Chapter 13

Amino Acids
and Proteins
 Introduction
 The three major groups of biological polymers are
polysaccharides, proteins and nucleic acids
 Proteins have many diverse functions; they are major
components of the following biomolecules
 Enzymes and hormones which catalyze and regulate biological
reactions
 Muscles and tendons which provide the body with means for movement
 Hemoglobin which carries oxygen to all parts of the body
 Antibodies they are integral parts of the immune system

 All proteins are polyamides

 Their monomeric units are one of about 20 - amino acids

Chapter 13 2
 Proteins have several levels of structure
 Primary structure refers to the exact sequence of amino acids along a
protein chain
 Secondary and tertiary structures refer to the further bending and
folding of the primary structure
 Quaternary structure refers to the aggregation of more than one
polyamide chain
 All amino acids except glycine are chiral and have the L
configuration (as related to glyceraldhyde) at the 
carbon

Chapter 13 3
 Amino acids
 Structure and Names
 22 amino acids but only 20 amino acids comprise the
building blocks for synthesis of proteins
 The remaining 2 amino acids are derived by modification
after biosynthesis of the protein
 Hydroxyproline and cystine are synthesized from proline and cysteine,
respectively, after the protein chain has been synthesized
 Cysteine is oxidized under mild conditions to the
dissulfide cystine
 The reaction is reversible
 This linkage is important in maintaining the overall shape of a protein

 Essential Amino Acids

 Essential amino acids are not made by higher animals
and must be part of the diet
 There are 8 essential amino acids for adult humans (see Table 24.1)

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Chapter 13 5
Chapter 13 6
Chapter 13 7
 Amino Acids as Dipolar Ions
 In the dry solid state amino acids exist as dipolar ions
(zwitterions)
 In aqueous solution an equilibrium exists between the dipolar
ion, the cationic and the anionic forms of the amino acid
 The predominant form depends on the pH of the solution

 At low pH the amino acid exists primarily in the cationic form

 At high pH the amino acid exists primarily in the anionic form
 At some intermediate pH called the pI (isoelectric point), the
concentration of the dipolar ion is at a maximum and the
concentrations of anionic and cationic forms are equal
 Each individual amino acid has a characteristic pI (see Table
24.1)
 Entire proteins also have a characteristic pI

Chapter 13 8
 The amino acid alanine has a neutral side chain and can
be used to illustrate the fundamental behavior of an
amino acid at various pHs
 At low pH alanine exist as the cation
 pKa1 of alanine (for ionization of the carboxylic acid proton) is 2.3,
considerably lower than the p Ka of a normal carboxylic acid

 pKa2 of alanine (for ionization of a proton from the protonated amino

group) is 9.7

 The isoelectric point, pI, for alanine is the average of the two p Ka
values i.e. (pKa1 + pKa2)/2

Chapter 13 9
 When base is slowly added to fully protonated alanine, a pH is reached
where half of the carboxylic acid groups are deprotonated
 This pH of 2.3 is the value of pKa1
 The Henderson-Hasselbach equation predicts this result

 As more base is added, the pI is reached and the molecule is

electrically neutral; this point is reached when exactly one equivalent
of base is added
 As more base is added and pH 9.7 is reached, half of the the aminium
groups will be deprotonated
 Addition of more base will eventually produce only the anionic amino
acid

Chapter 13 10
 Lysine, which contains a basic side-chain, has a more
complex equilibrium
 The pI for lysine will be high because of the presence of two basic
groups
 The pI for lysine is the average of the monocation (p Ka2) and the dipolar
ion (pKa3)

Chapter 13 11
 Synthesis of -Amino Acids
 The first three methods result in racemic mixtures of
amino acids
 Direct Ammonolysis of an -Halo Acid
 Yields tend to be poor in this reaction

 From Potassium Phthalimide

 This is a variation of the Gabriel synthesis and yields are
usually high

Chapter 13 12
 Polypeptides and Proteins
 Enzymes polymerize amino acids by forming amide
linkages
 The polymer is called a peptide and the amide linkages
are called peptide bonds or peptide linkages
 Each amino acid in the peptide is called an amino acid
residue
 Proteins can contain one or more polypeptide chains and
other associated molecules or metal ions

Chapter 13 13
 Polypeptides are customarily written with the N-terminal
residue to the left
 Three letter or one letter abbreviations are usually used as a short
hand to indicate the sequence of a polypeptide

Chapter 13 14
 Primary Structure of Polypeptides and
Proteins
 The sequence of amino acids in a polypeptide is called its
primary structure
 Several methods exist to elucidate the primary structure of peptides

 Edman Degradation
 Edman degradation involve sequential cleavage and
identification of N-terminal amino acids
 Edman degradation works well for polypeptide sequence
analyses up to approximately 60 amino acid residues
 The N-terminal residue of the polypeptide reacts with phenyl
isothiocyanate
 The resulting phenylthiocarbamyl derivative is cleaved from the
peptide chain
 The unstable product rearranges to a stable phenylthiohydantoin (PTH)
which is purified by HPLC and identified by comparison with PTH
standards

Chapter 13 15
 Automated amino acid sequencing machines use the
Edman degradation and high performance liquid
chromatography (HPLC)
 One Edman degradation cycle beginning with a picomolar amount of
polypeptide can be completed in approximately 30 minutes
 Each cycle results in identification of the next amino acid residue in
the peptide

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 Sanger N-Terminal Analysis
 The N-terminal end of the polypeptide is labeled with 2,4-
dinitrofluorobenzene and the polypeptide is hydrolyzed
 The labeled N-terminal amino acid is separated from the mixture and
identified

 The Sanger method is not as widely used as the Edman

method
Chapter 13 17
 Complete Sequence Analysis
 The Sanger and Edman methods of analysis apply to short
polypeptide sequences (up to about 60 amino acid
residues by Edman degradation)
 For large proteins and polypeptides, the sample is
subjected to partial hydrolysis with dilute acid to give a
random assortment of shorter polypeptides which are
then analyzed
 The smaller polypeptides are sequenced, and regions of overlap among
them allow the entire polypeptide to be sequenced
 Example: A pentapeptide is known to contain the
following amino acids:

 Using DNFB and carboxypeptidase, the N-terminal and C-terminal

amino acids are identified

 The pentapeptide is subjected to partial hydrolysis and the following

dipeptides are obtained

 The amino acid sequence of the pentapeptide must be:

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 Examples of Polypeptide and Protein
Primary Structure
 Oxytocin and Vasopressin
 Oxytocin stimulates uterine contractions during
childbirth
 Vasopressin causes contraction of peripheral blood
vessels and a resultant increase in blood pressure
 The two polypeptides are nonapeptides and differ in only 2 amino acid
residues

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Chapter 13 20
 Insulin
 Insulin is a hormone which regulates glucose metabolism
 Insulin deficiency in humans is the major cause of diabetes mellitus
 The structure of bovine insulin (shown below) was determined in 1953
by Sanger
 Human insulin differs from bovine insulin at only three amino acids in
its sequence

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 Polypeptide and Protein Synthesis
 Laboratory synthesis of polypeptides requires
orchestration of blocking and activating groups to
achieve selective amide bond formation
 Amino groups must be blocked until their reactivity as a nucleophile is
desired
 Carboxylic acid groups must be activated for acyl substitution at the
appropriate time
 Amino groups are usually blocked using one of the
following:
 A benzyloxycarbonyl group (a “Z” group)
 A di-tert-butyloxycarbonyl group (a “Boc” group)
 An 9-fluorenylmethoxycarbonyl group (an “Fmoc” group)

 Methods for installing and removing Z, Boc, and Fmoc

groups are shown below:

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 Methods for installing and removing Z, Boc, and Fmoc
groups are shown below:

Chapter 13 23
 Carboxylic acid groups are usually activated by
conversion to a mixed anhydride:
 Ethyl chloroformate can be used

Chapter 13 24
 An Example of Laboratory Peptide
Synthesis:
 Synthesis of Ala-Leu

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 Automated Peptide Synthesis
 Solid Phase Peptide Synthesis (SPSS) was invented by R.
B. Merrifield, for which he earned the Nobel Prize in 1984
 SPSS involves ‘growing’ a peptide on a solid polymer bead
by sequential cycles of amide bond formation
 The peptide is cleaved from the bead when the synthesis
is complete
 SPSS is used in commercial peptide synthesis machines
 Peptides dozens of residues in length can be synthesized automatically
 A landmark example is synthesis of ribonuclease, having 124 amino
acid residues

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Chapter 13 27
 Secondary, Tertiary, and Quaternary
Structures of Proteins
 Secondary Structure
 The secondary structure of a protein is defined by local
conformations of its polypeptide backbone
 These local conformations are specified in terms of regular folding
patterns such as helices, pleated sheets, and turns
 The secondary structure of a protein is determined by the
sequence of amino acids in its primary structure
 Key to secondary structure is that peptide bonds assume
a geometry in which all 6 atoms of the amide linkage are
trans coplanar

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  Coplanarity results from contribution of the second
resonance form of amides, in which there is considerable
N-C double bond character

 The carbon with attached R groups between the amide

nitrogen and the carbonyl group has relatively free
rotation and this leads to different conformations of the
overall chain
 Two common secondary structure are the -
pleated sheet and the -helix
 In the -pleated sheet, a polypeptide chain is in an
extended conformation with groups alternating from side
to side

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 The extended polypeptide chains in -pleated sheets form
hydrogen bonds to adjacent polypeptide chains
 Slight bond rotations are necessary between amide groups to avoid
unfavorable steric interactions between peptide side chains, leading to
the pleated structure
 The -pleated sheet is the predominant structure in silk fibroin

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 The -helix is the most important protein secondary
structure
 -Helices in a polypeptide are right-handed with 3.6
amino acid residues per turn (See figure 24.11 page 1198)
 The amide nitrogen has a hydrogen bond to an amino acid carbonyl
oxygen that is three residues away
 The R groups extend away from the axis of the helix

 -Helices comprise the predominant secondary structure

of fibrous proteins such as myosin (in muscle) and -
keratin (in hair and nails)
 There are other secondary structures that are more
difficult to describe
 Examples are coil or loop conformations and reverse turns or  bends

Chapter 13 31
 Tertiary Structure
 The tertiary structure of a protein is the three-
dimensional shape which results from further folding of
its polypeptide chains
 This folding is superimposed on the folding caused by its secondary
structure
 In globular proteins, the folding in tertiary structures
exposes the maximum number of polar (hydrophilic) side
chains to the aqueous environment, making most globular
proteins water soluble
 The folding also serves to enclose a maximum number of nonpolar
(hydrophobic) side chains within the protein interior
 Tertiary structures are stabilized by forces including
hydrogen bonding, disulfide bonds, van der Waals forces,
and ionic attractions

Chapter 13 32