Amino AcidsProtein Roles1. Many proteins function as enzymes, the biochemical catalysts. Enzymes catalyse nearly all

reactions that occur in living organisms.2. Some proteins bind other molecules for storage and transport. For example, myoglobin binds

oxygen in skeletal and cardiac muscle cells, and haemoglobin binds and transports oxygen and carbon dioxide in red blood cells.

3. Some proteins, such as tubulin, actin and collagen provide support and shape to cells and hence to tissues and organisms.

4. Assemblies of proteins can do mechanical work, such as the movement of flagella, the separation of chromosomes at mitosis and the contraction of muscles.

5. Many proteins play a role in decoding information in the cell. Some are involved in translation, whereas others play a role in regulating gene expression by binding to nucleic acids.

6. Some proteins are hormones, which regulate biochemical activities in target cells or tissues; other proteins serve as receptors for hormones.

7. Some proteins have highly specialised functions. For example, antibodies defend vertebrates against bacterial and viral infections, and toxins, produced by bacteria, can kill larger organisms.

These roles fall into four major functional groups:

Binding – carrier protein or enzyme bind a ligand or a substrate. Nearly all proteins have a binding function

Catalysis – enzymes Switching – many proteins, especially in cell membranes, that bind things that come from

the outside of the cell. Receptors then change shape and contact something inside the cell which switching something on inside the cell which sends a signal to the nucleus

Structural – hold cells in 3d shape

Proteins are made of Amino AcidsAmino acids can condense together to form peptides. Once you form a peptide bond/amide bond, it is very difficult to break – very strong. If the string of amino acids is larger than 30, it is capable of starting to fold up into a 3d shape, and so it is called a protein. If it is less than 30 amino acids, it is not capable of forming a 3d shape, and so is called a peptide.

General Structure of Amino AcidsThere is commonality in all amino acids – they all have:

a central α carbon an α amino group an α carboxylate group a hydrogen atom an R group

The difference between amino acids is the R group. The chemical character of the amino acid is therefore given by the R group.

Acid/Base Chemistry of Amino AcidsThe acidity of the carboxylic acids is due to the stability of the carboxylate anion relative to the acid. The charge of the carboxylate resides on two equivalent electronegative oxygen atoms. This is represented by a weighted average of the simple structures, resonance or canonical forms.

The relationship between these two canonical forms can be shown using curly arrows which are used in organic chemistry to denote the movement of pairs of electrons. The negative charge is shared between the two oxygen atoms.

Amino acids are zwitterionic – they have both an acidic group and a basic group. An acid donates protons and a base accepts protons. A carboxylate group is an acidic group (it has a tendency to lose its H to become COO-) and an amino group is a basic group (it has a tendency to accept an H to become NH3

+). This occurs in a pH dependant manner:

At pHs above the pK of an acidic side chain, the side chain is always negatively charged. At pHs below the pK of a basic side chain, the side chain is always positively charged.

pK is a dissociation constant. Anything that is capable of ionisation has a pK. The carboxylate group has a pK of about 2.5. This means that at a pH of 2.5, the carboxylate group is 50% COOH and 50% COO- and H+ - it has dissociated by 50%. The pH scale is logarithmic so if we go from pH 2.5 to pH 3.5, there is a tenfold change in the ionisation of the side chain, 4.5 is a one hundredfold change etc. So at pH 7, there will be 100 000 fewer protons around than there would be at 2.5. Hence, it remains 99.999% COO- at pH 7 as there are so few protons around. The converse is true of a basic group at pH 7 (when the amino group has a pK of 9.5) – there are more protons around. So, under physiological conditions, an amino acid never has COOH and NH2 groups – it always has COO- and NH3

+ groups. At pH 7 the acid has lost a proton and the base has gained a proton – they are charged (although the charges cancel each other out) and are therefore zwitterions.

Some R groups are also capable of being ionised. This is important in the role of R groups in protein structure and function. For example, histones bind DNA because DNA is strongly negatively charged and histones contain a lot of lysine which is a basic amino acid – it has a side chain that is positively charged at pH 7 so the positive charge of the histone binds the negative charge of the DNA.

pKa values vary somewhat depending on the precise molecular structure and the environment in which the acid-base chemistry is taking place.

For example, the carboxylic acid group of glycine is more acidic than a simple carboxylic acid since the corresponding carboxylate anion is more stable in the presence of the adjacent, positively charged, ammonium ion.

The effect of environment on pKa is particularly important in non-polar conditions such as the interior of a protein.

The table on the right (do not need to learn) shows that every amino acid has an α amino group and an α carboxylate group. The pKs of these groups are all very similar (between 1.8 and 2.5 for acidic carboxylate group and between 8.7 and 10.7 for basic amino group). Only some amino acids are capable of having their side chains ionised. Under physiological conditions, the acidic amino acids, with a pK less than 7 (aspartic acid & glutamic acid) will be negatively charged and the basic ones, with a pK greater than 7 (e.g. lysine and arginine) will be positively charged. Histidine has a pK which varies between pH 6 and pH 7, therefore it can be either basic or acidic at neutral pH which makes it very special. It is often present in the binding sites of enzymes where it plays a role in acid/base chemistry.

When put into a protein, the α amino group and the α carboxylate group form the peptide bond and therefore there charge can be ignored – there is no charge associated with a peptide bond. The only charge that has any significance for a protein in solution is the charge on the R groups.

Stereochemistry of Amino AcidsAmino acids exhibit stereochemistry – they can either be the D or the L form (D isomer rotates the plane of polarised light to the right, an L to the left). The two forms are non-superimposable mirror images of each other (stereoisomers).

In nature, almost all amino acids are in the L form. This means that it is easy for the protein to fold up when they are all in the same configuration.

A Survey of the α-Amino Acids of ProteinsThe following paragraphs take a look at the structure of the amino acids by group, and also some of the important amino acid derivatives used in the body. Amino Acids can be divided into a number of chemically distinct groupings. The side chains have differing reactivity and differing roles in proteins. The groups are:

hydrocarbon side chains carboxylic acid side chains amide side chains acyclic with basic N containing side chains hydroxyl functional groups sulphur containing side chains nitrogen heterocycles and proline

Amino Acids with Aliphatic R Groups

These are:

GLYCINE ALANINE VALINE LEUCINE ISOLEUCINE

These amino acids build in complexity. The simplest is glycine and it has a very important role to play in proteins. It is very small so is found at points in the polypeptide where there is a turn. Small amino acids such as glycine are found in these regions as large amino acids with bulky side chains would restrict the ability of the protein to turn. All these amino acids, particularly the larger ones (with longer hydrocarbon side chains) are hydrophobic. So, if there was a stretch of these amino acids in the primary sequence of a protein, and the protein folded up, these would be found on the inside of the protein, away from the water. If they were on the outside of a protein, they could be forming a binding site for something else that is hydrophobic.

Because both the α and β carbon atoms of isoleucine are asymmetric it has two chiral centres and therefore four possible stereoisomers:

Acidic R Groups and their Amide Derivatives

These are:

ASPARTATE GLUTAMATE ASPARGINE GLUTAMINE

Aspartate and glutamate confer negative charge on proteins because their side chains are ionised at pH 7. Under physiological conditions acidic side chains exist as the conjugate base, hence the suffix -ate.

These amino acids have either acidic side chains or polar side chains. Aspartate and glutamate have a tendency to lose an electron so they are negatively charged at pH 7. This means they are basic at pH 7. If there is one of these groupings in the active site of an enzyme it acts as a base – it would want to grabs protons back or attract a positive charge. It could also be involved in base catalysis where it abstracts a proton from something to make that group nucleophilic so it can attack a susceptible bond. If there is an appropriately positioned basic group next to water, it can take one of the protons from water, forming the nucleophile OH-.

Asparagine and glutamine are not charged – they are polar and can form hydrogen bonds. This means they are very important structurally – they can form hydrogen bonds to other groups, forming a network. A single hydrogen bond is very weak, but hundreds become a significant force.

Amino Acids with Basic R Groups

These are:

HISTDINE LYSINE ARGININE

The side chains of both lysine and arginine are positively charged at physiological pH contributing basicity to proteins, arginine is the most basic of the 20 amino acids.

They are positively charged because they have a pK which is higher than the pH at which they sit. As you take the pH down, they are protonated and positively charged. If they were in the active site, they could bind a negatively charged group or could be involved in acid catalysis.

Histidine is very important – it has the capability of losing a proton and gaining a proton – it can act as an acid or a base. In some enzyme active sites, histidine sits there and in one part of a reaction mechanism it donates a proton and in another part it will, once it has lost a proton, it will become a base and can accept a proton. It is therefore involved in acid/base chemistry in the active sites of enzymes. Histidine can also bind metal ions . It particularly likes to bind zinc. About 30% of all of the proteins in your body require zinc or a metal ion to work. Haemoglobin wouldn’t carry oxygen without iron in its active centre. Enzymes that hydrolyse proteins wouldn’t be able to do this without zinc in their active site. This is because zinc loves to bind water and if zinc binds water, it reduces the pK of water from 14.7 to 7, so water can be ionised at a much lower pH. This makes water a really good nucleophile in the presence of zinc – it is 50% OH - (and H+). This means it can take part in hydrolysis reactions.

Protonated and neutral forms of histidine are both physiologically important. This evenly balanced equilibrium underpins histidine’s key role in biological acid/base chemistry.

Side Chains with Alcohol and Sulphur Groups

These are:

METHIONINE CYSTEINE SERINE THREONINE

Cysteine is important as if there are two close together in a peptide, they can form strong covalent (disulphide bridges) between the –SH groups. This gives the protein stability. Proteins that exist in extremely nasty environments like hot sulphur springs can survive at 100°c due to the strong disulphide bridges that gives them tight structures. The disulphide bond is the ONLY covalent bond that exists in the tertiary structure of proteins between R groups. All the other R groups interact non-covalently.

Methionine is always found as the first amino acid in proteins. In bacteria it is formyl-methionine (it has a functional group on it that gives it polarity). As it is aliphatic (and hence hydrophobic) it is found buried deep inside proteins, away from water. It has a thiol group (sulphur), protected by methylation. In some metabolic processes, this amino acid becomes very important.

Serine and threonine have a free hydroxyl group in their side chain. They are important in metabolic processes as that hydroxyl group is capable of phosphorylation (being phosphorylated) – it loses the H and becomes OP. This is very important when it comes to activating enzymes for metabolic processes. Because of this, and because they are hydrophobic, they tend to be exposed on the outside of proteins.

Side Chains containing Nitrogen Heterocycles

These are:

PROLINE PHENYLALANINE TYROSINE TRYPTOPHAN

These amino acids with aromatic side chains are very hydrophobic. They are found on the inside of proteins. The hydrophobic effect results in hydrophobic amino acids such as these being sequestered away from water – it is the driving force for protein folding.

Proline is a special amino acid – it has no free amino group. The R group makes contact covalently with the amino group. This forms a ring structure. This is very important in protein structure – it is called a helix breaker – at the end of a helix there is always a proline as this stops helix formation between free amino and carboxylate groups. The presence of an extra alkyl group rather than a hydrogen atom on the amide nitrogen of proline not only precludes formation of a stabilising hydrogen bond with the usual carbonyl group but it also introduces a steric repulsion with that group. Proline often acts to terminate α-helices and is therefore called a helix breaker.

Hydrophobic Character of Amino AcidsThe more hydrophobic amino acids have a tendency to be sequestered away from the solvent towards the centre of protein molecules. This provides one of the major driving forces for protein folding.

Hydrophilic residues tend to interact with the solvent more easily via hydrogen bonding and thus can be on the outside of proteins.

The table on the right (do not need to learn) shows the hydrophobicity of the amino acids – the larger the number, the more hydrophobic the amino acid.

The group at the top are very hydrophobic and are therefore found on the inside of proteins. The group in the middle can be found either in the middle or outside – they are neither strongly hydrophobic nor strongly hydrophilic. Finally, the group at the bottom are very hydrophilic and are likely to be found on the outside of proteins, towards the water.

The Peptide BondProteins are linear polymers of L-α-amino acids linked via a peptide (amide) bond. They can form hydrogen bonds which affects secondary structure – the ONLY bond that affects secondary structure.

The nitrogen of the peptide bond forms a planar trigonal shape because a partial double bond is formed with the adjacent carbon.

The stereochemistry requires that the carbonyl carbon, nitrogen, oxygen, hydrogen atoms and the two neighbouring α-carbons are all constrained to lie in a plane.

Under physiological conditions this effectively restricts the peptide bond to one of two configurations: cis or trans.

To minimise steric crowding due to close proximity of bulky groups on the carbonyl carbon and the nitrogen the trans form is favoured, except where proline is involved.

Proline is a Unique Amino AcidThe steric preferences of proline are different from the other amino acids.

It has an alkyl chain rather than a hydrogen atom as the second substituent on nitrogen.

For amide bonds except those involving the nitrogen of proline there is a thermodynamic preference for the trans configuration (α-carbon substituents to be as far apart as possible) – the peptide therefore has a slight curve to it. As they are all the same form, they are flexible and can fold logically.

In amides containing proline the cis form is not dramatically disadvantaged.

In proteins about 10% of all peptide bonds involving proline are cis. This can induce quite sharp kinks in protein structure

The Shape of Polypeptides is defined by the Rotation about the C-N & C-C bonds

All atoms around the peptide bond lie in a planar conformation. There is no rotation around the peptide bond

Therefore, the shape of the polypeptide is defined by rotation about the C-N and C-C bonds.

This rotation is described by the torsion angles phi between C-N and psi between C-C.

If all psi and phi angles are the same, the peptide assumes a repeated structure.

For certain combinations of angles this can take the form of a helical structure (α-helix) or a beta sheet structure.

Clearly, the peptide structure exhibits flexibility and this is important for the complex structure of proteins.

The R groups are on alternating sides (opposite directions) as we go down the chain as otherwise the protein would not be able to fold correctly due to steric hindrance.

The Ramachandran PlotThe physical size of atoms and groups of atoms limits the possible psi and phi torsion angles that the backbone of the polypeptide chain can adopt without causing protruding R groups to bump into each other. Ramachandran devised a way of plotting these allowed psi and phi angles. The red patches on the right are those combinations of psi and phi angles that are allowed as they do not result in steric hindrance. The pink areas are allowed with a little relaxation of steric hindrance.