Lecture 10

Amino Acids and Peptides

We have been talking about amino acids for a while now, and we already know some of their properties (acid/base properties, chirality), and what types of macromolecules they form (proteins). Now we will start studying them in more detail.

We have to keep in mind that this part will be pretty descriptive. We need to learn some properties of the monomers that make up proteins in order to study them.

General Structure

We start by going over the general structure of these biologically important monomers. As we have seen earlier, amino acids have two functional groups connected to an sp3 carbon atom, an amine and a carboxylic acid. Since the amine is 'a' (one atom away) with respect to the carboxylic acid and vice-versa, natural amino acids are called a-amino acids, and the sp3 carbon is normally referred to as the a-carbon, or Ca. Of the two remaining groups/atoms, one is always a hydrogen atom. Again, this hydrogen is called the a-hydrogen, or Ha.

The fourth group is the one that varies and gives amino acids their differenciating properties, and we will usually call it the side chain. Before going into the different side chains present in natural amino acids, we will discuss something we already know. Since in 19 of the 20 cases we will see there will be four different substituents in the Ca carbon, it will be a chiral center.

The absolute configuration of all amino acids, except cysteine, is S (we'll see why). However, we normally refer to amino acids as to being either L or D. These are related to the levo and dextro configurations, but not of the amino acids themselves, but of glyceraldehyde. What we do is compare the abosulte configurations of the amino acid and glyceraldehyde: If they correspond to the L form of glyceraldehyde, we call them L-amino acids, if they correspond to the D form of glyceraldehyde, they are D-amino acids:

Now, all natural amino acids have L configuration, which means that their side chains all point to the same side. If we consider the atoms we have in the different side chains, this means, as we mentioned before, that all amino acids except cysteine have R absolute configuration. Why?

As we said before, the presence of chirality in the Ca carbon means that the amino acids are chiral, and therefore all the molecules that contain amino acids will have sides. The most important case are proteins.

Amino acid side chains

Amino acids can be classified according to the chemical nature and size of their side chain, which is ultimately what gives different chemical and structural properties to the 20 different natural amino acids. Try to have a table with their structures when going over this:

1) Non-polar aliphatic side chains: These include Glycine, Alanine, Valine, Leucine, Isoleucine, and Proline. These are all hydrocarbons, and are highly hydrophobic residues. Their only difference (very important, by the way) is their size and shape. Glycine is the smallest amino acid, and has no chiral center. Since it has no proper side chain, it will be the one that imposes the least steric restrictions around it. A protein behaves really funny around Glycine, because it becomes really flexible at that point. On the other hand, we have proline, in which the side chain is cyclized to the amine nitrogen forming a secondary amine. The cycle in Proline makes it really rigid, imposing a conformational constraint to the protein it forms part of. Also note than in Valine, Leucine, and Isoleucine we have a second chiral center.

Warning: Many books say that proline is an imino acid. THIS IS NOT RIGHT. An imine group is C=N.

2) Non-polar aromatic side chains: These include Phenylalanine, Tyrosine, and Tryptophan. They are all hydrophobic, and they they will participate in hydrophobic interactions. More importantly, they will participate in strong p-p interactions when the aromatic groups are stacked on one another. Tyrosine and Tryptophan have polar groups (the -OH and the N), which makes them slighlty more polar than Phenylalanine, and allows them to participate in hydrogen bonds. Since they have conjugated p-systems, these amino acids absorb UV light more strongly than other amino acids, whose only chromophore is the carbonyl group, and can be detected easily that way.

3) Polar uncharged side chains: These are Serine, Threonine, Cysteine, Methionine, Arginine, and Glutamine. They all have groups that can participate in hydrogen bonding with water (either as donors or acceptors), and are therefore hydrophilic and, to a certain point, soluble in water. The -OH makes Serine and Threonine poolar. The -SH makes Cysteine polar, and the R-S-R sulfur makes Methionine polar. Finally, the aminde groups in Asparagine and Glutamine makes them polar.

The thiol group (-SH) in Cysteine can get oxidized with O2, and form a disulfide bridge between two Cysteine residues (-S-S-). This is called a Cystine group.

4) Charged positive side chains: They include Lysine, Arginine, and Histidine. Lysine has an amino group at carbon e, Arginine bears a guanidino group, and Histidine an imidazole ring. Their pKR indicate that they all have positive charges at pH 7, which makes them good bases/acids and very polar. They have the ability to participate in strong ionic interactions with water and salt bridges with negativelly charged groups.

5) Charged negative side chains: These are Aspartate and Glutamate (or Aspartic and Glutamic acid). These two have carboxylic acid (-COOH) groups at the end of their side chains. The pKR of these groups indicate that they will normally be negativelly charged at pH 7.0, making them very polar and soluble in water. They can act as acid/bases, form hydrogen bonds, form salt bridges with positivelly charged groups, and act as nucleophiles in certain reactions.

We give the carbon atoms of the side chains names depending on how far they are from the a-carbon using the Greek alphabet (a, b, g, d...). For Lysine:

-CaH-CbH2-CgH2-CdH2-CeH2-NH3+

Now, since we will be using amino acids pretty heavily, there will come a certain point in which we cannot spend the time to draw out every one of them. We have to use a code to represent them in a fast and unambiguous way. We have to schemes. A three-letter code, and a one-letter code. The more biochemistry you study, the more you will learn these codes. You will also realize that although it is harder to remember, the one-letter code is the one most commonly used when writing the sequence of a protein with hundreds of amino acids for obvious reasons. Here is the code, translation, and silly mnemonic rules to help you digest it...
 
Side chain
three-letter
one-letter
mnemonic
Glycine
Gly
G
G-lycine
Alanine
Ala
A
A-lanine
Valine
Val
V
V-aline
Leucine
Leu
L
L-eucine
Isoleucine
Ile
I
I-soleucine
Proline
Pro
P
P-roline
Phenylalanine
Phe
F
F-enilalanine
Tyrosine
Tyr
Y
t-Y-rosine
Tryptophan
Trp
W
W-ryptophan
Serine
Ser
S
S-erine
Threonine
Thr
T
T-hreonine
Cysteine
Cys
C
C-ysteine
Methionine
Met
M
M-ethionine
Asparagine
Asn
N
asparagi-N-e
Glutamine
Gln
Q
Q-lutamine
Aspartate
Asp
D
a-D-spartate
Glutamate
Glu
E
glutamat-E
Lysine
Lys
K
K is next to L
Arginine
Arg
R
a-R-ginine
Histidine
His
H
H-istidinde

Sometimes it is tricky to identify between Asp and Asn, or Gln and Glu. In those cases in which the identity is in doubt, we call them Asx (B) and Glx (Z).

Acid / base propertires

We saw this when we discussed polyprotic acids and bases: amino acids will behave as weak acids, with two distinct pKa values. We saw the other day that depending on the pH we will have different proportions of the two ionizable groups protonated or deprotonated. We also saw the pH titration profile for alanine, and for the most part, all amino acids have a very similar pH profile.

Also, there was a certain region in the pH profile in which the total charge of the amino acid was zero: The number of negative charges on the COO- end were exacly balanced with the positive NH3+ charges, and the species existed as a zwitterion. This was the isoelectric point of the amino acid, and it is basically the mid point between the two pKa values. How can you calculate it?

Now, we have to remember that there are several amino acids that have a third easily ionizable group: Aspartate, Glutamate, Histidine, Lysine, and Arginine. We call the pKa value of the side chain ionizable group the pKR. The first two have pKR values well below 7 (they are COOH groups), histidine is near 6, and Arginine and Lysine are positively charged and have pKR values well above 7. Normally, we do not consider Serine, Threonine, or Tyrosine because they are far worst bases (the pKas for the -OH groups are > 10). What is the approximate pI of the later five amino acids?

Chromatographic separation of amino acids

The variation in the ionization state of an amino acid with pH is a property that we can actually put to good use. Since at different pHs amino acids will have different net charges depending on their pKas, they will bind differently to an ion exchange resin. The material in a resin is basically a polymer (polymer beads) to which charged groups are bound covalently. If the groups are positive, (usually -NH(CH2CH3)2+), the resin will bind anions and we call it an anion echange resin. If they are negativelly charged (usually -SO3- or -COO-), the resin will bind cations, and it is called a cation exchange resin. In order to separate a mixture of amino acids, we first load the resin into a column, and put the mixture at the top. We initially set the pH so that all the amino acids in our mixture are fully ionized. In that way, they will all bind tightly to groups of opposite chagre in the resin.

Now, we start the flow of our eluant solution, and we gradually change its pH. This is called a pH gradient. What will happen is that depending on their pKa values, the charges of the different amino acids will change as a function of pH. This has the effect of changing the net charge of the different amino acids in the mixture. Amino acids will less total charge at a certain pH will bind less tighly than others and will spend more time in solution, eluting faster from the columns, while those that are charged at that pH will remain attached to the column beads coming out latter, therefore separating different amino acids in the mixture.

Instead of making a pH gradient, we can do a salt gradient. Basically we increase gradually the concentration of salt (NaCl) of the eluant. The more salt, the more ions (Na+ and Cl-) that will be competing for the resin groups. These ions will butt out less chargeds species first, doing effectivelly the same thing than a pH gradient. We do this when changes in pH can screw up our samples.

Prepared by Guillermo Moyna, 1999.