Biochemistry

What the exam asks

Expect a table of pKa values and a pH, and expect the question to be a charge question wearing a biochemistry costume. The commonest items are: give the net charge of an amino acid or short peptide at a stated pH, work out an isoelectric point, predict which way something moves in an electrophoresis gel, and classify a described interaction by which level of protein structure it belongs to. The trap that catches most candidates is calling the zwitterion 'neutral' and then reasoning as though nothing is charged. Net zero is a sum, not a description of the molecule: it has a full positive and a full negative on it, which is why amino acids are solids with high melting points and dissolve happily in water. The second trap is averaging all three pKa values to find an isoelectric point. Only two of them count, the pair on either side of the form with no net charge, and picking that pair correctly is the entire question.

This is chemistry applied to the molecules life is built from, and the exam treats it as chemistry rather than as biology. An amino acid is not memorised, it is analysed: it has an acid group, a base group, and possibly an ionisable side chain, and everything asked about it comes from comparing the pH to those pKa values.

That comparison is the whole topic and it transfers straight from acids and bases. Below a group's pKa the group holds its proton; above it the group has let go. Apply that to each ionisable group in turn, add up the charges, and you have the net charge at any pH the stem cares to name. Do it in reverse, asking which pH makes the charges cancel, and you have the isoelectric point. No calculation heavier than averaging two numbers is ever required.

Proteins are then built by joining amino acids through the peptide bond, an amide made by condensation, and the levels of structure are a hierarchy of what holds them together: covalent backbone, then backbone hydrogen bonds, then side chain interactions folding one chain, then several chains assembling. Knowing which level a described interaction belongs to answers most protein questions.

Carbohydrates and lipids get lighter treatment. Carbohydrates are about the anomeric carbon and the glycosidic bond; lipids are defined by not dissolving in water, and the shape of a fatty acid tail explains almost everything they do.

What to hold

• An amino acid has an amino group, a carboxyl group, a hydrogen and a side chain on the same alpha carbon, and only the side chain differs between them.
• Nineteen of the twenty standard amino acids are chiral; glycine is not, because its side chain is a hydrogen, giving its alpha carbon two identical groups.
• The alpha carboxyl group has a pKa around 2 and the alpha amino group around 9 to 10, so at any ordinary pH the first is deprotonated and the second is protonated.
• At physiological pH an amino acid with a neutral side chain is a zwitterion: negative carboxylate and positive ammonium at once, net charge zero, not uncharged.
• The rule for any ionisable group is that pH below its pKa means protonated and pH above its pKa means deprotonated, so a carboxyl loses its charge as pH drops and an amine loses its charge as pH rises.
• The isoelectric point is the pH at which net charge is zero, and it is the average of the two pKa values that flank the neutral form, not the average of every pKa in the molecule.
• For an amino acid with a neutral side chain the isoelectric point is the average of the carboxyl and amino pKa values, which lands near 6.
• An acidic side chain drags the isoelectric point down, because the flanking pair is now the two lowest pKa values; a basic side chain drags it up for the mirror reason.
• Histidine is the only side chain with a pKa near physiological pH, which is why it appears wherever a protein needs to gain or lose a proton at working pH.
• In electrophoresis, a protein at a pH above its isoelectric point is negatively charged and moves towards the anode; below it, the protein is positive and moves towards the cathode; at it, the protein does not move.
• The peptide bond is an amide formed by condensation, losing a water, and it is planar with restricted rotation because the nitrogen lone pair delocalises into the carbonyl.
• Primary structure is the sequence held by covalent peptide bonds, secondary structure is helices and sheets held by hydrogen bonds along the backbone, tertiary is one chain folded by side chain interactions, and quaternary is several folded chains assembled.
• Denaturation destroys the folding but not the sequence, because the peptide bonds are covalent and the interactions holding the fold are not.
• A sugar cyclises to create a new stereocentre at the anomeric carbon, giving alpha and beta forms that differ at that one carbon and so are diastereomers.
• A reducing sugar is one whose anomeric carbon is still free to open; lock every anomeric carbon into a glycosidic bond, as sucrose does, and the sugar is non-reducing.
• A cis double bond puts a kink in a fatty acid tail that stops neighbouring chains packing tightly, which is why unsaturated fats melt lower than saturated ones of the same length.