Biomolecules

Living matter is made of the same elements as rocks, air and water. If you take any living tissue and put it through a chemical procedure that reports the elements it is built from, you get a list such as carbon, hydrogen, oxygen, nitrogen, sulphur, phosphorus, calcium, magnesium, sodium, potassium, chlorine, iron and zinc. This is called elemental analysis, and it tells you which elements are present and in what proportion. What it cannot tell you is how those elements are joined, and that is the far more interesting question. So a second kind of analysis is used, one that looks for whole compounds rather than bare elements.

The method is easy to picture. Take a piece of living tissue, say a bit of vegetable or liver, put it in a mortar and grind it with trichloroacetic acid until you get a thick slurry. Now strain the slurry through a fine cloth or filter paper. You end up with two fractions. What passes through is the filtrate, also called the acid-soluble pool. What stays behind on the filter is the retentate, or the acid-insoluble fraction.

The acid-soluble pool holds thousands of small organic compounds with molecular weights running from about 18 to around 800 daltons. These are the micromolecules: amino acids, nucleosides and nucleotides, simple sugars, fatty acids, glycerol and various organic acids. Inorganic ions such as sodium, potassium, calcium, magnesium, chloride, phosphate and sulphate are also in this fraction.

The acid-insoluble fraction holds the giants: proteins, nucleic acids and polysaccharides. These are the biomacromolecules, with molecular weights of about ten thousand daltons and above. Lipids turn up in this fraction too, which surprises many students, because a single lipid molecule usually weighs well under 800 daltons and is not really a macromolecule at all. The reason is mechanical. Lipids in a cell are built into membranes. When you grind the tissue you smash those membranes into fragments, and the fragments close up into tiny water-insoluble bags called vesicles. These vesicles are far too big to pass through the filter, so the lipids get held back along with the true macromolecules.

If you weigh out what a living tissue contains, water is by far the largest part, roughly 70 to 90 per cent of the wet weight. Proteins come next at about 10 to 15 per cent, nucleic acids around 5 to 7 per cent, carbohydrates about 3 per cent, lipids about 2 per cent and inorganic ions about 1 per cent. Notice that carbohydrates and lipids look small on this list even though they matter enormously, because they are constantly being used up and remade.

The compounds a cell makes fall into two groups. Primary metabolites are the ones with clear, known roles in normal working: amino acids, sugars, nucleotides, fatty acids, glycerol and so on. They are found in every cell of every organism, whether the cell is in a mango leaf or in your kidney. Secondary metabolites are a different matter. When you analyse a plant, a fungus or a microbe, you find alkaloids such as morphine and codeine, coloured pigments such as carotenoids and anthocyanins, terpenoids, essential oils such as lemon grass oil, toxins such as abrin and ricin, lectins such as concanavalin A, drugs such as vinblastin and curcumin, and polymeric substances such as rubber and gums. Their role inside the producing organism is not always obvious, though many of them help in defence against grazers, in attracting pollinators or in fighting off other microbes. What is beyond doubt is their usefulness to us, since a large share of our medicines, dyes, spices and scents comes from this group.

An amino acid is best thought of as a substituted methane. Take a central carbon atom and hang four things on it: an amino group written as -NH2, an acidic carboxyl group written as -COOH, a hydrogen atom and a fourth group called the R group or side chain. Because the amino group and the carboxyl group are attached to the very same carbon, and that carbon is called the alpha carbon, these are known as alpha amino acids. Only the R group changes from one amino acid to the next, and just 20 different R groups are used to build proteins.

Three examples show how little the R group has to change. In glycine the R group is simply a hydrogen atom, so glycine is the smallest and simplest of them all. In alanine the R group is a methyl group, -CH3. In serine the R group is -CH2OH, and that single oxygen and hydrogen make serine able to form hydrogen bonds that alanine cannot.

The chemical nature of an amino acid depends entirely on this side chain. If the R group carries an extra carboxyl group the amino acid is acidic, as in glutamic acid. If it carries an extra amino group the amino acid is basic, as in lysine. If the side chain has neither, the amino acid is neutral, as in valine. Some side chains contain a benzene ring and these are the aromatic amino acids: phenylalanine, tyrosine and tryptophan.

Amino acids are also ionisable. Both the amino group and the carboxyl group can gain or lose a proton, and which form you see depends on the pH of the solution. At the pH found inside cells the carboxyl group has given up its proton and carries a negative charge, while the amino group has taken up a proton and carries a positive charge. The molecule therefore carries a positive and a negative charge at the same time while its overall charge is zero. A particle like this is called a zwitterion, and it explains why amino acids behave like salts, dissolving well in water and having high melting points. Our bodies can manufacture many amino acids but not all of them; the ones we cannot make have to come ready-made from what we eat.

Now to the second family. A nucleotide has exactly three parts: a nitrogenous base, a pentose sugar and a phosphate group. If you take only the first two, that is, a base joined to a sugar with no phosphate, you have a nucleoside. Add the phosphate to a nucleoside and you are back to a nucleotide. Keeping these two words apart is worth a few minutes of your time because they are confused constantly.

The nitrogenous bases come in two shapes. Purines have two fused rings, and there are two of them: adenine and guanine. Pyrimidines have a single ring, and there are three: cytosine, uracil and thymine. DNA uses adenine, guanine, cytosine and thymine. RNA uses uracil in place of thymine, keeping the other three. The sugar is ribose in RNA and deoxyribose in DNA, the difference being one oxygen atom that deoxyribose lacks at its second carbon.

The naming follows a pattern. The nucleosides are adenosine, guanosine, cytidine, uridine and thymidine. The matching nucleotides are adenylic acid, guanylic acid, cytidylic acid, uridylic acid and thymidylic acid. Nucleotides are not only bricks for nucleic acids; several of them work on their own. Adenosine triphosphate, written ATP, is the energy currency of the cell, and nucleotide-based molecules such as NAD, NADP and FAD carry hydrogen and electrons from one reaction to another.

Carbohydrates are the sugars and their polymers. The simplest are the monosaccharides, which cannot be broken into anything smaller that is still a sugar. Glucose has the formula C6H12O6, six carbon atoms with an aldehyde group, so it is called an aldohexose. Fructose has the same formula but carries a ketone group instead, making it a ketohexose. Two compounds with the same formula and different arrangements like this are isomers, and their chemistry is noticeably different even though a chemical test for carbon, hydrogen and oxygen would not tell them apart. Ribose is a monosaccharide with five carbons, and galactose is another six-carbon sugar.

Join two monosaccharides and you get a disaccharide. Sucrose, ordinary table sugar, is glucose joined to fructose. Lactose, the sugar of milk, is glucose joined to galactose. Maltose is glucose joined to glucose. In each case the two units are held by a glycosidic bond, a bridge made through an oxygen atom, and a molecule of water is released when the bond forms.

Lipids are a much looser family. They are grouped together not by a shared structure but by a shared behaviour: they do not dissolve in water, and they do dissolve in organic solvents such as ether or chloroform. Most of them are small enough that a single molecule is not a macromolecule at all.

The commonest building block of a lipid is a fatty acid. A fatty acid is a carboxyl group with a long hydrocarbon chain attached to it. Palmitic acid has 16 carbon atoms counting the one in the carboxyl group. Arachidonic acid has 20 carbon atoms, again counting the carboxyl carbon. If every carbon in the chain is joined to the next by a single bond, the fatty acid is saturated. If there is at least one carbon to carbon double bond in the chain, it is unsaturated. Each double bond puts a permanent kink in the chain, so unsaturated chains cannot pack tightly against one another.

Glycerol is trihydroxy propane, that is, a three-carbon molecule with a hydroxyl group on each carbon. Fatty acids can be joined to these hydroxyl groups by esterification, releasing water each time. Attaching one fatty acid gives a monoglyceride, two gives a diglyceride and three gives a triglyceride. Triglycerides are what we ordinarily call fats and oils. The only real difference between a fat and an oil is the melting point: oils melt at a lower temperature and so stay liquid even in winter, and this happens because their fatty acids are largely unsaturated and their kinked chains cannot pack closely.

Some lipids carry a phosphorus-containing group. In a phospholipid, two of glycerol's hydroxyl groups hold fatty acids while the third holds a phosphate group that is itself attached to another small molecule. Lecithin, found in cell membranes, is the standard example. The importance of a phospholipid is that one end of it is attracted to water while the long fatty tails are repelled by it. Put many such molecules in water and they line up automatically, tails inward and heads outward, forming the double layer that every biological membrane is built on. Not all lipids follow the fatty acid pattern; some, such as cholesterol, are built from fused carbon rings instead.

A protein is a polymer of amino acids, but not a polymer of one repeating unit. Since up to 20 different amino acids can appear in any order, a protein is a heteropolymer, and that variety is exactly what allows proteins to do so many different jobs. Cellulose, by contrast, is a homopolymer because it is made of glucose alone.

The amino acids are held together by the peptide bond. It forms when the carboxyl group of one amino acid reacts with the amino group of the next, releasing a molecule of water. Two amino acids joined this way make a dipeptide, three make a tripeptide, and a long chain of them is a polypeptide. Every polypeptide chain therefore has two different ends. At one end sits the amino acid whose amino group is still free, called the N terminal. At the other sits the amino acid whose carboxyl group is still free, the C terminal. By convention the sequence is always written starting from the N terminal.

The primary structure is simply this sequence: which amino acids are present and in what order, counting from the N terminal end. It sounds like a small thing, yet it decides everything that follows, because the chain folds up in the only way that its particular sequence of side chains permits.

The secondary structure appears when short stretches of the chain settle into a regular repeating shape. The commonest is the right-handed helix, which you can imagine as a spiral staircase: the chain coils round and round, and each turn is pinned to the next by hydrogen bonds between the oxygen of one backbone carbonyl group and the hydrogen of a backbone amide group further along the same chain. Another regular shape is the pleated sheet, in which stretches of the chain lie side by side like folded paper and are pinned together by hydrogen bonds running across from one stretch to its neighbour. A protein does not usually take one of these shapes from end to end; only certain portions of the thread are folded this way, and the rest is irregular.

The tertiary structure is the shape of the whole chain once it has folded back on itself, helices, sheets, irregular loops and all, into a compact three-dimensional lump. A rough picture is a ball of wool, though a real protein folds into one precise arrangement rather than a random tangle. Four kinds of force hold it: hydrogen bonds, ionic attractions between oppositely charged side chains, the huddling together of water-hating side chains in the interior away from the surrounding water, and strong covalent disulphide bridges formed between the sulphur atoms of two cysteine residues. Tertiary structure matters more than any other level for function, because folding brings side chains that were far apart in the sequence close together in space, and that is how pockets such as an enzyme's active site come into being.

The quaternary structure exists only in proteins that are built from more than one polypeptide chain. Each chain is then called a subunit, and the quaternary structure describes how the subunits are arranged relative to one another. Adult human haemoglobin is the standard example: it consists of four subunits, two of the alpha type and two of the beta type, packed together into one working molecule.

Because the higher levels of structure depend on weak bonds, heat or a strongly acidic or alkaline environment can break them. The chain then loses its shape and its activity, even though the peptide bonds of the primary structure may survive intact. This is denaturation, and you see it every time the clear part of an egg turns white and solid on a pan. Proteins do an enormous range of work: collagen is the most abundant protein in the animal world and forms the material between cells, RuBisCO is the most abundant protein in the whole biosphere, trypsin is an enzyme, insulin is a hormone, antibodies fight infectious agents, receptor proteins handle sensory reception such as smell and taste, and the transporter GLUT-4 lets glucose move into cells.

A polysaccharide is a long thread of monosaccharides joined one after another by glycosidic bonds. Like a polypeptide, such a chain has two different ends: the end that still carries a free sugar group able to act as a reducing agent is called the reducing end, and the other is the non-reducing end. Five of these polymers are worth knowing well, and remarkably three of them are made of nothing but glucose.

Cellulose is a homopolymer of glucose. Its chains are straight, with no complex coiling, so they lie side by side and are pinned together into tough fibres. That is why cellulose is the chief material of the plant cell wall, and why cotton fibre and paper are largely cellulose. Humans cannot digest it, because our digestive enzymes cannot attack the particular kind of glycosidic linkage that joins its glucose units, so for us it passes through as roughage.

Starch is the storage polysaccharide of plants and is also built from glucose, but its chains coil into helices. This coiling has a neat consequence you can see in the laboratory: iodine molecules slip into the hollow of the helix and the mixture turns blue-black. Cellulose, having no such helix, has nowhere to trap iodine and gives no blue colour. So the iodine test is really a test of shape, not of chemical formula.

Glycogen is the storage polysaccharide of animals, again a glucose polymer, but much more highly branched than starch. It is stored mainly in the liver and in muscle, and the many branch ends mean many places where glucose can be released quickly when it is needed. Inulin breaks the pattern: it is a polymer of fructose, not glucose. Chitin breaks it in another way, since it is made of a chemically modified sugar, the amino sugar N-acetylglucosamine, and it forms the hard exoskeleton of arthropods such as insects, prawns and crabs.

The nucleic acids are polynucleotides. To build one, nucleotides are joined through their phosphate groups: a phosphate links the third carbon of one sugar to the fifth carbon of the sugar of the next nucleotide, forming a phosphodiester bond. Repeat this and you get a backbone of sugar, phosphate, sugar, phosphate all the way along, with the nitrogenous bases projecting sideways from each sugar. Because every link runs between a third carbon and a fifth carbon, the chain has direction, and the two ends are named the five prime end, where the fifth carbon of the sugar is free, and the three prime end, where the third carbon is free.

The famous double helix of DNA, proposed by Watson and Crick, is its secondary structure. Two polynucleotide chains coil around a common axis to form a right-handed helix, and they run antiparallel, meaning one chain runs in the five prime to three prime direction while the other runs the opposite way. The chains are held together by hydrogen bonds between bases that stick inward from the two backbones. The pairing is strict: adenine pairs only with thymine through two hydrogen bonds, and guanine pairs only with cytosine through three hydrogen bonds. A purine always faces a pyrimidine, which keeps the width of the helix constant along its whole length. In the common form of DNA there are roughly ten base pairs in one complete turn of the helix, and one full turn measures about 3.4 nanometres, so consecutive base pairs are stacked about 0.34 nanometres apart.

The strict pairing rule has a consequence that is hard to overstate: if you know the sequence of one strand, you know the sequence of the other. The two strands are complementary, and that is what makes it possible for a cell to copy its DNA accurately. RNA is usually a single chain, but it often folds back on itself so that stretches of its own sequence pair up, producing hairpin-shaped regions and giving RNA molecules definite three-dimensional shapes of their own.

Almost every enzyme is a protein. The exception is worth remembering: certain RNA molecules also act as catalysts, and a nucleic acid working as an enzyme is called a ribozyme. Since enzymes are proteins, they have secondary and tertiary structure, and it is precisely that folding which makes them work. When the chain folds, it leaves a crevice or pocket on the surface of the molecule, and this pocket is the active site. The substrate, the molecule the enzyme acts on, fits into this pocket. A key point that follows straight away is that the active site is not simply a run of neighbouring amino acids in the sequence; it is built largely from side chains that lay far apart along the chain and were brought together only by folding. Destroy the folding and the active site disappears, which is why a denatured enzyme is dead as a catalyst.

The rate of a reaction means the amount of product formed in a given time. Rates respond strongly to temperature: as a rule of thumb, the rate of a chemical process doubles or falls to half when the temperature is changed by 10 degrees, a relationship known as the temperature coefficient or Q10.

How much difference does an enzyme make? Consider the reaction in which carbon dioxide combines with water to give carbonic acid. Left to itself in solution, this reaction produces roughly 200 molecules of carbonic acid in an hour. Add the enzyme carbonic anhydrase and about 600000 molecules are produced every second. That is somewhere near ten million times faster, and it is why our blood can pick up carbon dioxide in the tissues and dump it in the lungs quickly enough to keep pace with breathing. Inorganic catalysts are useful, but they come nowhere near this.

To understand where that speed comes from, think about what has to happen for reactant to become product. The reactant cannot simply slide into the product state. It first has to pass through an unstable in-between arrangement called the transition state, which has a higher energy than either the starting material or the finished product. The extra energy the reactant must gather in order to reach that peak is the activation energy. At ordinary temperatures only a very small fraction of molecules happen to have that much energy at any moment, so the uncatalysed reaction crawls.

An enzyme does not push the reactant over the peak, and it does not supply energy. What it does is provide a different path with a lower peak. By holding the substrate in a strained position, bringing reacting groups into exactly the right alignment and steadying the transition state with its own side chains, the enzyme lowers the activation energy. A far greater share of the substrate molecules now have enough energy to make it across, and the reaction races. Two things an enzyme cannot do are also worth stating clearly. It does not change the overall energy difference between reactants and products, and it does not shift the point of equilibrium; it only makes both the forward and the reverse reaction reach that point sooner.

The actual sequence of events runs like this. First the substrate binds to the active site, forming an enzyme substrate complex, usually written as ES. This binding is not passive: the active site adjusts its shape slightly so that it grips the substrate more closely, an adjustment known as induced fit. Next, the enzyme's side chains act on the substrate, breaking and making bonds and producing an enzyme product complex. Finally the product is released, leaving the enzyme unchanged and immediately ready to take another substrate molecule. Because the enzyme comes out exactly as it went in, a very small amount of enzyme can process an enormous quantity of substrate.

Enzymes are also strikingly specific. A given enzyme usually acts only on one substrate or on a small family of closely related ones, because both the shape and the chemical character of the active site must match the substrate. This specificity is what allows thousands of different reactions to go on side by side in the same cell without interfering with one another.

Temperature. Enzyme activity climbs as the temperature rises, because molecules move faster and substrate meets active site more often. It reaches a maximum at the optimum temperature and then falls off sharply. The fall is not a slowing down but a breakdown: above the optimum the weak bonds holding the tertiary structure begin to break, the enzyme loses its shape, the active site is destroyed and the enzyme is denatured. This damage is usually permanent. Low temperature behaves quite differently. It does not destroy the enzyme; it only makes molecules sluggish, so activity is low but returns when warmth returns, which is exactly why food keeps in a refrigerator. Most enzymes in the human body work best near normal body temperature, while enzymes of bacteria living in hot springs stay perfectly active at temperatures that would wreck ours.

pH. Every enzyme has an optimum pH at which it is most active, and activity falls on either side of it. The reason is that the side chains at the active site are ionisable groups; change the acidity and they gain or lose protons, their charges change, and the substrate no longer binds properly. Extreme pH denatures the enzyme outright. The optimum matches the place the enzyme works: pepsin, which acts in the stomach, works best around pH 2, while trypsin, which acts in the alkaline intestinal fluid, works best around pH 8. Salivary amylase prefers a value close to neutral, near pH 6.8.

Substrate concentration. Start with a fixed amount of enzyme and add substrate gradually. At first the velocity of the reaction rises almost in step with the amount of substrate, because plenty of active sites are lying free. As more sites become occupied, each further addition helps less, so the rise flattens. Eventually every active site is busy the moment it becomes free, the enzyme is saturated, and the reaction reaches its maximum velocity, usually written as Vmax. Beyond this point adding more substrate makes no difference at all, and the only way to go faster is to add more enzyme.

Inhibitors. A chemical that binds an enzyme and shuts down its activity is an inhibitor. In competitive inhibition the inhibitor is so similar in structure to the real substrate that it fits into the active site and sits there, blocking it. The classic example is malonate, which closely resembles succinate and so inhibits the enzyme succinic dehydrogenase. Because inhibitor and substrate are fighting over the same pocket, the outcome depends on numbers: add plenty of substrate and it wins the competition, so the enzyme can still reach its maximum velocity, it just needs more substrate to get there. Several medicines against bacterial disease work in exactly this way.

In non-competitive inhibition the inhibitor does not resemble the substrate and does not go near the active site. It binds somewhere else on the enzyme and changes the enzyme's shape, and with it the shape of the active site. The substrate may still be able to bind, but the reaction no longer proceeds properly. Since the two molecules are not competing for one pocket, flooding the system with substrate does not rescue the enzyme, and the maximum velocity itself is reduced.

Classification. Enzymes are sorted into six classes, numbered one to six, according to the kind of reaction they carry out. Oxidoreductases catalyse oxidation and reduction between two substrates. Transferases move a group, other than hydrogen, from one substrate to another. Hydrolases break bonds using water, including ester, ether, peptide and glycosidic bonds. Lyases remove groups without using water, leaving behind double bonds. Isomerases carry out isomerisation, whether optical, geometric or positional. Ligases join two molecules together, forming bonds such as carbon to oxygen, carbon to sulphur or carbon to nitrogen. Each class is divided further into subclasses, and every enzyme ends up with a number of four digits.

Cofactors. Many enzymes cannot work as bare protein. They need a non-protein partner called a cofactor, and only when the two are together is the enzyme active. The protein part alone is the apoenzyme; apoenzyme plus cofactor together make the holoenzyme. There are three kinds of cofactor. Prosthetic groups are organic and are bound tightly and permanently to the apoenzyme; haem is the prosthetic group of peroxidase and catalase, the enzymes that break hydrogen peroxide down into water and oxygen. Coenzymes are organic too, but their association with the apoenzyme is only brief, lasting through the moment of catalysis; many of them are built around vitamins, and NAD and NADP both contain the vitamin niacin. Metal ions form coordination bonds with side chains at the active site and with the substrate at the same time, holding the two in place; zinc is the metal cofactor of the protein-digesting enzyme carboxypeptidase. Remove the cofactor from any of these and the protein that is left behind, however intact, cannot catalyse anything.