonine contain a second stereocenter
5. Three and one-letter codes

44. Uncommon Amino Acids
• Each derived from
a common amino
acid by a
modification
• hydroxylysine and
hydroxyproline are
found only in a
few connective
tissues
such as collagen
• thyroxine is found
only in the thyroid
gland

45. Uncommon Amino Acids
• Each derived from
a common amino
acid by a
modification
• hydroxyproline
and hydroxlysine
are found only in a
few connective
tissues
such as collagen
• thyroxine is found
only in the thyroid
gland

46. Uncommon Amino Acids
Plant cell wall
Collagen
Collagen
Myosin
Prothrombin
Ca2+ binding protein
Elastin

47. Other Amino Acids
(not constituents of proteins) : Metabolites
Key intermediate in
biosynthesis of Arg
and in urea cycle
Urea

49. Amino Acids can act as Acids and Bases
Cationic
species
Zwitterion Anionic
species
[OH-] [OH-]

50. Ionization of Amino Acids
• In an amino acid, carboxyl group (-) and
amino group (+) are charged at neutral pH.
• In free amino acids, a-carboxyl, and α-amino
groups have titratable protons. Some side chains
do as well

51. Ionization of Amino Acids
• Remember, amino acids without charged groups on side chain
exist in neutral solution as zwitterions with no net charge
+1 0 -1

52. -COOH
Imidazole
-SH
-OH
pK 2~3
pK 9~10
ε-amino
guanidinium

53. Titration of Amino Acids
• When an amino acid is titrated, the titration curve represents the reaction of
each functional group with the hydroxide ion
+1
0
-1
= 2.34 + 9.69
2
= 6.02

54. The characteristic pH at which the net
electric charge is zero is called the
isoelectric point or isoelectric pH,
designated pI.
For glycine, which has no ionizable
group in its side chain, the isoelectric
point is simply the arithmetic mean
of the two pKa values:
+1
+1
0
0
-1
-1
Titration of Alanine with NaOH
2.34 9.60

55. pI = (2.19+4.25)/2 = 3.22
+1 0 -1 -2
+1
0
-1
-2
2.19 4.25 9.67

56. pI = (6.0+9.17)/2 = 7.59
+2
+1
+1
+2 0
0
-1
-1
1.82 6.0 9.17

57. Titration of Histidine with NaOH

58. Acidity: a-COOH Groups
• The average pKa of an a-carboxyl group is 2.19,
which makes them considerably stronger acids than
acetic acid (pKa 4.76)
• the greater acidity of the amino acid carboxyl group is
due to the electron-withdrawing inductive effect of the
-NH3
+ group

59. Basicity: a-NH3
+ groups
• The average value of pKa for an a-NH3
+ group is
9.47, compared with a value of 10.76 for a 2°
alkylammonium ion

60. Basicity (cont’d)
Guanidine Group
• The side chain of arginine is a considerably stronger
base than an aliphatic amine
• basicity of the guanido group is attributed to the large
resonance stabilization of the protonated form relative
to the neutral form
Imidazole Group
• The side chain imidazole group of histidine is a
heterocyclic aromatic amine

61. Ionization vs pH
• Given the value of pKa of each functional group, we
can calculate the ratio of each acid to its conjugate
base as a function of pH
• Consider the ionization of an a-COOH
• writing the acid ionization constant and rearranging
terms gives
[ a-COO H]
[ a-COO -
]
Ka =
[ H3O+
]
=
Ka
[ a-COO H]
[ a-COO -
]
[ H3O+
]
or
pKa = 2.00
aCOO
-
aCOOH + H3 O
+
+ H2 O
[ a-COO H]
[ a-COO -
]
Ka =
[ H3O+
]
=
Ka
[ a-COO H]
[ a-COO -
]
[ H3O+
]
or

62. Ionization vs pH (cont’d)
• substituting the value of Ka (1 x 10-2) for the hydrogen
ion concentration at pH 7.0 (1.0 x 10-7) gives
• at pH 7.0, the a-carboxyl group is virtually 100% in
the ionized or conjugate base form, and has a net
charge of -1
• we can repeat this calculation at any pH and
determine the ratio of [a-COO-] to [a-COOH] and the
net charge on the a-carboxyl at that pH
=
Ka
[ a-COOH]
[ a-COO-
]
[ H3O+
]
= 1.00 x 105
1.00 x 10-7
1.00 x 10-2
=

63. Ionization vs pH (cont’d)
• We can also calculate the ratio of acid to conjugate
base for an a-NH3
+ group; for this calculation,
assume a value 10.0 for pKa
• writing the acid ionization constant and rearranging
gives [ a-NH 2 ]
[ a-NH 3
+
]
Ka
=
[H 3 O+
]
+
pKa = 10.00
aNH2
aNH3
+
H3 O+
+ H2 O

64. Ionization vs pH
• substituting values for Ka of an a-NH3
+ group and
the hydrogen ion concentration at pH 7.0 gives
• at pH 7.0, the ratio of a-NH2 to a-NH3
+ is
approximately 1 to 1000
• at this pH, an a-amino group is 99.9% in the acid
or protonated form and has a charge of +1
[ a-NH 2 ]
[ a-NH 3
+
]
Ka
=
[H 3 O+
]
=
1.00 x 10-10
1.00 x 10-7
= 1.00 x 10-3

65. • We have calculated the ratio of acid to conjugate
base for an a-carboxyl group and an a-amino group
at pH 7.0
• We can do this for any weak acid and its conjugate
base at any pH using the Henderson-Hasselbalch
equation
[weak acid]
[conjugate base]
log
=
pH pKa +
Henderson-Hasselbalch Equation
Proton acceptor
Proton donor

66. Isoelectric pH
• Isoelectric pH, pI: the pH at which the majority of molecules
of a compound in solution have no net charge
• the pI for glycine, for example, falls midway between the pKa
values for the carboxyl and amino groups
• Isoelectric pH values for the 20 protein-derived amino acids are
given in ReferenceTables
pI = 1
2 ( pKa aCOOH + pKa aNH3
+
)
=
2
1 (2.35 + 9.78) = 6.06

67. pKa Values Express the Strengths
of Weak Acids
• The acid strengths of weak acids are expressed as
their pKa
• The imidazole group of histidine and the guanido
group of arginine exist as resonance hybrids with
positive charge distributed between both nitrogens
(his) or all three nitrogens (arg)
• The net charge on an amino acid is the algebraic
sum of all the positively and negatively charged
groups present

68. Typical range of pKa values for
ionizable groups in proteins
Dissociating Group pKa range
α-carboxyl 2.0-3.0
Non α-COOH of Asp or Glu 3.0-4.0
Imidazole of His 6.5-7.4
SH of Cys 8.5-9.0
OH of Tyr 9.5-10.5
α-Amino 9.0-10.0
ε-Amino of Lys 9.8-10.4
Guanidinium of Arg ~12.0

69. pKa Values Vary with the Environment
• The environment of a dissociable group affects its
pKa
• A polar environment favors the charged form :
R-COO- or R-NH3
+
• A non-polar environment favors the uncharged form :
R-COOH or R-NH2
A non-polar environment raises the pKa of a
carboxyl group (making it a weaker acid) but lowers
that of an amino group (making it a stronger acid)
• The presence of adjacent charged groups can
reinforce or counteract solvent effects

70. α-R Groups Determine the Properties
of Amino acids
• Glycine, the smallest amino acid, can be
accommodated in places inaccessible to other amino
acids, it often occurs where peptides bend sharply
• The hydrophobic R-groups typically occur primarily in
the interior of cytosolic proteins
• The charged groups stabilize specific protein
conformations via ionic interactions or salt bonds
• The OH (ser) and SH (cys) act as nucleophiles which
are good in catalysis
• The OH (ser, thr, tyr) participate in regulation of the
activity of enzymes responsible for phosphorylation
reactions

72. Test for Amino Acids - Ninhydrin
O
O
O
O
H
H
Indan-1,2,3-trione
- H2O
O
O
O
Ninhydrin
H2O
C
O
O
N C
O
O
Positive Test
Ruhemann’s Purple

73. Peptide Bonds
• Individual amino acids
can be linked by forming
covalent bonds.
• Peptide bond: the
special name given to
the amide bond between
the a-carboxyl group of
one amino acid and the
a-amino group of
another amino acid

74. Peptides and Proteins
Peptide bond

75. Peptides
• Peptides are compounds in which an amide bond
links the amino group of one a-amino acid and the
carboxyl group of another.
• An amide bond of this type is often referred to as a
peptide bond.
Peptide Bond

76. Alanine and Glycine
CH3
O
C
+
H
C O
–
H3N
O
C
H
H
C
H3N
+
O
–
Ala Gly

77. Alanylglycine
CH3
O
C
H3N
+
H
C
O
C
N
H
H
C O
–
H
• Two a-amino acids are joined by a peptide
bond in alanylglycine. It is a dipeptide.
Peptide bond

78. Alanylglycine
CH3
O
C
H3N
+
H
C
O
C
N
H
H
C O
–
H
Ala—Gly
AG
N-terminus C-terminus

79. Alanylglycine
CH3
O
C
H3N
+
H
C
O
C
N
H
H
C O
–
H
• The peptide bond
is characterized by
a planar geometry.

80. Geometry of Peptide Bond
• the four atoms of a peptide bond and the two alpha
carbons joined to it lie in a plane with bond angles of
120° about C and N
• to account for this geometry, a peptide bond is most
accurately represented as a hybrid of two contributing
structures (resonance structures)
• the hybrid has considerable C-N double bond
character and rotation about the peptide bond is
restricted

81. Resonance Structures
Pi electon
delocalization

82. Bond Length of peptide C-N is shorter
Bond angle is similar to sp2 hybrid

84. Peptides
• peptide: the name given to a short polymer of amino
acids joined by peptide bonds; they are classified by
the number of amino acids in the chain
• dipeptide: a molecule containing two amino acids
joined by a peptide bond
• tripeptide: a molecule containing three amino acids
joined by peptide bonds
• polypeptide: a macromolecule containing many
amino acids joined by peptide bonds
• protein: a biological macromolecule of molecular
weight 5000 g/mol or greater, consisting of one or
more polypeptide chains

85. Higher Peptides
• Peptides are classified according to the
number of amino acids linked together.
dipeptides, tripeptides, tetrapeptides, etc.
• Leucine enkephalin is an example of a
pentapeptide.

86. Dipeptide
• Artificial Sweetener, ex. in Diet Coke
• 200 times sweeter than sugar
• D-form a.a. substitution is bitter,
use Alatame instead
Aspartic acid
Phenylalanine

87. Peptides with Physiological Activity
Histidine

88. Glutathione
an important water-phase antioxidant and
essential cofactor for antioxidant enzymes
g-glutamic acid cysteine
glycine

89. S G Y A L
Ser – Gly- Tyr – Ala - Leu
Ser Gly
Tyr
Ala
Leu

90. Serine
Glutamate
Asparagine
Histidine
Lysine
Ser-Glu-Asn-His-Lys S E N H K
C-Terminal Amino Acid
N-Terminal
Amino Acid

91. +2 +1 0 -1
2.34
9.6
4.25
10.53
pI = (4.25+9.6)/2 =6.93
-2

92. Leucine Enkephalin
Tyr—Gly—Gly—Phe—Leu
YGGFL
a pentapeptide involved in regulating nociception in the body. The enkephalins are
termed endogenous ligands, as they are internally derived and bind to the body's
opioid receptors.

93. Peptides with Physiological Activity (cont’d)
Induces labor in
pregnant women
and controls
contraction of
uterine muscle.
Control of blood
pressure by
regulating
contraction of
Smooth muscle

94. Oxytocin
• Oxytocin is a cyclic nonapeptide.
• Instead of having its amino acids linked in an
extended chain, two cysteine residues are
joined by an S—S bond.
N-terminus
C-terminus
Ile—Gln—Asn
Tyr
Cys S S
Cys—Pro—Leu—GlyNH2
1
2
3
4 5
6 7 8 9
Disufide Linkage

95. Oxytocin
S—S bond
An S—S bond between two cysteines is
often referred to as a disulfide bridge.

96. Protein Size is Varied

97. Polypeptides have
characteristic amino
acid compositions

98. Some Proteins have chemical groups
other than amino acids
• Non-amino acid part = prosthetic group

99. Class Activity :
1. Determine the Isoelectric pH :
a. Phenylalanine
b. Lysine
c. Aspartic acid
2. Determine the Isoelectric pH of the peptide :
Tyr-Lys-Ala-Phe-Glu-Asn
3. Write the structure of the above hexapeptide.
4. What is its charge at physiological pH?

100. SUMMARY
• Both D-amino acids and non-α-amino acids occur in
nature, but only L-α-amino acids are present in
proteins
• All amino acids possess at least two weakly acidic
functional groups: R-NH3
+ and R-COOH
• The pk values of all functional groups of an amino
acid dictate its net charge at a given pH
• pHI is the pH at which the amino acid bears no net
charge
• The R-groups of amino acids determine their unique
biochemical function

101. SUMMARY
• Peptides are named for the number of amino acid
residues present, and as derivatives of the carboxyl
terminal residue.
• The primary structure of a peptide is its amino acid
sequence, starting from the amino terminal residue.
• The partial double bond character of the bond that
links the carbonyl carbon and the nitrogen of a
peptide renders the four atoms of the peptide bond
coplanar and restricts the number of possible peptide
conformation.

102. … hope you have learned something!
RAMON S. DEL FIERRO, Ph.D. (Tokyo)
All the Best!