Chapter 27: Amino Acids, Peptides and Proteins

-Amino Acids

The generic structure of an -amino acid

 The 20 naturally occurring -amino acids used by cells to synthesise proteins can be
generally represented by the generic formula shown above.
 The means the main difference between the various amino acids lies in the structure of
the "R" group.
 These 20 -amino acids can be sub-classified according to how the properties of other
functional groups in the "R" group influence the system.
o non-polar side chains (e.g. alkyl groups)
o polar (e.g. amides, alcohols)
o acidic (e.g. carboxylic acids, phenols)
o basic (e.g. amines)

Question :  Which common amino acid doesn't quite fit the generic formula ? 

Amino acids with non-polar side chains. The hydrophobic side chains are chemically
unreactive and tend to aggregate rather than be exposed to the aqueous environment, so they tend
to found on the interior of proteins.  Hydrophobic means "water hating" - remember "oil and
water don't mix" and "like dissolves like" - this is because non-polar hydrocarbons do not
interact with polar water molecules in an energetically favourable way - they would rather
interact with another non-polar hydrocarbon molecule : this it the hydrophobic effect - the
aggregation of non-polar systems in an aqueous environment.
(see also "micelles")
 

Amino acid Abbreviations Structural Formula 3D-representation

Glycine Gly (G)

Alanine Ala (A)

 Valine Val (V)

Leucine Leu (L)

Isoleucine Ile (I)

Methionine Met (M)

Proline Pro (P)

Phenylalanine Phe (F)

Tryptophan Trp (W)

Amino acids with polar side chains.  These are side chains can be involved in hydrogen
bonding interactions. Cysteine is important because of its ability to form disulfide bonds.

Amino acid Abbreviations Structural Formula 3D-representation

Asparagine Asn (N)

Glutamine Gln (Q)

 Serine Ser (S)

Threonine Thr (T)

Tyrosine Tyr (Y)

Cysteine Cys (C)

Amino acids with acidic side chains. These carboxylate group will be -ve at physiological pH.  
Often involved at the active sites of enzymes, in hydrogen bonding interactions and in acid/base
type reactivity.
 

Amino acid Abbreviations Structural Formula 3D-representation

Aspartic acid Asp (D)

Glutamic acid Glu (E)

Amino acids with basic side chains.  Often involved at the active sites of enzymes, in hydrogen
bonding interactions and in acid/base type reactivity (e.g. histidine)

Amino acid Abbreviations Structural Formula 3D-representation

Lysine Lys (K)

 Arginine Arg (R)

Histidine His (H)

Chapter 27: Amino Acids, Peptides, Proteins and Nucleic Acids

Structure and pKa of Amino Acids

 Even the simplest amino acids have both an acidic functional group, the carboxylic acid,
and a basic functional group the amine.
 Compounds that can behave as both acids and bases are said to be amphoteric.

Question: Can you think of a very simple, common compound that is amphoteric ? 
 

The equations that define acidity and basicity are: 

 

Remember that the lower the pKa, the stronger the acid. ( review ?)

From these expressions it is possible to derive the important Henderson-Hasselbalch equation :

  pKa = pH + log [HA] / [A-]

How does this equation help us ?

 It tells us that when the pH = pKa then log [HA] / [A-] = 0 therefore [HA] = [A-]   i.e.
equal amounts of the two forms, the acid and the conjugate base.
 If we make the solution more acidic, i.e. lower the pH, so pH < pKa, then log [HA] / [A-]
has to be > 0 so [HA] > [A-]. This makes sense as it tells us that a stronger acid will
cause the formation of HA, the protonated form.
 If instead we make the solution more basic, ie raise the pH, so pH > pKa and log [HA] /
[A-] has to be < 0 so [HA] < [A-]. This makes sense as it tells us that a stronger base will
cause the formation of A- , the deprotonated form.
IMPLICATIONS :

 Typical simple carboxylic acids, RCO2H,  have a pKa of about 5, and typical simple
ammonium ions, RNH3+ have a pKa of about 9.
 Therefore, since the acid is the stronger acid (lower pKa) the amino acid will exist in the
zwitterionic form where the acid has protonated the amine in neutral aqueous solution
(or normal physiological pH).

These principles can be extended to poly acidic / basic systems (such

as amino acids) by thinking of each pKa value in turn.
This information will let you decide which structure of an acid or
base will dominate at a particular pH.

Let's look at an example.

To the left are the processes for the amino acid HISTIDINE which has
an extra basic group.

It has three acidic groups of pKa's 1.82 (carboxylic acid), 6.04 (pyrrole
NH) and 9.17 (ammonium NH).
Histidine can exist in the four forms shown, depending on the solution
pH, from acidic pH (top) to basic pH. (bottom).

Starting from the top, we can imagine that as we add base, the most
acidic proton is removed first (COOH), then the pyrrole NH then
finally the amino NH. These takes us through each of the forms in turn.

At pH < 1.82, A is the dominant form.

In the range 1.82 < pH < 6.02 B is the dominant form.

In the range 6.02 < pH < 9.17 C is the dominant form, and

when pH > 9.17, D is the major form in solution.

 
 
 

Isoelectronic point, pI
  The isoelectronic point or isoionic point is the pH at which the amino acid does not
migrate in an electric field.
 This means it is the pH at which the amino acid is neutral, i.e. the zwitterion form is
dominant.
 A table of pKa and pI values can be found on the next page.
 The pI is given by the average of the pKas that involve the zwitterion, i.e. that give the
boundaries to its existence.

There are 3 cases to consider....

 neutral side chains

These amino acids are characterised by two pKas : pKa1 and pKa2 for the carboxylic acid and
the amine respectively.
The isoelectronic point will be halfway between, or the average of, these two pKas, i.e.   pI = 1/2
(pKa1 + pKa2). This is most readily appreciated when you realise that at very acidic pH (below
pKa1) the amino acid will have an overall +ve charge and at very basic pH (above pKa2 ) the
amino acid will have an overall -ve charge.  For the simplest amino acid, glycine, pKa1= 2.34
and pKa2 = 9.6, pI = 5.97.

The other two cases introduce other ionisable groups in the side chain "R" described by a third
acid dissociation constant, pKa3

 acidic side chains

The pI will be at a lower pH because the acidic side chain introduces an "extra" negative charge.
So the neutral form exists under more acidic conditions when the extra -ve has been neutralised. 
For example, for aspartic acid shown below, the neutral form is dominant between pH 1.88 and
3.65, pI is halfway between these two values, i.e.   pI = 1/2 (pKa1 + pKa3),  so pI = 2.77.
  basic side chains

The pI will be at a higher pH because the basic side chain introduces an "extra" positive charge.
So the neutral form exists under more basic conditions when the extra +ve has been neutralised. 
For example, for histidine, which was discussed on the previous page, the neutral form is
dominant between pH 6.00 and 9.17, pI is halfway between these two values, i.e.  pI = 1/2
(pKa2 + pKa3),  so pI = 7.59.

Chapter 27: Amino Acids, Peptides and Proteins

Table of pKa and pI values

 The pKa values and the isoelectronic point, pI, are given below for the 20 -amino acids.
 pKa1= -carboxyl group, pKa2 =-ammonium ion, and pKa3 = side chain group.

Amino acid pKa1  pKa2 pKa3    pI

Glycine 2.34  9.60   ---   5.97
Alanine 2.34  9.69   ---   6.00
Valine 2.32  9.62   ---   5.96
Leucine 2.36  9.60   ---   5.98
Isoleucine 2.36  9.60   ---   6.02
Methionine 2.28  9.21   ---   5.74
Proline 1.99 10.60   ---   6.30
Phenylalanine 1.83  9.13   ---   5.48
Tryptophan 2.83  9.39   ---   5.89
Asparagine 2.02  8.80   ---   5.41
Glutamine 2.17  9.13   ---   5.65
Serine 2.21  9.15   ---   5.68
Threonine 2.09  9.10   ---   5.60
Tyrosine 2.20  9.11   ---   5.66
Cysteine 1.96  8.18   ---   5.07
Aspartic acid 1.88  9.60   3.65   2.77
Glutamic acid 2.19  9.67   4.25   3.22
Lysine 2.18  8.95 10.53   9.74
Arginine 2.17  9.04 12.48 10.76
Histidine 1.82  9.17   6.00   7.59

Terminology and Conventions for Peptides and Proteins

  Amines, -NH2 can react with carboxylic acids, RCO2H to form amides, RCONH2 
(review ?)
 Proteins are polymers of amino acids, linked by amide bonds i.e. proteins are just
polyamides.
 Peptides are "short" proteins.
 The amide bonds in peptides and proteins are also known as peptide bonds.
 A dipeptide has two amino acid units, e.g. Ala-Gly (AG),  a tripeptide three amino acid
units e.g. Val-Leu-Ala (VLA), and so on...
 The precise order of the amino acids in the peptide is the amino acid sequence.
 Peptides are named according to their amino acid sequence.
 By convention, the name is written so that the higher priority carboxyl group is to the
right and the amino end to the left.
 Note this "agrees" with the ideas of nomenclature where the higher priority group is used
at the "right hand end" of the name
e.g. 2-aminoethanoic acid,  H2NCH2CO2H
 The carboxyl group end is referred to as the C terminus.
 The amino group end is referred to as the N terminus.

 The order of the naming is important as the two structures are not the same but are
constitutional isomers.

Sequencing Peptides
 In order to determine which amino acids are present in a protein, the protein has to be
broken apart.
 This requires that the amide bonds are hydrolysed to the component amino acids (review
including mechanism ?)
  Chemical hydrolysis using 6M HCl / heat will break all the amide bonds (i.e. it is not
selective).
 The amino acids are then separated using chromatography (this tells us which amino
acids are present)
 Reaction with ninhydrin allows the molar ratios of the amino acids to be determined. (i.e.
how much is present)
 Enzymatic hydrolysis using proteases is more selective and can be used to break the
protein into fragments rather than individual amino acids.
As examples:
o Trypsin cleaves amides where the C=O is part of lysine or arginine.
o Chymotrypsin cleaves amides where the C=O unit has an aromatic side chain :
phenylalanine, tyrosine, tryptophan.
o Carboxypeptidases cleaves only at the C-terminus (this allows the C-terminus to
be identified)
 End group analysis means that a "molecular flag" is attached to the N-terminus so
allowing it to be recognised.
 Once this information has been collected, the pieces needed to be analysed and the
sequence carefully deduced.

Synthesis of Amino Acids

There are several ways in which -amino acids can be synthesised using reactions we have
already encountered:
 

 Nucleophilic
substitution of a-
halocarboxylic
acids

 Strecker synthesis
  Alkylation of an
acetamidomalonate

Reactions of Amino Acids

 Amino acids contain two functional groups : amines and carboxylic acids.
 So amino acids undergo the reactions characteristic of those functional groups: review
amines ? review carboxylic acids ?
 The most important reactions are fair as amino acid chemistry is concerned are the
reactions that are utilised in the formation of peptides and proteins.
 The reaction with ninhydrin is used as a visual indicator as a there is a colour change
(primary amines give a blue/purple product)

 Amines :
acylation to
form amides 

 Carboxylic
acids :
esterification 

 Ninhydrin
test

Peptide Synthesis : Protecting groups

 Protein synthesis is important for several reasons including:
o confirming the structure of natural proteins (e.g. for medical research etc.)
o to investigate how protein structure and function are controlled by the amino acid
sequence.

 However, it is not as straight forward as mixing the amino acids together to form the
amides.
 For example, a mixture of alanine, A and glycine, G would give a mixture of amides : A-
G, G-A, A-A and G-G,  plus higher polypeptides...)
 In order to control the coupling reaction, it is necessary to use protecting groups.
 By protecting the amine group of one component and the carboxylic acid group of the
other, a specific amide bonds can be formed.

 Therefore the sequence required is to:

o protect the amino group in the N-terminal amino acid and the carboxyl group in
the C-terminal amino acid
o couple the two amino acids by forming the new amide bond
o deprotect the termini of the new peptide (as and if required)
 By repeating the process, polypeptides can be grown one amino acid residue at a time, or
by building pieces and then joining those together.

 
Amine protecting groups
1.   benzoyloxycarbonyl groups (abbreviation = Z or Cbz in older literature)

protection

deprotecti
on
 2.   tert-butoxycarbonyl groups (abbreviation = Boc)

protection

deprotecti
on

Carboxyl protecting groups

1.   simple esters esp. methyl, and ethyl

protection

deprotecti
on

2.   benzyl esters (abbreviation = Bn)

deprotecti
on

Peptide Synthesis : Forming the peptide bond

 Trying to form the amide by simply reacting the amine and the carboxylic acid is not very
effective.
 In order to facilitate the process, a coupling reagent can be used to activate the process.
 A common coupling agent is N,N'-dicyclohexylcarbodiimide (DCCI) which reacts with
the carboxylic acid to form a reactive acylating reagent.
 During the coupling reaction the DCCI is converted to a urea that can be readily removed
(e.g. by chromatography)

 An alternative method is to use a reactive ester and react it with the amine via a typical
nucleophilic acyl substitution reaction.

 A important method of peptide synthesis, the Merrifield Method, (Nobel Prize 1984)
involves attaching the initial amino acid to an unreactive polymer and then "growing" the
peptide chain while it is still anchored to the polymer.  This methodology makes the
removal of excess reagents and by products etc. a simple case of washing them away. 
The same type of reactions are those described above are used.