Amino acids

importance
• In addition to providing the monomer units from which
the long polypeptide chains of proteins are synthesized,
the L-α-amino acids and their derivatives participate in
• cellular functions as diverse as nerve transmission and
the biosynthesis of porphyrins, purines, pyrimidines,
and urea.
• Short polymers of amino acids called peptides perform
prominent roles in the neuroendocrine system as
hormones, hormone-releasing factors, neuromodulators,
or neurotransmitters.
 IMPORTANCE
• While proteins contain only L-α-amino acids,
microorganisms elaborate peptides that
contain both D- and L-α-amino acids.
• Several of these peptides are of therapeutic
value, including the antibiotics bacitracin and
gramicidin A and the antitumor agent
bleomycin.
• Certain other microbial peptides are toxic.
 IMPORTANCE
• The cyanobacterial peptides microcystin and nodularin
are lethal in large doses, while small quantities
promote the formation of hepatic tumors.
• Neither humans nor any other higher animals can
synthesize 10 of the 20 common L-α-amino acids in
amounts adequate to support infant growth or to
maintain health in adults.
• Consequently, the human diet must contain adequate
quantities of these nutritionally essential amino acids.
PROPERTIES OF AMINO ACIDS
 The Genetic Code Specifies
20 L-α-Amino Acids
• Of the over 300 naturally occurring amino
acids, 20 constitute the monomer units of
proteins.
• While a nonredundant three-letter genetic
code could accommodate more than 20
amino acids, its redundancy limits the
available codons to the 20 L-α-amino acids
listed in the Table below:
L- α-Amino acids present in proteins.
With Side Chains Containing
Hydroxylic (OH) Groups
With Side Chains Containing Sulfur
Atoms
Disulfide bonds from cysteine
With Side Chains Containing Acidic
Groups or Their Amides
With Side Chains Containing Basic
Groups
Containing Aromatic Rings
Absorption of ultraviolet light by
aromatic amino acids
 Absorption of ultraviolet light by
aromatic amino acids.
• Comparison of the light absorption spectra of the aromatic
amino acids tryptophan and tyrosine at pH 6.0.
• The amino acids are present in equimolar amounts (10-3 M)
under identical conditions.
• The measured absorbance of tryptophan is as much as four
times that of tyrosine.
• Note that the maximum light absorption for both
tryptophan and tyrosine occurs near a wavelength of 280
nm.
• Light absorption by the third aromatic amino acid,
phenylalanine (not shown), generally contributes little to
the spectroscopic properties of proteins.
Classification on the basis of polarity
Imino Acid
 Modified amino acids
• Some proteins contain additional amino acids that
arise by modification of an amino acid already present
in a peptide.
• Examples include conversion of peptidyl proline and
lysine to 4-hydroxyproline and 5-hydroxylysine; the
conversion of peptidyl glutamate to
γ-carboxyglutamate; and the methylation,
formylation, acetylation, prenylation, and
phosphorylation of certain aminoacyl residues.
• These modifications extend the biologic diversity of
proteins by altering their solubility, stability, and
interaction with other proteins.
Uncommon amino acids; both found in
collagen
Found in blood clotting protein,
prothrombin
Found in fibrous protein, elastin
Key intermediates in biosynthesis of
arginine and urea in the cycle
 Only L-α-Amino Acids Occur in
Proteins
• With the sole exception of glycine, the α-
carbon of amino acids is chiral.
• Although some protein amino acids are
dextrorotatory and some levorotatory, all
share the absolute configuration of
L-glyceraldehyde and thus are L-α-amino
acids.
 Steric relationship of the stereoisomers of
alanine to the absolute configuration of L- and
D-glyceraldehyde
Stereoisomerism in -amino acids.
Stereoisomerism in amino acids
D and L alanine
• The two stereoisomers of alanine, L- and D-alanine, are
nonsuperimposable mirror images of each other
(enantiomers). (b, c) Two different conventions for
showing the configurations in space of stereoisomers.
• In perspective formulas (b) the solid wedge-shaped bonds
project out of the plane of the paper, the dashed bonds
behind it.
• In projection formulas (c) the horizontal bonds are
assumed to project out of the plane of the paper, the
vertical bonds behind.
• However, projection formulas are often used casually and
are not always intended to portray a specific
stereochemical configuration.
 Stereoisomerism in amino acids
• In these perspective formulas, the carbons are lined up
vertically, with the chiral atom in the center.
• The carbons in these molecules are numbered beginning
with the terminal aldehyde or carboxyl carbon (red), 1 to 3
from top to bottom as shown.
• When presented in this way, the R group of the amino acid
(in this case the methyl group of alanine) is always below
the carbon.
• L-Amino acids are those with the α-amino group on the left,
and D-amino acids have the α-amino group on the right.
 Occurrence of D- amino acids
• D-Amino acids that occur naturally include
free D-serine and D-aspartate in brain tissue,
• D-alanine and D-glutamate in the cell walls of
gram positive bacteria, and D-amino acids in
some non-mammalian peptides and certain
antibiotics.
 Amino Acids May Have Positive,
Negative, or Zero Net Charge
• Charged and uncharged forms of the ionizable
―COOH and ―NH3+ weak acid groups exist in
solution in protonic equilibrium:
 Dissociation of amino acids
• While both R—COOH and R―NH3+ are weak
acids, R—COOH is a far stronger acid than
R—NH3+.
• At physiologic pH (pH 7.4), carboxyl groups
exist almost entirely as R—COO− and amino
groups predominantly as R—NH3+.
Effect of pH on the charged state of
aspartic acid.
• Amino acids have characteristic titration
curves.
• Acid-base titration involves the gradual
addition or removal of protons.
Titration curve of glycine;-mono-
amino mono- carboxy
 Titration curve of glycine
• Shown above is the titration curve of 0.1 M
glycine at 25oC.
• The ionic species predominating at key points
in the titration are shown above the graph.
• The shaded boxes, centered at about pK1=
2.34 and pK2= 9.60, indicate the regions of
greatest buffering power.
Titration of glutamate
Titration of histidine
B does not exist under physiological
conditions
 pKa Values Express the Strengths
of Weak Acids
• The acid strengths of weak acids are
expressed as their pKa as shown before (see
table with pKas
• The imidazole group of histidine and the
guanidino group of arginine exist as resonance
hybrids with positive charge distributed
between both nitrogens (histidine) or all three
nitrogens (arginine)
Resonance hybrids
 Net charge of amino acids
• The net charge on an amino acid—the algebraic
sum of all the positively and negatively charged
groups present—depends upon the pKa values
of its functional groups and on the pH of the
surrounding medium.
• Altering the charge on amino acids and their
derivatives by varying the pH facilitates the
physical separation of amino acids, peptides, and
proteins.
 At Its Isoelectric pH (pI), an Amino
Acid Bears No Net Charge
• The isoelectric species is the form of a molecule
that has an equal number of positive and negative
charges and thus is electrically neutral.
• The isoelectric pH, also called the pI, is the pH
midway between pKa values on either side of the
isoelectric species.
• For an amino acid such as alanine that has only
two dissociating groups, there is no ambiguity.
Isoelectric pH
Isoelectric pH
 IMPORTANCE OF pI
• Similar considerations apply to all polyprotic acids (eg,
proteins), regardless of the number of dissociating
groups present.
• In the clinical laboratory, knowledge of the pI guides
selection of conditions for electrophoretic separations.
• For example, electrophoresis at pH 7.0 will separate two
molecules with pI values of 6.0 and 8.0 because at pH 8.0
the molecule with a pI of 6.0 will have a net positive charge,
and that with pI of 8.0 a net negative charge.
• Similar considerations apply to understanding
chromatographic separations on ionic supports such as
DEAE cellulose
 pKa Values Vary With the
Environment
• A polar environment favors the charged form
(R―COO− or R―NH3+)
• nonpolar environment favors the uncharged
form (R―COOH or R―NH2).
• A nonpolar environment thus raises the pKa of
a carboxyl group (making it a weaker acid)
• It lowers that of an amino group (making
it a stronger acid).
Typical range of pKa values for
ionizable groups in proteins.
 THE 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 of alanine, valine,
leucine, and isoleucine and the aromatic R groups
of phenylalanine, tyrosine, and tryptophan
typically occur primarily in the interior of cytosolic
proteins.
 THE α-R GROUPS DETERMINE THE
PROPERTIES OF AMINO ACIDS
• The charged R groups of basic and acidic amino
acids stabilize specific protein conformations via
ionic interactions, or salt bonds.
• Histidine plays unique roles in enzymatic catalysis.
• The primary alcohol group of serine and the
primary thioalcohol (―SH) group of cysteine are
excellent nucleophiles and can function as such
during enzymatic catalysis.
 THE α-R GROUPS DETERMINE THE
PROPERTIES OF AMINO ACIDS
• The ―OH groups of serine, tyrosine, and
threonine also participate in regulation of the
activity of enzymes whose catalytic activity
depends on the phosphorylation state of
these residues.
 Peptides Are Chains of Amino Acids
• Two amino acid molecules can be covalently joined
through a substituted amide linkage, termed a peptide
bond, to yield a dipeptide.
• Such a linkage is formed by removal of the elements
of water (dehydration) from the -carboxyl group of
one amino acid and the α-amino group of another.
• Peptide bond formation is an example of a
condensation reaction, a common class of reactions in
living cells.
Formation of a peptide bond by
condensation.
 Formation of a peptide bond
• The α-amino group of one amino acid (with R2
group) acts as a nucleophile to displace the
hydroxyl group of another amino acid (with R1
group), forming a peptide bond (shaded in
yellow).
• Amino groups are good nucleophiles, but the
hydroxyl group is a poor leaving group and is
not readily displaced.
• At physiological pH, the reaction shown does
not occur to any appreciable extent.
 Pentapeptide
serylglycyltyrosylalanylleucine, or
Ser–Gly–Tyr–Ala–Leu.
 Pentapeptide

• serylglycyltyrosylalanylleucine, or Ser–Gly–
Tyr–Ala–Leu.
• Peptides are named beginning with the
aminoterminal residue, which by convention
is placed at the left.
• The peptide bonds are shaded in yellow; the R
groups are in red.
tripeptide
 Naming of peptides
• Amino acids present in peptides are called
aminoacyl residues and are named by replacing
the -ate or -ine suffixes of free amino acids with -
yl (eg, alanyl, aspartyl, tyrosyl).
• Peptides are then named as derivatives of the
carboxyl terminal aminoacyl residue.
• For example, Lys-Leu-Tyr-Gln is called lysyl-leucyl-
tyrosyl-glutamine.
• The -ine ending on glutamine indicates that its α-
carboxyl group is not involved in peptide bond
formation.
Peptides Can Be Distinguished by
Their Ionization Behavior
 Alanylglutamylglycyllysine

• This tetrapeptide has one free α-amino group,

one free α-carboxyl group, and two ionizable
R groups.
• The groups ionized at pH 7.0 are in red.
 The Peptide Bond Has Partial
Double-Bond Character
• Although peptides are written as if a single bond
linked
the α-carboxyl and α-nitrogen atoms, this bond in fact
exhibits partial double-bond character:
• There thus is no freedom of rotation about the bond
that connects the carbonyl carbon and the nitrogen of
a peptide bond.
• Consequently, all four atoms are coplanar.
• The imposed semirigidity of the peptide bond has
important consequences.
Partial double bond character of
peptide bonds
Dimensions of a fully extended
polypeptide chain.
 Characteristics of a peptide bond
• The unshaded atoms are the α-carbon atom, the
α-hydrogen atom, and the α-R group of the particular
amino acid.
• Free rotation can occur about the bonds that connect the α-
carbon with the α-nitrogen and with the α-carbonyl carbon
(blue arrows).
• The extended polypeptide chain is thus a semirigid
structure with two-thirds of the atoms of the backbone
held in a fixed planar relationship one to another.
• The distance between adjacent α-carbon atoms is 0.36
nm (3.6 Å).
 Some peptides of biological
importance
• Aspartame (Nutrasweet) –dipeptide, artificial
sweetener.
• Oxytocin (9 residuals) stimulate uterine
contractions.
• Bradykinin (9 residues) inhibits inflammation
of tissues
• Thyrotropin releasing factor (3 residues)
stimulate release of thyrotropin
 Some examples of small polypeptides
and oligopeptides
• Insulin – 2 polypetides; one has 30 residues,
the other 21.
• Glucagon; 29 residues, opposes action of
insulin
• Corticotropin- 39 residues, stimulates the
adrenal cortex.
Molecular Data on Some Proteins
 Polypeptides Have Characteristic
Amino Acid Compositions-2 examples
 Amino acid composition by acid
hydrolysis
• *In some common analyses, such as acid
hydrolysis, Asp and Asn are not readily
distinguished from each other and are together
designated Asx (or B).
• Similarly, when Glu and Gln cannot be
distinguished, they are together designated Glx
(or Z).
• In addition, Trp is destroyed.
• Additional procedures must be employed to
obtain an accurate assessment of complete amino
acid content.
 Some Proteins Contain Chemical
Groups Other Than Amino Acids
• Some proteins contain permanently
associated chemical components in addition
to amino acids;
• These are called conjugated proteins.
• The non–amino acid part of a conjugated
protein is usually called its prosthetic group.
• Conjugated proteins are classified on the basis
of the chemical nature of their prosthetic
groups. See the following slide.
Conjugated Proteins
 There Are Several Levels of Protein
Structure
• Four levels of protein structure are commonly defined.
• A description of all covalent bonds (mainly peptide bonds and
disulfide bonds) linking amino acid residues in a polypeptide chain
is its primary structure.
• The most important element of primary structure is the sequence
of amino acid residues.
• Secondary structure refers to particularly stable arrangements of
amino acid residues giving rise to recurring structural patterns eg
α- helix.
• Tertiary structure describes all aspects of the three-dimensional
folding of a polypeptide.
• When a protein has two or more polypeptide subunits, their
arrangement in space is referred to as quaternary structure e.g.
hemoglobin.
Levels of structure in proteins.
 PROTEINS & PEPTIDES MUST BE
PURIFIED PRIOR TO ANALYSIS
• Classic approaches exploit differences in relative solubility
of individual proteins as a function of pH (isoelectric
precipitation), polarity (precipitation with ethanol or
acetone), or salt concentration (salting out with ammonium
sulfate).
• Chromatographic separations partition molecules between
two phases, one mobile and the other stationary.
• For separation of amino acids or sugars, the stationary
phase, or matrix, may be a sheet of filter paper (paper
chromatography) or a thin layer of cellulose, silica, or
alumina (thin-layer chromatography; TLC).
Column Chromatography- R=reservoir,
C=column-(stationary phase),
F=fraction collector
 Partition Chromatography
• Separation depend on the relative affinity of different
proteins for a given stationary phase and for the
mobile phase.
• Proteins that interact more strongly with the
stationary phase are retained longer.
• The length of time that a protein is associated with the
stationary phase is a function of the composition of
both the stationary and mobile phases.
• Optimal separation of the protein of interest from
other proteins thus can be achieved by careful
manipulation of the composition of the two phases.
Size Exclusion Chromatography
 Size Exclusion Chromatography
• A: A mixture of large molecules (diamonds) and small
molecules (circles) are applied to the top of a gel
filtration column.
• B: Upon entering the column, the small molecules
enter pores in the stationary phase matrix from which
the large molecules are excluded.
• C: As the mobile phase flows down the column, the
large, excluded molecules flow with it while the small
molecules, which are temporarily sheltered from the
flow when inside the pores, lag farther and farther
behind.
 SEPARATION AND PURIFICATION
TECHNIQUES
• Ion Exchange Chromatography
• Hydrophobic Interaction Chromatography
• Affinity Chromatography
• Reversed-Phase High-Pressure
Chromatography
• Polyacrylamide Gel Electrophoresis (PAGE)
• Isoelectric Focusing (IEF)
Determination of the amino acid
sequence of a polypeptide
 Steps in sequencing a polypeptide.
(a) Identification of the amino-terminal residue can be the
first step in sequencing a polypeptide. Sanger’s method for
identifying the amino-terminal residue is shown above.
(b) The Edman degradation procedure reveals
the entire sequence of a peptide. Labels and removes only
the N-terminal residue from a peptide. All other peptide
bonds left intact.
• For shorter peptides, this method alone readily yields the
entire sequence, and step (a) is often omitted.
• Step (a) is useful in the case of larger polypeptides, which
are often fragmented into smaller peptides for sequencing.