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CHEMISTS could argue that they have done more than anyone this century to meet the world’s demand for a better food supply, more protection against illness, and greater comfort. But we know that the cost to the environment of such improvements is great. Now chemists are searching for new, environmentally friendly ways of making the molecules that improve our quality of life. Some of us think that enzymes might be the answer.

Most useful molecules that chemists make – drugs and pesticides, for example – are organic ones, made up largely of carbon and hydrogen atoms. Chemists build them up in a series of steps, starting from simple, readily available materials. But without the help of a catalyst, one or more of these steps would go along painfully slowly, often so slowly as to be useless. In the past, chemists used compounds including heavy metals such as tin and mercury as catalysts. Now they realise that such metals sometimes have a devastating effect on soils, rivers and seas, and they are looking for less harmful catalysts to replace them.

This is where enzymes come in. Enzymes are nature’s catalysts: plants, animals and microorganisms all rely on them. Virtually all biochemical reactions depend on an enzyme. They speed up the splitting apart or the stitching together of molecules, and catalyse cycles of reactions that regulate the consumption and production of energy. Without them, our bodies would grind to a halt. Biochemists have already identified and classified about two-thirds of the 7000 or so enzymes on Earth. Enzymes catalyse many kinds of changes in organic molecules, which is why chemists would like to exploit them for non-biological reactions.

You can often tell what enzymes do from their names – most of them are named after the molecule they act upon, and the ending ‘-ase’ shows that they catalyse its break-up. Proteases, for example, split up proteins; dehydrogenases remove hydrogen; and esterases catalyse the breaking up of esters into their component carboxylic acid and alcohol. Under certain conditions, however, enzymes can also catalyse the reverse reaction. An enzyme that biologists already exploit in this way is DNA polymerase, which catalyses the building up from smaller units of deoxyribonucleic acid (DNA), the molecule that carries the genetic code. Molecular biologists use DNA polymerase to help them to copy DNA segments millions of times.

Enzymes are true catalysts. They work by lowering the amount of energy needed for a reaction, and emerge unchanged at the end of it. How they achieve this remarkable feat has a lot to do with their structure. All enzymes are large protein molecules that are arranged in chains of amino acids folded in a complex way. The most important part of the molecule is a particular place on the surface, called the active site. When an enzyme meets a molecule whose shape fits its active site, the enzyme can hold it in the most efficient way for the molecule to react. Some researchers think the enzyme does this by subtly altering the shape of the particular molecule – its substrate – that it acts upon. Many enzymes have flexible loops at the active site, whose position changes depending on the shape of the substrate. Other researchers think the substrate fits into the active site of the enzyme exactly, rather like a key into a lock.

The eventual outcome is the same: once the substrate molecule has reacted, it has a different shape. Now it no longer fits the active site, and the enzyme releases it as a product (see Figure 1). In the 1960s, many chemists, including the Nobel laureate Vladimir Prelog, began to realise that such a mechanism could be extremely useful. An enzyme will bind only to a particular type of molecule – the one that fits into its active site. They might be able to use enzymes as precise tools for making molecules. When they looked more closely, the chemists found that enzymes have other qualities that give them a distinct edge over artificial catalysts.FIG-mg17133501.jpg

Most enzymes work under very mild conditions – temperatures of around 35 Degree C, atmospheric pressure and neutral acidity. Not only do enzyme-based reactions need less complex equipment, they also save energy. For example, to make methanol, a useful starting material for many reactions, chemists have to combine molecules of methane with molecules of oxygen. To do this they have to use a two-step process. The second step involves temperatures of 450 to 500 Degree C, pressures of around 300 atmospheres and a catalyst such as chromium/zinc oxide. Even then, they manage to convert only about 15 per cent of the gases that reach the second step into methanol. Nature has no such problems – an enzyme called mono-oxygenase will bring about the reaction easily. No wonder chemists began to look for ways of using enzymes as alternative catalysts for industrial processes.

Sometimes, enzymes are indispensable precisely because they will work under mild conditions, as researchers into penicillin-G found. Penicillin-G can kill bacteria because it has a square ‘ring’ of atoms called a beta-lactam ring, which has other atoms – including nitrogen, sulphur and oxygen – attached (see Figure 2). This ring interferes with the mechanism by which bacteria make cell walls, so that they are weaker and burst more easily. As long ago as the 1950s, scientists working for the British company Beechams and other pharmaceuticals firms realised that they could make antibiotics that are even more potent, if only they could replace one of the groups attached to the ring with a more complex one. The problem was how to do it without disturbing the sensitive beta-lactam ring. Enzymes extracted from the microorganism Escherichia coli came to the rescue. They catalysed the removal of the unwanted group and the scientists ‘sewed’ the new one on in its place, producing the anti-bacterial compound ampicillin that is familiar to anyone who has visited their doctor with a chest or bladder infection. Jack Baldwin and a team at Oxford University have taken the science even further. Using enzymes, Baldwin’s group can now make natural and new penicillins from simple tripeptides (a tripeptide is a string of three amino acids joined together). These mega-penicillins will help to control bacterial infections such as blood poisoning that are otherwise difficult to treat.FIG-mg17133502.jpg

For the fine (high-purity) chemicals and the pharmaceuticals industries, enzymes have one quality above all that makes them ideal – stereoselectivity. This is the ability to pick out from a mixture those molecules that are either ‘left-handed’ or ‘right-handed’ . It is the basis of a method for making artificial hormones. The family of hormones called prostaglandins act as molecular ‘traffic controllers’ in many reactions in the human body. One such hormone is prostaglandin-I2; people who have too little of it in their bodies suffer from heart disease. But doctors cannot prescribe a dose of this hormone because it is unstable and breaks down before it has chance to work. In the future, they may be able to prescribe an artificial hormone that is stable and acts in exactly the same way in the body as a natural prostaglandin. To be safe, this artificial hormone must mimic the natural one so closely that not even molecules of its mirror image must contaminate it.

To make such pure, artificial prostaglandin-I2, chemists start from a reaction that gives them an equal mixture of an ester (an organic salt formed from an acid and an alcohol) and its mirror image. They add an enzyme extracted from the pancreas of pigs, called porcine pancreatic lipase, and water. The enzyme separates the ‘left-handed’ ester molecules from the ‘right-handed’ ones – almost impossible to do with chemicals – by catalysing the transformation of ‘right-handed’ molecules of ester into molecules of alcohol. The specificity of the enzyme guarantees that any remaining ester molecules are purely ‘left-handed’. Chemists then convert either of these pure products, ester or alcohol, in a further series of steps into artificial prostaglandins (see Figure 3).FIG-mg17133503.jpg

Alexander Klibanov and his colleagues at the Massachusetts Institute of Technology did a lot to popularise the idea that chemists could make pure, chiral compounds like the left-handed ester by persuading the enzyme to work backwards. This is not as improbable as it sounds. The ability of enzymes to promote reactions going in either direction reflects their role as catalysts for chemical reactions in nature. Enzymes have been evolving for so long that it is not surprising to find that nature uses them in the most economical way. What is remarkable is that enzymes behave in the same way when they act on artificial molecules – some esters are never seen outside a chemistry laboratory.

ÐÓ°ÉÔ­´´s have known since the turn of the century that pancreatic lipase will catalyse not only the hydrolysis of an ester to an alcohol, but also the reverse reaction: the combination of an alcohol and a carboxylic acid to give molecules of ester, if there is little water. In 1984, Klibanov reacted a mixture of left- and right-handed carboxylic acid molecules with an alcohol, in a trace of water, adding the enzyme yeast lipase. The enzyme catalysed the combination of the right-handed acid molecules with the alcohol to give an ester, but the left-handed ones were unchanged. The chemists could convert both the ester and the pure, left-handed acid molecules into more exotic compounds.

Food chemists often rely on enzymes to catalyse the key step in a series of reactions. One such step is the formation of a peptide bond, which is catalysed by the group of enzymes called peptidases. Peptide bonds are carbon-nitrogen-carbon bonds that link amino acid units together to make larger dipeptides, oligopeptides and finally proteins. Aspartame, a dipeptide that food chemists use widely because of its sweet taste, and better known to most people as Nutrasweet, could not be made without the help of the peptidase thermolysin. In this case, commercial success owes a lot to luck – its intense sweetness came to light only by accident. Although the British company ICI made the peptide first, it was a chemist working for the American pharmaceuticals company Searle who discovered its most interesting property. This chemist had the bad habit of licking his fingers before picking up filter papers. One day he licked off a tiny amount of Aspartame – serendipitous, but not recommended as the best way of testing new chemicals.

All these enzyme-catalysed reactions, or biotransformations, are easy to do. You do not need any special equipment and you can buy pure enzymes in a bottle. Some reactions are more tricky because they rely on enzymes that need ad ditives, extra ingredients that take part in the catalysis. These may be metal atoms such as iron or small, non-protein molecules called cofactors, or both. Many of the vitamins and other substances that we need in trace amounts as part of our diet end up in cofactors. In fact, it may be the cofactor that does most of the work. Catalase, the enzyme that catalyses the decomposition of hydrogen peroxide, has iron atoms as cofactors, and simple iron compounds on their own have a similar effect.

For these trickier reactions, chemists prefer to use complete microorganisms that have a ready-made enzyme system with cofactors included . The microorganism Thermoanaerobium brockii contains an alcohol dehydrogenase enzyme that, as its name suggests, catalyses the removal of hydrogen from alcohols. Under the right conditions, this enzyme also catalyses the reverse reaction: the production of an alcohol by adding hydrogen atoms to a ketone (ketones are molecules with a carbon-oxygen double bond). To do this it needs a molecule called dihydronicotinamide adenine dinucleotide phosphate, or NADPH. NADPH is one of the most common cofactors. Its main job is to supply hydrogen atoms and it becomes nicotinamide adenine dinucleotide phosphate (NADP) in the process.

Baker’s yeast and other microorganisms such as Thermoanaerobium brockii are particularly useful catalysts for reactions that involve adding hydrogen atoms. Yeast cells contain both the right enzymes and one of the nicotinamide adenine dinucleotide family of cofactors. Organic chemists use yeast to make large quantities of pure 3-hydroxybutanoate, one of a range of similar molecules that are popular building blocks because they are asymmetric, or chiral.

Chiral compounds are important starting materials in processes that chemists use to make natural molecules. Such molecules are often extremely complex and act in a specific way on biological systems. One example is coriolin, a compound made up of three connected carbon rings that is used to treat cancer. In the mid-1980s, Dee Brooks at Abbott Laboratories in Chicago made coriolin in a series of steps, starting from a five-carbon ring with two ketone groups – oxygen atoms attached by double bonds to carbon atoms. The two ketone groups were arranged symmetrically on opposite sides of the five-carbon ring. Simple baker’s yeast provided the right enzymes to catalyse the addition of a hydrogen atom to one of the ketone groups, transforming it into a hydroxyl group (see Figure 4).FIG-mg17133504.jpg

Chemists have at their fingertips thousands of substances whose job is to bring about just this transformation, but none of them will select without fail only one of the two oxygen atoms on the five-carbon ring. Having obtained their ring with only one double-bonded oxygen, chemists could go on to make coriolin. Once again, chemists use enzymes to target reactions at certain atoms to get the products they want.

Research into steroids has benefited most from the help of enzymes. Steroids are large, complex organic molecules based on four linked carbon rings. They include many hormones, the D vitamins and bile acids that contribute to the digestion of fats in the body. With the help of the microorganism Rhizopus arrhizus, researchers at the Upjohn Laboratories in Kalamazoo, Michigan, converted the steroid progesterone into a powerful drug to combat inflammation. They could not have done this without enzymes, because the conversion involves adding an oxygen atom at only one of several possible places. Chemicals that add oxygen atoms would never be this discriminating. But before chemists can apply the technique to the manufacture of other fine chemicals, they need to do more research. Even enzymes will add oxygen atoms to more than one site in molecules that are more complex than steroids.

When Steve Ley and his colleagues at Imperial College in London worked with Steve Taylor and others at ICI’s Teesside Laboratories, they took advantage of the superiority of microorganisms and their enzymes over chemicals to make copies of the molecules that act as chemical messengers in the central nervous system. First, the ICI team picked out its ‘bugs’ from soil by dropping the proposed starting material – benzene – on it. Those microorganisms that use benzene as an energy source multiplied rapidly and the scientists collected them and grew them in the laboratory. Then they fed these ‘tame’ bugs on benzene and harvested the product. The enzymes associated with the bugs added two hydroxyl (OH) groups to transform benzene into cyclohexa-3,5-diene-1,2-diol. Ley and his team now had a convenient and abundant source of the starting material they needed to make the messenger molecules.

George Whitesides of Harvard University in the US used aldolase from rabbit muscle to catalyse the formation of new carbon-carbon bonds. Because it brings about the breakdown of glucose, this enzyme catalyses the reaction that provides a muscle with the energy to move. Whitesides was particularly interested in aldolase because it makes sure that the bond that forms between the carbon atoms forms in such a way that they become chiral centres, which means that they give the molecule ‘handedness’. Such reactions lie at the heart of organic chemistry.

Industrialists are already using reactions like these as a better way of producing natural substances. For example, they use enzymes called isomerases to help them convert fructose into its more useful form, glucose. With more sophisticated techniques, they should be able to manufacture fine chemicals in a similar way, without the disadvantages of cost and damage to the environment. Biotechnologists and chemists are already well on the way to making enzymes on demand. Most of the recent research has concentrated on four areas. Some scientists are looking for new microorganisms, some for ways of using existing enzymes to make important molecules for the pharmaceuticals and agrochemicals industries. Others are studying how enzymes behave in unusual circumstances, such as when they are used in solvents other than water. And there is some interesting research going on in the area of mixed ‘pots’ of enzymes which can work together to produce a cascade of reactions in which the end product of one reaction becomes the starting material for the next. Each type of enzyme catalyses its particular step of the cascade without interfering with any other. If all this research comes to fruition, enzymes could pave the way to a greener chemistry, and improve our quality of life.

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1: CARBON COMPOUNDS POLISH UP THEIR IMAGE

CARBON atoms form up to four single bonds with other atoms or groups of atoms. When all four bonds are filled, the atoms arrange themselves in space so that they sit at the corners of a tetrahedron. This simple fact has far-reaching consequences. If the four atoms (or groups) are all different, they can be arranged in two different ways, giving molecules that are mirror images of each other. These molecules cannot be superimposed – they are ‘right-‘ or ‘left-handed’ and chemists call them chiral (from the Greek word kheir, for hand).

The resemblance of the right- and left-handed forms is deceptive: although physically and even chemically similar, they may have dramatically different effects on biological systems. Many drugs are complex organic molecules that have more than one chiral centre.

Their shape is crucial. When chemists copy natural molecules to use as drugs they have to be sure that their copies are not contaminated with any mirror images. The contamination of the safe compound thalidomide with its mirror image, a teratogen, had tragic consequences.

Sometimes the only way of separating a pair of chiral molecules is by using enzymes. Designers of drugs are finding enzymes very useful because they will pick out molecules of a particular chirality from a mixture of chiral molecules – called a racemic mixture or racemate – and leave their mirror images untouched.

Chirality is important for more than ensuring the purity and safety of drugs, however. It is the basis for many of the elegant methods that organic chemists use to build up complex molecules with centres of a particular chirality. The properties of a molecule often depend on its particular combination of chiral centres.

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2: ENZYMES AND THE CHEMICAL CONSUMER

CHEMISTS enlist the help of enzymes in two ways. The first is to take a microorganism, complete with all its enzymes, and feed it with the right starting material – bakers and brewers have been using this method for centuries. Baker’s yeast (Saccharomyces cerevisiae) contains an assortment of enzymes, including dehydrogenases and esterases, that catalyse the fermentation of sugar. There are advantages with this ‘whole cell’ method. Using an intact microorganism to do the work means that all the right ingredients are there – enzyme, cofactors, and metal ions, all conveniently packaged. But the results are not always reliable. For one thing, microorganisms are easily poisoned by solutions that become too concentrated. And microorganisms have a habit of pleasing themselves what they make out of the food the chemists give them, so the product that the chemists want may be only one of a complicated mixture. This brings the extra problem of separating the components of the mixture.

The second method is to remove the enzyme from its natural home inside a bacterium, a fungus or a mammalian cell, and apply it in concentrated form to the reaction. Some of the best sources of enzymes are the liver and the pancreas of animals, because these organs do the job of breaking down chemicals in the animal’s body. Once they have isolated an enzyme, biochemists support it on a mesh of larger, stable molecules to stop it breaking down. By filtering off the product molecules at the end of the reaction they can recover the enzyme, still lodged on the mesh, quite easily. Despite the slightly greater cost and the extra job of topping up the cocktail of cofactors and metal ions, this method often guarantees the chemists a product that is pure. ÐÓ°ÉÔ­´´s are fairly evenly divided as to which method is the best.

Stanley Roberts is a professor and Nick Turner is a lecturer in organic chemistry at the University of Exeter. Both do research in those areas of biotransformations that are concerned with the preparation of new pharmaceuticals, agricultural chemicals and fragrances.