Amino acids, proteins and DNA

In a nutshell

Amino acids, proteins and DNA are the molecules of life, and this topic treats them as organic chemistry: the functional groups you already know, behaving in ways you can predict.

An amino acid is just a molecule with both an acidic -COOH group and a basic -NH₂ group, so it reacts with both acids and bases. Link many together and you get a protein; swap in a sugar, a phosphate and a base and you get DNA.

Assumed knowledge: Carboxylic acids and derivatives (the amide link), Optical isomerism (3.3.7), Amines (3.3.11).

Core content

Amino acids: acid-base behaviour and zwitterions

An α-amino acid has an amine group (-NH₂) and a carboxylic acid group (-COOH) attached to the same carbon, with a side chain R: the general structure is H₂N-CH(R)-COOH.

The -COOH group is acidic (can donate H⁺) and the -NH₂ group is basic (can accept H⁺). Having both means an amino acid reacts with both acids and alkalis.

In the solid and in neutral solution the -COOH donates an H⁺ to the -NH₂ on the same molecule. This gives a dipolar ion called a zwitterion, with no overall charge but a positive and a negative end.

Move to acid (add H⁺): the -COO^- end is protonated, so you get the cation ⁺H₃N-CH(R)-COOH.

Move to alkali (add OH^-): the -NH₃⁺ end loses its H⁺, so you get the anion H₂N-CH(R)-COO^-.

Because a zwitterion is a genuine ion, solid amino acids are held together by strong electrostatic forces between oppositely charged ions, not by the weaker hydrogen bonding you would expect for a neutral molecule of similar size. That is why amino acids have relatively high melting points.

Still don't get it? · zwitterions and why amino acids melt high

Think of two sets of fridge magnets. A neutral molecule is like a magnet whose poles are weak, so a stack of them only clings together gently: that is hydrogen bonding. A zwitterion is like a magnet with a full strength north pole at one end and a full south pole at the other, so a stack of them grips hard: those are the electrostatic forces between full + and - charges.

Build it up a step at a time. (1) An amino acid has an acid end that likes to give away H+ and a base end that likes to take one. (2) The easiest thing to do is pass the H+ from its own -COOH to its own -NH2, giving a +NH3 end and a -COO- end: that is the zwitterion. (3) A zwitterion carries real + and - charges, so it is an ion, and ions attract each other with strong electrostatic forces. (4) Melting means pulling those ions apart, which needs a lot of energy, so the melting point is high.

Exam wording: in the marked answer you must say the amino acid is held by electrostatic forces between IONS (or ionic bonding), and that this is stronger than the hydrogen bonding in the non-ionic molecule you are comparing it with. If you call the zwitterion a 'molecule' or talk about 'intermolecular forces', you lose the marks.

Proteins: the peptide link and primary structure

Amino acids join by a condensation reaction: the -COOH of one and the -NH₂ of the next react, a molecule of water is lost, and a peptide link (-CO-NH-) forms. The peptide link is simply an amide group.

Two amino acids joined give a dipeptide; many joined give a polypeptide, and a protein is one or more polypeptide chains.

The primary structure is the sequence (order) of amino acids in the polypeptide chain, held together by the covalent peptide links.

Secondary structure

Local parts of the chain fold into regular shapes held in place by hydrogen bonds between the backbone groups. The hydrogen bonds form between the δ⁺ H of an N-H group and a lone pair on the δ^- O of a C=O group further along the chain.

AQA credits two secondary structures:

• the α-helix: the chain coils into a corkscrew, held by hydrogen bonds running along the coil.
• the β-pleated sheet: chains lie side by side and are held by hydrogen bonds between the strands.

Tertiary structure

The tertiary structure is the overall 3D folding of the whole chain, held in place by interactions between the R side chains of amino acids that end up near each other.

The spec names two of these interactions:

• Hydrogen bonds between polar R groups (for example two serine -OH groups).
• Sulfur-sulfur (disulfide) bridges: two cysteine side chains, each ending in -CH₂-SH, react to form a covalent -CH₂-S-S-CH₂- bridge.

Ionic interactions between a -COO^- and an -NH₃⁺ R group also help, but the two the mark scheme always rewards are hydrogen bonding and S-S bonds.

Hydrolysis of the peptide link

Heating a protein or peptide with concentrated hydrochloric acid breaks the peptide links by hydrolysis (water is added back), splitting the chain into its constituent amino acids.

Because the hydrolysis is done in acid, the amino acids come off in their protonated form (-NH₃⁺ and -COOH). In alkaline hydrolysis they come off as the carboxylate (-NH₂ and -COO^-).

Separating amino acids: thin-layer chromatography

A mixture of amino acids (for example from hydrolysing a protein) is separated and identified by thin-layer chromatography (TLC).

• A spot of the mixture is placed on a pencil baseline near the bottom of a TLC plate, and the plate stands in a solvent whose level is below the baseline (if the solvent is above the line it dissolves the spots off the plate).
• The solvent rises up the plate, carrying each amino acid a different distance according to how strongly it is held by the stationary phase.
• Amino acids are colourless, so the spots are located by spraying with ninhydrin (spots turn coloured) or by viewing under ultraviolet (UV) light.

Each amino acid has a characteristic R_f value, found by comparing how far the spot moves with how far the solvent front moves:

$R_f = \frac{\text{distance moved by the amino acid spot}}{\text{distance moved by the solvent front}}$

R_f has no units and is always less than 1. An unknown is identified by matching its R_f against known values measured in the same solvent.

Enzymes and stereospecific active sites

Enzymes are proteins that act as biological catalysts. Each enzyme has an active site, a hollow in its folded (tertiary) structure, into which a substrate molecule binds to form an enzyme-substrate complex.

The active site is stereospecific: it has a precise 3D shape, so if the substrate is chiral, only one enantiomer has the correct arrangement of groups to fit and bind. The other enantiomer, being a non-superimposable mirror image, does not fit, so only one optical isomer is catalysed.

Still don't get it? · why only one enantiomer fits

Think of your hands and a right-handed glove. Your two hands are mirror images, yet only the right hand slides into a right-handed glove; the left hand simply will not fit no matter how you turn it. The active site is the glove; the two enantiomers of the substrate are your two hands.

Step by step. (1) A chiral molecule has two forms that are mirror images and cannot be laid on top of one another, exactly like left and right hands. (2) The active site is a fixed 3D shape with its binding groups in fixed positions. (3) Only the enantiomer whose groups line up with those binding positions can sit in the site and bind. (4) The mirror-image enantiomer has its groups in the 'wrong' places, so it cannot bind.

Exam wording: say the active site is stereospecific and only one enantiomer has the correct 3D shape / arrangement of groups to fit (bind to) the active site, so only that enantiomer reacts.

Drugs as enzyme inhibitors

A drug can work as an enzyme inhibitor by binding to the active site and blocking it, so the substrate can no longer enter and the enzyme-catalysed reaction is stopped.

Because the shape of the active site decides what binds, computers are used to model active sites and help design drug molecules that fit them.

The structure of DNA

DNA is built from nucleotides. The structures of the phosphate ion, the sugar 2-deoxyribose (a pentose) and the four bases adenine, cytosine, guanine and thymine are given to you in the Chemistry Data Booklet.

A nucleotide is a phosphate ion bonded to a 2-deoxyribose sugar, which is in turn bonded to one of the four bases.

A single DNA strand is a polymer of nucleotides joined by covalent bonds between the phosphate of one nucleotide and the 2-deoxyribose of the next. This builds a sugar-phosphate-sugar-phosphate backbone with the bases attached to the sugars.

DNA exists as two strands wound into a double helix. The strands are complementary: the bases pair up specifically, A always with T and C always with G.

The two strands are held together by hydrogen bonding between the base pairs:

• adenine with thymine (A-T): 2 hydrogen bonds
• cytosine with guanine (C-G): 3 hydrogen bonds

Because A only pairs with T and C only with G, the base sequence on one strand fixes the sequence on the other, which is what 'complementary' means.

Cisplatin as an anticancer drug

Cisplatin is a platinum(II) complex, cis-[Pt(NH₃)₂Cl₂], used as an anticancer drug. It is square planar, with the two Cl ligands in cis positions.

Cisplatin prevents DNA replication in cancer cells by a ligand replacement (substitution) reaction with DNA: a chloride ligand is replaced and a coordinate (dative covalent) bond forms between the platinum and a nitrogen atom on a guanine base.

Once bound, cisplatin holds the DNA so that the strands cannot separate, so the DNA cannot replicate and the cancer cell cannot divide. The water-substitution step that activates it can be written as:

cis-[Pt(NH₃)₂Cl₂] + H₂O → [Pt(NH₃)₂Cl(H₂O)]⁺ + Cl^-

Adverse effects and the balance of risk: cisplatin also binds to DNA in healthy cells, especially fast-dividing ones such as hair follicles and bone marrow, killing them and causing side effects such as hair loss and a lowered immune response. These can be limited by giving small doses and targeting the drug at the tumour. Society has to weigh the benefit (an effective cancer treatment) against the adverse effects.

Worked examples

Example 1: calculating an R_f value.

On a developed chromatogram, an amino acid spot has moved 3.6 cm from the baseline and the solvent front has moved 8.0 cm from the baseline. Calculate the R_f value.

$R_f = \frac{\text{distance moved by the spot}}{\text{distance moved by the solvent front}}$

$R_f = \frac{3.6 \text{ cm}}{8.0 \text{ cm}}$

$R_f = 0.45$

Both distances are measured from the baseline to the centre of the spot and to the solvent front. The answer has no units (the cm cancel) and is quoted to 2 significant figures to match the data. A value greater than 1 means a measuring or method error, because a spot cannot travel further than the solvent.

Example 2: drawing an amino acid in acid and in alkali (a diamino acid).

Lysine is 2,6-diaminohexanoic acid, H₂N-(CH₂)₄-CH(NH₂)-COOH. Deduce its structure in acidic and in alkaline solution.

1. Identify every group that can gain or lose H⁺: two basic -NH₂ groups and one acidic -COOH group.
2. In acid (excess H⁺), every basic group is protonated and the acid group stays as -COOH: $^{+}\text{H}_3\text{N-(CH}_2)_4\text{-CH(NH}_3^{+})\text{-COOH}$ Both -NH₂ groups become -NH₃⁺, giving an overall charge of 2+. Forgetting the second -NH₃⁺ is the single most common lost mark here.
3. In alkali (excess OH^-), the acid group is deprotonated to -COO^- and both amine groups stay as -NH₂: $\text{H}_2\text{N-(CH}_2)_4\text{-CH(NH}_2)\text{-COO}^{-}$ giving an overall charge of 1-.

The method is general: in acid, protonate every basic group; in alkali, deprotonate every acidic group.

Common exam mistakes

• Explaining the high melting point by calling the zwitterion a "molecule" or citing "intermolecular forces". The mark needs ionic bonding / electrostatic forces between ions, described as stronger than the hydrogen bonding in the non-ionic molecule being compared.
• Protonating only one amine group of a diamino acid (lysine) in acid. In acid both -NH₂ groups become -NH₃⁺, so the ion carries a 2+ charge.
• Drawing the peptide link with an extra oxygen between the carbon and nitrogen. The link is -CO-NH-, a carbonyl carbon bonded straight to the nitrogen, not -CO-O-NH-.
• Giving the repeating unit of a polymer or an anhydride when a dipeptide is asked for. A dipeptide is two amino acid residues joined by one peptide link, with the end groups shown.
• Writing "hydration" instead of "hydrolysis" for breaking the peptide link. Hydrolysis adds water to split the bond.
• Drawing the disulfide bridge as a dashed bond, or adding an extra H to the sulfur. The S-S bridge is a covalent bond (solid line), formed between two -CH₂-SH side chains to give -CH₂-S-S-CH₂-.
• For secondary-structure hydrogen bonding, stopping at "there is a hydrogen bond" without saying it forms between the δ⁺ H of N-H and a lone pair on the δ^- O of C=O, and drawing the N-H...O as linear. Calling it lone-pair "donation" describes a coordinate bond and is wrong.
• Getting the base-pair hydrogen bond counts the wrong way round: A-T has 2, C-G has 3. Also drawing thymine in the wrong orientation when pairing it with adenine.
• In a nucleotide, copying the sugar from the data sheet and omitting the phosphate (or drawing two phosphates), or leaving out the CH₂ that links the sugar to the phosphate.
• Spelling trap: the DNA base is thymine (not "thiamine", which is vitamin B1).
• For cisplatin, describing the Pt-N bond as ionic or ordinary covalent. It is a coordinate (dative covalent) bond to a nitrogen of guanine, and the two Cl must be cis.

Key definitions

• Zwitterion: a dipolar ion that has both a positive charge (-NH₃⁺) and a negative charge (-COO^-) on the same molecule, with no overall charge.
• Peptide link (peptide bond): the -CO-NH- (amide) link formed between two amino acids in a condensation reaction, with the loss of a molecule of water.
• Primary structure: the sequence (order) of amino acids in a polypeptide chain.
• Secondary structure: the folding of the polypeptide chain into an α-helix or β-pleated sheet, held in place by hydrogen bonds between the N-H and C=O groups of the backbone.
• Tertiary structure: the overall three-dimensional folding of the whole polypeptide chain, held by hydrogen bonds and sulfur-sulfur (disulfide) bonds (and ionic interactions) between the R groups.
• Hydrolysis (of a peptide): breaking the peptide link by adding a molecule of water, re-forming the constituent amino acids.
• R_f value: the distance moved by a component divided by the distance moved by the solvent front (measured from the baseline).
• Stereospecific active site: an active site with a fixed 3D shape that binds to only one enantiomer of a chiral substrate.
• Nucleotide: a phosphate ion bonded to a 2-deoxyribose sugar, which is bonded to one of the four bases (adenine, cytosine, guanine or thymine).
• Complementary strands: the two DNA strands whose base sequences match through specific pairing (A with T, C with G).

Specification

• I can state that amino acids have both acidic and basic properties, including forming zwitterions.
• I can draw an amino acid as a zwitterion and the ions it forms in acid solution and in alkaline solution.
• I can describe a protein as a sequence of amino acids joined by peptide links, and explain the importance of hydrogen bonding and sulfur-sulfur bonds.
• I can identify primary, secondary (α-helix and β-pleated sheet) and tertiary structure in diagrams, and explain how they are maintained.
• I can draw a peptide formed from up to three amino acids, and draw the amino acids formed by hydrolysis of a peptide.
• I can describe how amino acids are separated and identified by thin-layer chromatography using ninhydrin or UV light, and calculate R_f values.
• I can state that enzymes are proteins and explain why a stereospecific active site binds only one enantiomeric form of a substrate or drug.
• I can explain the principle of a drug acting as an enzyme inhibitor by blocking the active site, and that computers help design such drugs.
• I can describe a nucleotide, and a single DNA strand as a sugar-phosphate backbone of nucleotides with attached bases.
• I can explain how hydrogen bonding between base pairs holds the two complementary strands of the DNA double helix together.
• I can explain why cisplatin prevents DNA replication, and why such drugs can have adverse effects.

Related notes

• Carboxylic acids and derivatives (the amide link and its hydrolysis behind the peptide link)
• Bonding (hydrogen bonds and coordinate bonds)
• Optical isomerism (3.3.7): chirality, which underlies the stereospecific active site
• Amines (3.3.11): the basic -NH₂ group that makes amino acids react with acids
• Transition metals (3.2.5): cisplatin as a Pt(II) complex, ligand substitution and coordinate bonds
• Chromatography (3.3.16): the chromatography principles behind TLC and R_f values