Amino acids, proteins, enzymes and DNA

Amino acids

Hair, egg white and muscle are made largely of protein, and every protein is a chain of amino acids. Living things build their proteins from twenty amino acids, all with the same skeleton: an amine group, NH₂, and a carboxylic acid group, COOH, bonded to the same carbon atom. The general formula is H₂NCH(R)COOH, and the amino acids differ only in the side chain, R.

Glycine, the simplest, has R = H and is aminoethanoic acid. Alanine has R = CH₃ and is 2-aminopropanoic acid. Serine has CH₂OH, cysteine CH₂SH, lysine a chain ending in a second NH₂ group, and aspartic acid a second COOH group. A question gives the structure of any amino acid it uses, so the skill is recognising the pattern, not learning the side chains.

In every amino acid except glycine, the central carbon has four different groups attached: H, NH₂, COOH and R. It is a chiral centre, and the amino acid exists as two enantiomers that rotate plane-polarised light in opposite directions. Glycine has two hydrogen atoms on that carbon, so it has no chiral centre and is not optically active. Proteins in living things are built from one enantiomer of each amino acid only.

Amino acid

A compound with an amine group and a carboxylic acid group; in the α-amino acids that make proteins both are on the same carbon atom.

Zwitterions and the isoelectric point

Glycine is a white crystalline solid that decomposes above about 230 °C without melting. Propanoic acid, with almost the same Mr, is a liquid that freezes at −21 °C. The difference is that solid glycine is ionic. The COOH group donates a proton and the NH₂ group accepts one, within the same molecule, giving ⁺H₃NCH₂COO⁻, an ion with a positive and a negative charge and no overall charge, called a zwitterion. The solid is a lattice of zwitterions held by strong attractions between opposite charges, so a great deal of energy is needed to break it down, and the charged groups make glycine dissolve well in water.

In solution the form an amino acid takes depends on the pH. In acid the COO⁻ group takes a proton back, and the amino acid becomes a positive ion, ⁺H₃NCH(R)COOH. In alkali the NH₃⁺ group loses its proton, and the amino acid becomes a negative ion, H₂NCH(R)COO⁻. Between the two there is a pH at which the amino acid is present almost entirely as the zwitterion, with no overall charge: the isoelectric point.

⁺H₃NCH₂COO⁻(aq) + H⁺(aq) → ⁺H₃NCH₂COOH(aq)In acid: the carboxylate group accepts a proton.

⁺H₃NCH₂COO⁻(aq) + OH⁻(aq) → H₂NCH₂COO⁻(aq) + H₂O(l)In alkali: the ammonium group loses a proton to hydroxide.

Describe the drawing

Three displayed structures of alanine stacked from low pH at the top to high pH at the bottom. At low pH, in acid, the amine group is NH₃⁺ and the acid group COOH, overall charge plus one. At the isoelectric point, about pH 6, the zwitterion has NH₃⁺ and COO⁻, overall charge zero. At high pH, in alkali, the amine is NH₂ and the acid group COO⁻, overall charge minus one. Downward arrows read minus H⁺ from COOH and then minus H⁺ from NH₃⁺, with plus H⁺ arrows back up, and the central carbon in each is starred as the chiral centre.

For glycine and alanine the isoelectric point is close to pH 6. An amino acid with an extra COOH in its side chain, such as aspartic acid, has a lower isoelectric point, about pH 2.8, because a lower pH is needed to keep its second acid group protonated. Lysine, with an extra NH₂, has an isoelectric point of about pH 9.7.

A drawn answer must show the charges on the right atoms: the positive charge on the nitrogen of NH₃⁺ and the negative charge on an oxygen of COO⁻. A zwitterion drawn with the charges but with the H still on the COOH group is a different species and does not earn the mark.

Zwitterion

An ion with both a positive and a negative charge and no overall charge, as in ⁺H₃NCH(R)COO⁻.

Isoelectric point

The pH at which an amino acid exists as the zwitterion and has no overall charge.

Peptides and proteins

The amine group of one amino acid can react with the carboxylic acid group of another, losing a molecule of water. The new –CONH– group is a peptide link, the same amide link as in nylon, and the reaction is a condensation. Two amino acids joined this way make a dipeptide, which still has a free NH₂ at one end and a free COOH at the other, so more amino acids can join on.

Describe the drawing

Glycine, H₂N–CH₂–C(=O)–OH, and alanine, with its amine group drawn as H–N–H and a CH₃ side chain, side by side. The OH of glycine's acid group and one H of alanine's amine group are ringed together in coral, with an arrow down to H₂O, water eliminated. Below, the dipeptide gly-ala is drawn in full, H₂N–CH₂–C(=O)–N(H)–CH(CH₃)–COOH, with the C=O and N–H group enclosed in a cyan box labelled peptide link, and the free NH₂ and COOH ends labelled N-terminal and C-terminal.

Two different amino acids can join in two ways. Glycine's COOH can react with alanine's NH₂, giving H₂NCH₂CONHCH(CH₃)COOH, or alanine's COOH can react with glycine's NH₂, giving H₂NCH(CH₃)CONHCH₂COOH. These are different compounds, gly-ala and ala-gly. A mixture of the two amino acids can also give gly-gly and ala-ala, so four dipeptides are possible in all.

A protein is a polypeptide chain of fifty or more amino acids, and its structure is described at three levels. The primary structure is the sequence of amino acids in the chain. The secondary structure is the way the chain coils into an α-helix or lies side by side in a β-pleated sheet, held in place by hydrogen bonds between the N–H of one peptide link and the C=O of another. The tertiary structure is the folding of the whole chain into a three-dimensional shape.

The tertiary structure is held by bonds between the side chains. Hydrogen bonds form between groups such as OH and NH₂. Ionic bonds form between an NH₃⁺ on one side chain and a COO⁻ on another. Disulfide bridges, covalent S–S bonds, form between the side chains of two cysteine residues. Weaker van der Waals forces hold hydrocarbon side chains together inside the fold. An answer about tertiary structure must name hydrogen bonding and the sulfur–sulfur bonds; the ionic bonds and van der Waals forces complete the picture.

Heating a protein under reflux with 6 mol dm⁻³ hydrochloric acid for about a day hydrolyses every peptide link, the reverse of the condensation. In the acid the amino acids come out as their positive ions. A drop of the mixture is spotted on a thin-layer chromatography plate beside drops of known amino acids, and the plate is stood in a solvent. Amino acids are colourless, so the dried plate is sprayed with ninhydrin, which turns them purple, or viewed under ultraviolet light. Each amino acid is identified by matching its Rf value, the distance it moved divided by the distance the solvent moved, with a known one on the same plate.

Peptide link

The –CONH– group that joins two amino acids, formed by a condensation reaction that eliminates water.

Primary structure

The sequence of amino acids in a polypeptide chain.

Secondary structure

The coiling of a polypeptide chain into an α-helix or its folding into a β-pleated sheet, held by hydrogen bonds between peptide links.

Tertiary structure

The folding of the whole polypeptide chain into a three-dimensional shape, held by hydrogen bonds, ionic bonds and disulfide bridges between side chains.

Enzymes and drugs

Enzymes are proteins that catalyse reactions in living cells. Each has an active site, a pocket in its tertiary structure lined with the side chains of a few amino acids. A substrate binds in the site by the same interactions that hold the protein's fold: hydrogen bonds between OH, NH and C=O groups, ionic attractions between charged groups such as NH₃⁺ and COO⁻, and van der Waals forces between hydrocarbon parts. Held there, the substrate forms an enzyme–substrate complex in which the bonds that must break are strained and the groups that react are brought together, so the reaction has a lower activation energy.

The simplest picture of this is the lock and key model: the active site has a shape that only its substrate fits, as only one key fits a lock, and a molecule of a different shape is not held and does not react.

The active site is chiral, because the protein is built from one enantiomer of each amino acid. A substrate is usually held at three or more points at once, each group on its chiral centre matched to a binding group in the site. The mirror image of the substrate can match at most two of those points at a time, with the third group pointing the wrong way, so it does not bind properly and is not converted. Enzymes are therefore stereospecific: the enzyme that oxidises lactic acid in muscle acts on one of its two enantiomers and leaves the other untouched.

Use 'stereospecific' and 'the active site is chiral' in an answer. 'The wrong enantiomer is the wrong shape' does not say why the site can tell two molecules with identical bonds apart.

A drug can work by blocking an active site. A molecule shaped enough like the substrate to bind in the site, with groups placed to make the same hydrogen bonds and ionic attractions, but which does not react, occupies the site and stops the substrate binding. The enzyme is inhibited. The ACE inhibitors that lower blood pressure work this way: they bind the active site of the enzyme that makes a hormone which narrows blood vessels. Because the site is chiral, a chiral drug usually has to be the right enantiomer, and the other may do nothing or something harmful.

Chemists now design such molecules by computer before any of them is made. The three-dimensional structure of the enzyme, often found by X-ray crystallography, is loaded into modelling software. Candidate molecules are fitted into the active site on screen, and each is scored on how well its shape fills the pocket and how many hydrogen bonds and ionic attractions it can make with the side chains there. Only the best-scoring candidates are synthesised and tested, which saves years of making compounds that would never have fitted. Modelling cannot show whether a molecule is absorbed, reaches the enzyme or is toxic, so laboratory and clinical testing still follow.

Active site

The region of an enzyme, formed by its tertiary structure, to which the substrate binds and where the reaction is catalysed.

Stereospecific active site

An active site that can bond to only one enantiomeric form of a substrate or drug, because the site itself is chiral.

DNA, and how cisplatin acts on it

DNA is a polymer of nucleotides. Each nucleotide has three parts: a phosphate group, a sugar called 2-deoxyribose, and a base, one of adenine, cytosine, guanine and thymine. The phosphate of one nucleotide is joined to the sugar of the next by a condensation reaction, so a single strand has a backbone of sugar and phosphate groups in turn, with a base hanging from every sugar.

Two strands wind round each other in a double helix, with the bases on the inside. The bases pair by hydrogen bonding: adenine with thymine through two hydrogen bonds, and cytosine with guanine through three. Only these pairs fit, because only they line up hydrogen bond donors opposite acceptors at the right distance. The sequence of one strand therefore fixes the sequence of the other, which is how DNA is copied when a cell divides.

Describe the drawing

Two base pairs, one above the other, each held between a grey sugar–phosphate backbone on the left and another on the right, deoxyribose drawn as pentagons and phosphate as discs marked P. At the top adenine, a fused six- and five-membered ring, faces thymine, a six-membered ring, and two dashed hydrogen bonds join them: the N–H of adenine's NH₂ group to a C=O of thymine, and the N–H of thymine to a ring nitrogen of adenine. Below, guanine faces cytosine across three dashed hydrogen bonds: guanine's C=O to an N–H of cytosine's NH₂, guanine's ring N–H to a ring nitrogen of cytosine, and an N–H of guanine's NH₂ to cytosine's C=O. Each base is bonded through a ring nitrogen to its deoxyribose.

Cisplatin is the square planar complex cis-[Pt(NH₃)₂Cl₂]. Blood contains a high concentration of chloride ions, so the chloride ligands stay in place as the drug travels. Inside a cell the chloride concentration is far lower, and the two chloride ligands are replaced, first by water and then by nitrogen atoms on DNA. The platinum bonds to a nitrogen on each of two neighbouring guanine bases on the same strand, in a ligand substitution reaction.

Describe the drawing

Two panels. Above, cisplatin drawn square planar with platinum at the centre, two ammonia ligands on neighbouring corners and two chloride ligands on the other two, and beside it transplatin with its chlorides on opposite corners, marked trans: inactive. An arrow notes that inside the cell the chlorides are replaced by water and then by a nitrogen of guanine. Below, a short length of one DNA strand with two neighbouring guanines, each joined to a deoxyribose on a sugar–phosphate backbone that bends sharply between them; the platinum, still carrying its two ammonia ligands, is bonded by amber coordinate bonds to nitrogen N7 of each guanine at neighbouring corners of its square.

The bound platinum kinks the DNA strand, so the strands cannot be separated and copied properly. The cell cannot replicate its DNA, cannot divide, and dies. Cancer cells divide rapidly, so they are affected most. The trans isomer has its two chlorides opposite each other and cannot reach two neighbouring guanines on one strand, so only the cis isomer is used as a drug.

Cisplatin also acts on healthy cells, worst on those that divide quickly, such as hair follicles and the lining of the gut, and it damages the kidneys. Patients lose hair and suffer sickness. The dose is kept as low as will treat the cancer, given in courses with breaks between them, and the risk of the side effects is weighed against the much greater risk of the cancer left untreated.

Nucleotide

The monomer of DNA, made of a phosphate group, the sugar 2-deoxyribose and one of four bases.

Other boards: electrophoresis, esters and the trans isomer

Cambridge examines a second separation of amino acids, which uses the charge on each one instead of how strongly it is held by a TLC plate. In electrophoresis a strip of paper or a slab of gel is soaked in a buffer solution of fixed pH, the mixture is spotted in the middle, and a potential difference is applied across it. An amino acid that is positive at that pH moves towards the negative electrode; one that is negative moves towards the positive electrode; one at its isoelectric point has no overall charge and stays where it was spotted.

How far each moves depends on its charge and its size. A larger charge moves faster, and a larger ion, such as a peptide, moves more slowly than a small amino acid with the same charge. Choosing the pH of the buffer sets the charges, so a pH between the isoelectric points of two amino acids sends them in opposite directions. The strip is dried and sprayed with ninhydrin, as a TLC plate is, and the same method separates the fragments of DNA in genetic fingerprinting.

Electrophoresis

The separation of charged species, such as amino acids in a buffer of fixed pH, by their movement towards the oppositely charged electrode when a potential difference is applied.

OCR also examines the ester of an amino acid. The carboxylic acid group keeps the reactions of its family, so glycine heated with ethanol and concentrated sulfuric acid gives ethyl aminoethanoate. In that acid mixture the amine group is protonated, so the ester forms as its salt, ⁺H₃NCH₂COOCH₂CH₃, and the free ester is released by adding alkali.

H₂NCH₂COOH(s) + CH₃CH₂OH(l) ⇌ H₂NCH₂COOCH₂CH₃(l) + H₂O(l)With concentrated sulfuric acid as the catalyst, written for the neutral molecules.

Edexcel asks why the drug is supplied as the cis isomer alone rather than as a mixture with the trans. Transplatin does not stop the cancer cells dividing, but it is still a platinum compound that cells take up and the kidneys must clear. In a mixture it would add to the side effects without adding to the treatment, and the dose of the active isomer would be uncertain. The synthesis is designed to give the cis isomer, and each batch is checked for the trans form.