1

Amino acids

1.1 Basic structure of amino acids

Amino acids are compounds which have a carboxy group at one end and an
amino group at the carbon atom next to the carboxy group, the so called
-carbon (see g. 1.1). Several amino acids contain additional acidic or basic
groups.
The carboxy group will donate a proton to the amino group, so that an amino
acid (in the absence of other acids or bases) will carry both a negative and a
positive charge, making the whole molecule appear uncharged (zwitter-ion).
The simplest amino acid is glycine, where R is a hydrogen atom. Since the
-carbon carries only 3 dierent ligands (carboxy group, amino group and
hydrogen), it is not enantiomeric. Thus glycine is not chiral, unlike all other
amino acids which carry 4 dierent ligands on the -carbon. We thus dis-
tinguish D- and L-amino acids, only L-amino acids occur in proteins and
in mammalian metabolism. D-amino acids do occur in bacterial cell walls
(murein) however.

1.2 The isoelectric point

From your chemistry lessons you know how to determine the pKa of an acid or
base, the pH at which half of the molecules are charged. A compound which
can act as both acid and base (like an amino acid) has another important
property: The isoelectric point pI, which is the pH at which the number of
positive charges on the molecule is the same as the number of negative charges.
At the pI the molecule would therefore appear uncharged. At this pH the
molecules ability to interact with water is lowest, and therefore its solubility.
4 1 Amino acids

O OH O O
C C
+
H2N C H H3N C H
R R

NH2
O
H2N C C C C C C'
H2 H2 H2 H2 H OH

CO H N
Figure 1.1. Top: Basic structure of an amino acid. Amino acids can form
zwitter-ions. Middle: Nomenclature of carbon atoms, using lysine as example. The
Carboxy-carbon is designated C, the following carbon atoms are labelled with the
letters of the Greek alphabet. Sometimes the last C-atom is called , irrespective of
the chain length. Bottom: In l-amino acids if the -carbon is placed on the paper
plane, with the hydrogen facing you, the remaining substituents read CORN.

How can we get the isoelectric point? Looking at the titration curve of glycine
(see g. 1.3, top) we see that below pK1 most of the molecules bear one positive
charge at the amino group, the carboxy group is uncharged.
Above pK2 it is the other way round, the carboxy group bears a (negative)
charge and the amino group is uncharged. Right in the middle between pK1
and pK2 there is an inection point in the titration curve, this is the pI.
Thus we remember: In a chemical which has one acidic and one basic group,
pI is the average of the two pK values:
1
pI = (pK1 + pK2 ) (1.1)
2
 1.2 The isoelectric point 5
O O
C
+
H 3N CH
H
Glycine (Gly, G)

O O O O O O O O
C C C C
+ +
H 3N
+
CH H 3N
+
CH H3N CH H3N CH

C H C OH C SH C Se
H2 H2 H2 H2

Alanine (Ala, A) Serine (Ser, S) Cysteine (Cys, C) Selenocystein (Sec, U)

O O O O
O O O O O O C C
C C C +
+ + H3N CH
+
H3N
+
CH H 3N CH H3N CH
H3N CH
CH2 CH2
HC CH3 HC OH CH2
C C S CH3
CH3 CH3 C H2
O O H2N O

Valine (Val, V) Threonine (Thr, T) Aspartic acid (Asp, D) Asparagine, (Asn, N) Methionine (Met, M)

O O O O O O O O O
O C
C C C C
+ +
H3N
+
H3N CH H 3N
+
CH H 3N CH H 3N
+
CH CH
CH2 HC CH3 CH2 CH2 CH2
HC CH3 CH2 CH2 CH2 CH2
CH3 CH3 C C CH2
O O O NH2
NH
C
Leucine (Leu, L) Isoleucine (Ile, I) Glutamic acid (Glu, E) Glutamine (Gln, Q) +
H2N N H2
Arginine (Arg, R)
O O O O
C C
+ +
H 3N CH H3N CH
CH2 CH2
CH2 CH2
CH2 CH2
CH2 CH2
+
N H3 NH
Lysine (Lys, K) C O

N CH3

O O O O O O
C C C
+ + + Pyrrolysine (Pyl, O)
H3N CH H 3N CH H 3N CH
CH2 CH2 CH2

NH

O
Phenylalanine (Phe, F) Tyrosine (Tyr, Y) Tryptophan (Trp, W)

O O O O
C C
H
+ C H3N
+
CH
H2N
CH2

NH
+
Proline (Pro, P) N
H Histidine (His, H)

Figure 1.2. The 22 amino acids encoded by genes. Once incorporated into proteins,
amino acids may be further modied. Pyrrolysine has been found only in bacteria,
it is encoded by the amber stop codon UAG. Selenocysteine is encoded by the
UGA opal stop-codon (see g. A.1 on page 338). Acidic groups marked red, basic
groups blue. Note that Thr and Ile have a chiral - in addition to the -carbon. Pyl
has two chiral carbon atoms in the ring.
6 1 Amino acids
pI = 5.97
_ _
HO O pK1 = 2.34 O O pK2 = 9.60 O O
+ C + C C
H3N CH2 H3N CH2 H2N CH2

glycine
12

10

pH
6

0
0 0.5 1 1.5 2
OH-Equivalents (mol/mol)

pI = 3.22
_ _ _
HO O O O O O O O
C C C C
+ + +
H3N CH pK1 = 2.19 H3N CH H3N CH H2N CH
pKR = 4.25 pK2 = 9.67
CH2 CH2 CH2 CH2
CH2 CH2 CH2 CH2
C C C C
HO O HO O _O O _O O

Glutamate
12

10

8
pH

0
0 0.5 1 1.5 2 2.5 3
OH-Equivalents (mol/mol)

pI = 7.59
_ _ _
HO O O O O O O O
C C C C
+ + +
H3N CH pK1 = 1.82 H3N CH pKR = 6.00 H3N CH pK2 = 9.17 H2N CH
CH2 CH2 CH2 CH2
H H H H
C N C N C N C N
CH CH CH CH
C N C N C N C N
H + H H + H H H

Histidine
12

10

8
pH

0
0 0.5 1 1.5 2 2.5 3
OH-Equivalents (mol/mol)

Figure 1.3. Titration curves of glycine, glutamic acid and histidine. At the iso-
electric point amino acids carry an equal number of positive and negative charges,
thus they have no net charge. The isoelectric point can be calculated as the average
between the pKa -values on each side of that uncharged form.
 1.3 The one-letter code 7

But how do we do it when there are 3 or more ionisable groups?

First we write down the various forms of the molecule, with the corresponding
pKa values between them. Then we count the positive and negative charges on
each form and identify the electrically neutral one. The pI can be calculated
as the average between the pKs on either side of it, just as it is done for
molecules with only 2 ionisable groups.
Glutamic acid for example (see g. 1.3, middle) at very low pH carries only one
positive charge at the amino group (1st form). As the pH increases beyond
2.19, more and more protons are lost from the carboxy group on C. This
(2nd ) form carries one positive and one negative charge, and is the neutral
one. Beyond pH 4.25, a second proton is lost from the terminal carboxy group
(3rd form, one positive, two negative charges) and beyond pH 9.67 a further
proton is lost from the amino group (4th form, no positive and 2 negative
charges). Thus the pI is calculated as the average of 2.19 and 4.25, which is
3.22.
The rational with histidine (see g. 1.3, bottom) is similar. The electrically
neutral form is the 3rd , and the pI is the average of 6.00 and 9.17, which is
7.59.

1.3 The one-letter code

In most cases amino acid names are abbreviated with the rst three letters of
their names. These abbreviations are easy to remember, however they use up
unnecessary memory in computer data bases. The 20 common amino acids
can be encoded by the 26 letters of the roman alphabet (leaving space for
some rare amino acids), and then each amino acid in a protein sequence uses
up only 1 rather than 3 bytes of storage space. Unfortunately, many amino
acids start with the same letter (like Ala, Arg, Asp and Asn) thus we can not
simply use the rst letter to encode them.
The following list should help you to remember one-letter codes:
Amino acids with a unique rst letter: Cys, His, Ile, Met, Ser, Val
Where several amino acids start with the same letter, common amino acids
are given preference: Ala, Gly, Leu, Pro, Thr
Letters other than the 1st letter are used for Asn (asparagiN), Arg
(aRginine), Tyr (tYrosine)
Similar sounding names: Asp (asparDic acid), Glu (glutEmate), Gln
(Qtamine), Phe (Fenylalanine)
The remaining amino acids have letters that do not occur in their name:
Lys (K close to L), Trp (W reminds of double ring), Sec (U), Pyl (O)
8 1 Amino acids

The letter X is used for any amino acid, J for Ile or Leu, the hyphen (-)
for gaps.
Certain sequences of amino acids occur in several dierent proteins, were
they serve a special function. Such conserved motives are usually named
after their 1-letter amino acid abbreviations. Thus you may encounter KDEL-
motives or DEATH-ATPases.

1.4 Biological function of amino acid variety

You may now ask why there are so many dierent amino acids. The answer
is that these dierent molecules have dierent properties, that let them serve
dierent functions in proteins (see also table 1.1).
One dierence you have already learned about: There are amino acids whose
side chains can bear positive or negative charges, while other side chains
are always uncharged. Charged side chains have dierent pKr , which can be
inuenced strongly by neighbouring amino acids, for example Cys (pKr = 5
10), His (pKr = 410) and the carboxylic acid group of Glu and Asp (pKr =
47). This is important for proton transfer reactions in the catalytic centre of
proteins. Ionisable groups also form the ionic bonds (salt bridges) which
stabilise protein tertiary structure (see page 23).
Asp, Glu and His residues can chelate bivalent metal ions like Fe, Zn and Ca.
This is important for enzymes with metal co-factors, in haemoglobin and in
some regulatory proteins like calmodulin. Some amino acids are hydrophilic
(= water friendly) because they carry polar groups (-COOH, -NH2 , -OH, -
SH). Other amino acids are hydrophobic (= water fearing, fat friendly),
with long aliphatic (Ile, Leu, Val) or aromatic (Phe, Trp) side chains. If these
residues point into the solution, they force water molecules into a local struc-
ture of higher order (i.e. lower entropy), which is unfavourable. Burying
these residues into the interior of the protein avoids this penalty, this is the
molecular basis for hydrophobic interactions.
Some amino acids have small side chains (like glycine), others very big, bulky
ones (like tryptophan). The small hydrogen residue of Gly not only ts into
tight spaces (see section on collagen (page 202) for an important example),
but because it has no -carbon it can assume secondary structures (see page
16) that are forbidden for all other amino acids.
Proline has its nitrogen in a ring structure, which makes the molecule very
sti, limiting the exibility of protein chains.
The SH-group of Cys, the unprotonated His and the OH-group of Ser and Thr
are nucleophiles which are essential residues in the active centre of enzymes.
Table 1.1. Properties of the 21 amino acids encoded in a mammalian genome. Post-translational modication may change these
properties considerably. The helix propensity measures the energy by which an amino acid destabilises a poly-alanine helix.
Amino acid 3-letter 1-letter MW pK1 (-COOH) pK2 (NH3 + ) pK3 (Side group) pI Hydropathy Abundance (%)
Alanine Ala A 89 2.34 9.69 - 6.01 +1.8 9.0
Arginine Arg R 174 2.17 9.04 12.48 10.76 -4.5 4.7
Asparagine Asn N 132 2.02 8.08 - 5.41 -3.5 4.4
Aspartic acid Asp D 133 1.88 9.60 3.65 2.77 -3.5 5.5
Cysteine Cys C 121 1.96 8.18 10.28 5.07 +2.5 2.8
Glutamic acid Glu E 147 2.19 9.67 4.25 3.22 -3.5 6.2
Glutamine Gln Q 146 2.17 9.13 - 5.65 -3.5 3.9
Glycine Gly G 75 2.34 9.60 - 5.97 -0.4 7.7
Histidine His H 155 1.82 9.17 6.00 7.59 -3.2 2.1
Isoleucine Ile I 131 2.36 9.68 - 6.02 +4.5 4.6
Leucine Leu L 131 2.36 9.60 - 5.98 +3.8 7.5
Lysine Lys K 146 2.18 8.95 10.53 9.74 -3.9 7.0
Methionine Met M 149 2.28 9.21 - 5.74 +1.9 1.7
Phenylalanine Phe F 165 1.83 9.13 - 5.48 +2.8 3.5
Proline Pro P 115 1.99 10.96 - 6.48 -1.6 4.6
Selenocysteine Sec U 168 2.16 9.40 5.20 3.68 rare
Serine Ser S 105 2.21 9.15 13.60 5.68 -0.8 7.1
Threonine Thr T 119 2.11 9.62 13.60 5.87 -0.7 6.0
Tryptophan Trp W 204 2.38 9.39 - 5.89 -0.9 1.1
Tyrosine Tyr Y 181 2.20 9.11 10.07 5.66 -1.3 3.5
Valine Val V 117 2.32 9.62 - 5.97 +4.2 6.9
1.4 Biological function of amino acid variety
9
10 1 Amino acids

Some amino acids confer properties to the protein which can be used in the
laboratory: Met binds certain heavy metals which are used in X-ray structure
determination and reacts with cyanogene bromide (BrCN) to cleave the
protein at specic sites. Cys and Lys are easily labeled with reactive probes.
Aromatic amino acids, in particular Trp absorb UV-light at 280 nm, this can
be used to measure protein concentration. In addition they show uorescence,
which can be used to measure conformational changes in proteins.

Exercises

1.1. Which of the following statements is/are true about amino acids?
1) All protein forming amino acids are chiral.
2) Threonine has two chiral centres.
3) Only L-amino acids occur in living organisms.
4) All amino acids have a pI-value.

1.2. Dene the isoelectric point of a compound.

1.3. Glycine is chiral

because
the -carbon contains only 3 dierent substituents.

1.4. Connect the following amino acids with their 1-letter code:
1) Valine A) D
2) Tryptophane B) V
3) Aspartic acid C) W
4) Tyrosine D) X
E) Y

1.5. Connect the following properties with amino acids:

1) hydrophobic A) Tryptophane
2) positively charged B) Serine
3) small C) Lysine
4) polar D) Glycine
5) aromatic E) Glutamine

1.6. Lysine has the pKa -values 2.18 (carboxy-group), 8.95 (-amino group)
and 10.53 (-amino group). The pI is .
 1.4 Biological function of amino acid variety 11

1.7. The following compound is a dipeptide,

because
it contains two covalently linked amino acids
COOH COOH
H2N CH H2N CH
C S S CH2
H2