AMINO ACIDS

1. Amino acids

2. Introduction
• All poly/peptides are polymers of amino
acids
• Naturally, there are more than 300 known
amino acids
• Only 20 amino acids occur in (mammalian)
proteins through protein synthesis. They are
also called
– basic/primary/standard/proteinogenic
– They are almost all L-α-amino acids

3. Introduction
• In addition to the 20 standard amino acids
coded by DNA, 2 additional amino acids
are also DNA coded  in a non-standard
manner.
– Selenocysteine: in some proteins is coded by
the stop codon UGA
– Pyrrolysine: used by some methanogenic
bacteria in methane producing enzyme
systems. Coded for by UAG.

4. Introduction
• The standard amino acids have a wide
range of biological functions
–Monomers of proteins through the
standard genetic code-protein synthesis
– Glycine and glutamate are also
neurotransmitters.
–Tyrosine is involved in the formation of
thyroid hormone etc

5. Introduction
– Standard amino acids also serve as
precursors of other molecules
• Tryptophan: precursor of the
neurotransmitter serotonin
• Glycine: is one of the reactants in the
synthesis of porphyrins e.g. heme
• Arginine: used in the synthesis of (the
hormone) nitric oxide

6. Introduction
• Other amino acids in proteins are usually
formed by post-transcriptional
modification of proteins, i.e. after
translation.
• Such modifications are often important for
protein function

7. Non std aa: functions and
occurrence
• Occur as free or in combined states.
• Commonly formed as modifications of std aa
– E.g taurine is formed by decarboxylation of
cysteine
– Dopamine is made from tyrosine
– Hydroxyproline (in collagen) is made from
proline (post transcriptionally)

8. Non std aa: functions and
occurrence
• They serve specialized functions,
– e.g. dopamine and γ-amino butyric acid
(GABA) are neurotransmitters
• They usually occur in the metabolic pathways for
standard amino acids, e.g.
– Ornithine and citruline: occur in the urea cycle
(part of aa breakdown).
– Carnitine is used in lipid transport within a cell
etc.

9. Non std aa: functions and
occurrence
• Hundreds of other non proteinogenic
α- amino acids have been identified in
nature
–E.g in carbonaceous chondrites
(the primitive Murchison meteorite
(>79 aa)),
–in plants (nisin or alamethicin) etc

11. Structure of amino acids

12. Structure of amino acids
• Under normal physiological pH, std aa
(except proline), have
– an amino group (--NH2) and
– a carboxylic acid (--COOH)
• Both the amino and the carboxylic acid
groups are attached to the same carbon,
the tetrahedral α-carbon
(refer α-amino acids!).

13. Structure of amino acids
• Amino acids are distinguished from one
another by their R groups, attached to the α-
carbon
– One exception is glycine (gly) where the R is
hydrogen
• The 4th substitution is usually hydrogen
• The α-carbon is chiral (except gly), ie is
asymmetrical due to having 4 different groups
attached (-NH2, -COOH, -R and -H)

14. Structure of amino acids
• The side chain R is specific
for an amino acid and it
confers chemical properties
to an aa (weak acids or
bases, hydrophiles/-phobes)

15. Structure of amino acids
• Understanding the R groups of aa is
important in order to devise:
– Methods of aa analysis
– Methods of aa purification
– Characterization and Identification of proteins
• e.g. aromatic aa absorb UV light at 275-280nm. Trp has a maximal
peak at 280nm etc. proteins can be sequenced and distinguished as
in Functional Genomics.

16. Nomenclature of amino acids

17. Nomenclature
Amino Acid 3 letter
code
1 letter
code
Amino Acid 3 letter
code
1 letter
code
Glycine Gly G Threonine Thr T
Alanine Ala A Cysteine Cys C
Valine Val V Tyrosine Tyr Y
Leucine Leu L Asparagine Asn N
Isoleucine Ile I Glutamine Gln Q
Methionine Met M Aspartic Acid Asp D
Proline Pro P Glutamic Acid Glu E
Phenyl
Phe F Lysine Lys K
alanine
Tryptophan Trp W Arginine Arg R
Serine Ser S Histidine His H

18. Classification

19. Classification
• R polarity:
1. hydrophilic (if R is polar)
Interact with aqueous
environment, Involved in H-bond
formation. Found on exterior
parts of proteins & reactive sites
of enzymes
2. hydrophobic (if R is non-polar)
Reside in the interior of
proteins,They do not ionizedo
not participate in H-bond
formation

20. Hydrophobic
and Neutral
at pH=7
Hydrophilic
and Neutral
at pH=7

21. Classification
• Acid base reaction: Depends on
ionizability of
1. # of Amino groups
2. # of Carboxylic groups
3. R group properties

22. Classification
Acidic
(Two Carboxylic Groups)
Basic
(more than one basic group)

23. Amino acid charge
At physiological pH (around 7.4) the
carboxyl group will be unprotonated and
the amino group will be protonated.
-COOH <--------> -COO- + H+
-NH3+ <---------> -NH2 + H+

24. Amino acid charge
• An amino acid with
no ionizable R-group
would be
electrically neutral
at this pH (ie + and
– charge balanced).
• This species is
called a zwitterion.

25. Amino acid charge
• As in other organic acids, the acidic
strength of the carboxyl, amino and
ionizable R-groups in amino acids is
defined by the pK.

26. Amino acid charge
• The net charge of an amino acid will thus be the
summation of all the charges in the amino acid at a
specified pH
• Amino acids have different charges at different pH
value of the aqueous medium around
• When the net charge of an amino acid or protein is
zero the pH will be equivalent to the isoelectric
point: pI.

27. Amino acid charge
NOTE:
• At its isoelectric pH (pI) an amino acid
bears no net charge (The net – and +
charge balances)

28. Titration curve of leucine
pI = pK1 + pK2
2

29. pI = pK2 + pK3
2
Titration of Histidine

30. NOTE THAT, YOU
• Know the structure of an amino acid.
• Know why amino acids are optically active.
• Know why some amino acids are called
neutral.
• Know why some amino acids are called
acidic and others basic, hydrophobic/phillic.
• Know a zwitterion
• Know the definition of an isoelectric point and
how this point is calculated for amino acids.

31. Peptides
and
Peptide Bonds

32. Definitions
– Peptide bond=the bond between 2 amino acids
– Peptide=a molecule made of the union of aa joined by
peptide bonds
• Di-ppd= 2 amino acid residues/1 peptide bond
• Tri-ppd= 3 amino acid residues/2 peptide bonds
• Tetra--=? Etc
– Residue=an amino acid in a peptide whose carboxyl
group has been used in a peptide bond
• Named by replacing the –ate or ine end of aa with –yl
• Eg alanine=alanyl, cycteine=cyctenyl etc
aspartate= ?
Also called aminoacyls

33. Peptides
IMPORTANCE
• Hormones eg insulin
• Neurotransmitters/modulators eg
• Drugs (antibiotics) eg valinomycin
• Beverage sweeterners eg aspartame
• Some mediate tumor development

34. • Naming of peptides
• Peptides are named as derivatives of the amino
acid with intact –COOH
– Eg: Alanyl-aspartyl-histidyl-valine
– It means the whole residue sequence is a derivative
of valine (valine has an intact –COOH in the above
sequence)
– Conventinal presentation of peptides: N---------C,
– Ie N terminus on the left and the C terminus on the
right

35. Peptide bond formation
• Condensation reaction that result in
polymerization of aapeptideprotein
• It involves the alfa amino group of one aa
and the alfa carboxyl group of the other,
with elimination of a water molecule

36. H2O

37. Electron Resonance Stability of
peptide bonds
• The presence of a
carbonyl group in the
peptide bond allows
for ERS
• The peptide bond
shows rigidity, ie has
a partial double bond
character

39. Characteristics of the peptide
bond
• Due to partial double bond, there is no
freedom of rotation around C—N
• All 4 atoms of the bond lie on the same
plane- it is coplanar
– There is free rotation in the rest of the bonds
in the peptide backbone
– Thus 2/3 of atoms in a peptide are co-planar
while 1/3 have free rotation
• Peptides are therefore semi-rigid

40. Abnormal peptide bonds
• Non alfa peptidyl bonds may occur in
peptides
– Eg in thyrotropin releasing hormone (TRH)
where the glutamate is made into a cycle
(pyroglutamate) and the carboxyl end is
‘amidated’ (pyrolyl carboxyl)
– In glutathione

41. Some peptides of biological
importance
• Glutathione
– A tripeptide containing glutamic acid, cystein
and glycine
– Present in RBC and other cells to maintain
optimal redox potentials
– Anhances/optimizes the action of enzymes
(eg glutathione reductase) by preventing
oxidation of their –SH to –SS

42. Some peptides of biological
importance
• Bradykinin and Kallidin
– 9 and 10 residues respectively
– From partial hydrolysis of plasma proteins by
snake venoms (hemolytic venoms)
– Powerful vasodepressors and cardiac
inhibitors

43. Some peptides of biological
importance
• Tyrocidin and Gramicidin
– Cyclic peptides, 10 aa each
– They form rings their terminal 2 amino acid
residues
– They are useful antibiotics

44. Some peptides of biological
importance
• Peptide hormones and factors
– Posterior Pituitary-oxytocin and vassopressin
– Hypothalamic hormones-somatostatin
– Intestinal hormones-gastrin
– Kidney and brain factors-hypertensin and
enkephalins

45. • PLEASE NOTE:
– Do you know what is a peptide and peptide
bond?
– Dou you know an amino acid and an amino
acid residue?
– Dou you know how to name residues and
peptides?
– Peptide bond formation?
– Do you know the features of a peptide bond?
– Do you know why peptides are semi-rigid?

46. The primary structure of
proteins

47. Primary structure
• Refers to
– the number and the order of amino acid
residues in the peptide/polypeptide
– This is the linear sequence of residues

48. Primary structure and biological
• Mutations
activity
– SNPs: DNA changechange in the order &
sometimes the number of amino acid
residues.
eg
1. Alcohol intolerance in many Asians
2. Susceptibility to malaria (K189M in the
ICAM-1 gene susceptibility to cerebral
malaria)

49. Primary structure Nomenclature
• Both 3 letter and single letter systems
used to determine primary structures of
ppds (remember aa nomenclature)
• In the 3 letter system, residues are joined
by a dash/line: Gly-Tyr-Ala-Glu-Lys
• In the 1 letter system the same ppd
becomes: GYAEK, ie no lines

50. Primary structure Nomenclature
• If uncertain about a residue, it is enclosed
in brackets and the possible residues
separated by a comma
– Thus Gly- (Tyr,Ala)-Glu-Lys

51. Charge on peptides
• Formation of the peptide bond involves the
loss of +1 and -1 =0 charge.
• Peptide bonds are neutral at any pH
• At pH 7.4, peptides are charged due to
– N-terminal group
– C-terminal group
– Basic or acidic R groups

52. Determination of primary structure
• Sanger worked with insulin in the 60s.
–Separated the 2 chains, A and B,
cleaved the peptides into smaller
peptides with overlapping sequences
– Using 1-fluoro-2,4-dinitrobenzene, he
could remove and identify each aa at a
time

53. Protein
mixture
Stages of sequencing
Proteins
Peptide
mixture
Peptides MS analysis
MS data
Identification
separation
digestion digestion
separation
Database search
algorithms

54. Stages of sequencing
• Purify peptides and asses and purify by
any suitable technique e.g. SDS-PAGE,
chromatography
• Homogeneity should be 90-95%

56. FAB-MS
Schematic Representation
Xenon
atoms
Helium atoms

57. Peptide fragmentation

58. From public online data banks

59. Applications of sequencing
To study
Protein functions, localization, modifications
and protein interactions
Also therapeutics and diagnostics

60. • DO YOU KNOW
– The biological implications of the primary
structure of proteins (peptides)
– Nomenclature of peptides?
– Basic steps of sequencing a peptide?
– Importance of peptide sequencing?
– The principle of FAB MS in peptide
sequencing?
• Then GOOD DAY!