by Isaac Asimov
In 1902, the German chemist George Lunge suggested that this sort of thing was the explanation of catalysis in general. In 1916, Irving Langmuir went a step farther and advanced an explanation for the catalytic action of substances, such as platinum, that are so nonreactive that they cannot be expected to engage in ordinary chemical reactions. Langmuir suggested that excess valence bonds at the surface of platinum metal would seize hydrogen and oxygen molecules. While held imprisoned in close proximity on the platinum surface, the hydrogen and oxygen molecules would be much more likely to combine to form water molecules than in their ordinary free condition as gaseous molecules. Once a water molecule was formed, it would be displaced from the platinum surface by hydrogen and oxygen molecules. Thus, the process of seizure of hydrogen and oxygen, their combination into water, release of the water, seizure of more hydrogen and oxygen, and formation of more water could continue indefinitely.
This process is called surface catalysis. Naturally, the more finely divided the metal, the more surface a given mass will provide, and the more effectively catalysis can proceed. Of course, if any extraneous substance attaches itself firmly to the surface bonds of the platinum, it will poison the catalyst.
All surface catalysts are more or less selective, or specific. Some easily absorb hydrogen molecules and will catalyze reactions involving hydrogen; others easily absorb water molecules and catalyze condensations or hydrolyses; and so on.
The ability of surfaces to add on layers of molecules (adsorption) is widespread and can be put to uses other than catalysis. Silicon dioxide prepared in spongy form (silica gel) will adsorb large quantities of water. Packed in with electronic equipment, whose performance would suffer under conditions of high humidity, it acts as a dessicant, keeping humidity low.
Again, finely divided charcoal (activated carbon) will adsorb organic molecules readily—the larger the organic molecule, the more readily. Activated carbon can be used to decolorize solutions, for it would adsorb the colored impurities (usually of high molecular weight), leaving behind the desired substance (usually colorless and of comparatively low molecular weight).
Activated carbon is also used in gas masks, a use foreshadowed by an English physician, John Stenhouse, who first prepared a charcoal air filter in 1853. The oxygen and nitrogen of air pass through such a mass unaffected, but the relatively large molecules of poison gases are adsorbed.
FERMENTATION
The organic world, too, has its catalysts. Indeed, some of them have been known for thousands of years, though not by that name. They are as old as the making of bread and the brewing of wine.
Bread dough, left to itself and kept from contamination by outside influences, will not rise. Add a lump of leaven (from a Latin word meaning “rise”), and bubbles begin to appear, lifting and lightening the dough. The common English word for leaven is yeast; possibly descended from a Sanskrit word meaning “to boil.”
Yeast also hastens the conversion of fruit juices and grain to alcohol. Here again, the conversion involves the formation of bubbles, so the process is called fermentation, from a Latin word meaning “boil.” The yeast preparation is often referred to as ferment.
It was not until the seventeenth century that the nature of leaven was discovered. In 1680, for the first time, a Dutch investigator, Anton van Leeuwenhoek, saw yeast cells. For the purpose, he made use of an instrument that was to revolutionize biology—the microscope. It was based on the bending and focusing of light by lenses. Instruments using combinations of lenses (compound microscopes) were devised as early as 1590 by a Dutch spectacle maker, Zacharias Janssen. The early microscopes were useful in principle, but the lenses were so imperfectly ground that the objects magnified were almost useless, fuzzy blobs. Van Leeuwenhoek ground tiny but perfect lenses that magnified quite sharply up to 200 times. He used single lenses (simple microscope).
With time, the practice of using good lenses in combinations (for a compound microscope is, potentially at least, much stronger than a simple one) spread, and the world of the very little opened up further. A century and a half after Leeuwenhoek, a French physicist, Charles Cagniard de la Tour, using a good compound microscope, studied the tiny bits of yeast intently enough to catch them in the process of reproducing themselves. The little blobs were alive. Then, in the 1850s, yeast became a dramatic subject of study.
France’s wine industry was in trouble. Aging wine was going sour and becoming undrinkable, and millions of francs were being lost. The problem was placed before the young dean of the Faculty of Sciences at the University of Lille, in the heart of the vineyard area. The young dean was Louis Pasteur, who had already made his mark by being the first to separate optical isomers in the laboratory.
Pasteur studied the yeast cells in the wine under the microscope. It was obvious to him that the cells were of varying types. All the wine contained yeast that brought about fermentation, but those wines that went sour contained another type of yeast in addition. It seemed to Pasteur that the souring action did not get under way until the fermentation was completed. Since there was no need for yeast after the necessary fermentation, why not get rid of all the yeast at that point and avoid letting the wrong kind make trouble?
He therefore suggested to a horrified wine industry that the wine be heated gently after fermentation, in order to kill all the yeast in it. Aging, he predicted, would then proceed without souring. The industry reluctantly tried his outrageous proposal and found, to its delight, that souring ceased, while the flavor of the wine was not in the least damaged by the heating. The wine industry was saved. Furthermore, the process of gentle heating (pasteurization) was later applied to milk, to kill any disease germs present.
Other organisms besides yeast hasten breakdown processes. In fact, a process analogous to fermentation takes place in the intestinal tract. The first man to study digestion scientifically was the French physicist Rene Antoine Ferchault de Réaumur. He used a hawk as his experimental subject and, in 1752, made it swallow small metal tubes containing meat; the tubes protected the meat from any mechanical grinding action, but they had openings, covered by gratings, so that chemical processes in the stomach could act on the meat. Réaumur found that when the hawk regurgitated these tubes, the meat was partly dissolved, and a yellowish fluid was present in the tubes.
In 1777, the Scottish physician Edward Stevens isolated fluid from the stomach (gastric juice) and showed that the dissolving process could be made to take place outside the body, thus divorcing it from the direct influence of life.
Clearly, the stomach juices contained something that hastens the breakdown of meat. In 1834, the German naturalist Theodor Schwarm added mercuric chloride to the stomach juice and precipitated a white powder. After freeing the powder of the mercury compound, and dissolving what was left, he found he had a very concentrated digestive juice. He called the powder he had discovered pepsin, from the Greek word meaning “digest.”
Meanwhile, two French chemists, Anselme Payen and Jean François Persoz, had found in malt extract a substance that could bring about the conversion of starch to sugar more rapidly than could acid. They called this diastase, from a Greek word meaning “to separate,” because they had separated it from malt.
For a long time, chemists made a sharp distinction between living ferments such as yeast cells and nonliving, or unorganized, ferments such as pepsin. In 1878, the German physiologist Wilhelm Kühne suggested that the latter be called enzymes, from Greek words meaning “in yeast,” because their activity was similar to that brought about by the catalyzing substances in yeast. Kühne did not realize how important, indeed universal, that term “enzyme” was to become.
In 1897, the German chemist Eduard Buchner ground yeast cells with sand to break up all the cells and succeeded in extracting a juice that he found could perform the same fermentative tasks that the original yeast cells could. Suddenly the distinction between the ferments inside and outside of cells vanished. It was one more breakdown of the vitalists’ semim
ystical separation of life from nonlife. The term “enzyme” was now applied to all ferments.
For this discovery Buchner received the Nobel Prize in chemistry in 1907.
PROTEIN CATALYSTS
Now it was possible to define an enzyme simply as an organic catalyst. Chemists began to try to isolate enzymes and find out what sort of substances they were. The trouble was that the amount of enzyme in cells and natural juices is very small, and the extracts obtained were invariably mixtures in which it was hard to tell what was an enzyme and what was not.
Many biochemists suspected that enzymes were proteins, because enzyme properties could easily be destroyed, as proteins could be denatured, by gentle heating. But, in the 1920s, the German biochemist Richard Willstätter reported that certain purified enzyme solutions, from which he believed he had eliminated all protein, showed marked catalytic effects. He concluded that enzymes were not proteins but relatively simple chemicals, which might, indeed, utilize a protein as a carrier molecule. Most biochemists went along with Willstätter, who was a Nobel Prize winner and had great prestige.
However, the Cornell University biochemist James Batcheller Sumner produced strong evidence against this theory almost as soon as it was advanced.
From jackbeans (the white seeds of a tropical American plant), Sumner isolated crystals that, in solution, showed the properties of an enzyme called urease, which catalyzes the breakdown of urea to carbon dioxide and ammonia. Sumner’s crystals showed definite protein properties, and he could find no way to separate the protein from the enzyme activity. Anything that denatured the protein also destroyed the enzyme. All this seemed to show that what he had was an enzyme in pure and crystalline form, and that enzyme was a protein.
Willstätter’s greater fame for a time minimized Sumner’s discovery. But, in 1930, the chemist John Howard Northrop and his co-workers at the Rockefeller Institute clinched Sumner’s case. They crystallized a number of enzymes, including pepsin, and found all to be proteins. Northrop, furthermore, showed that these crystals are pure proteins and retain their catalytic activity even when dissolved and diluted to the point where the ordinary chemical tests, such as those used by Willstätter, could no longer detect the presence of protein.
Enzymes were thus established to be protein catalysts. By now, some 2,000 different enzymes have been identified, and over 200 enzymes have been crystallized; all without exception are proteins.
For their work, Sumner and Northrop shared in the Nobel Prize in chemistry in 1946.
ENZYME ACTION
Enzymes are remarkable as catalysts in two respects—efficiency and specificity. There is an enzyme known as catalase, for instance, that catalyzes the breakdown of hydrogen peroxide to water and oxygen. Now the breakdown of hydrogen peroxide in solution can also be catalyzed by iron filings or manganese dioxide. However, weight for weight, catalase speeds up the rate of breakdown far more than any inorganic catalyst can. Each molecule of catalase can bring about the breakdown of 44,000 molecules of hydrogen peroxide per second at 0° C. The result is that an enzyme need be present only in small concentration to perform its function.
For this same reason, to put an end to life, it takes but small quantities of substances (poisons) capable of interfering with the workings of a key enzyme. Heavy metals, when administered in such forms as mercuric chloride or barium nitrate, react with thiol groups, which are essential to the working of many enzymes. The action of those enzymes stops, and the organism is poisoned. Compounds such as potassium cyanide or hydrogen cyanide place their cyanide group (–CN) in combination with the iron atom of other key enzymes and bring death quickly and, it is to be hoped, painlessly, for hydrogen cyanide is the gas used for execution in the gas chambers of some of our Western states.
Carbon monoxide is an exception among the common poisons. It does not act on enzymes primarily but ties up the hemoglobin molecule (a protein but not an enzyme), which ordinarily carries oxygen from lungs to cells but cannot do so with carbon monoxide hanging on to it. Animals that do not use hemoglobin are not harmed by carbon monoxide.
Enzymes, with catalase a good example, are highly specific: catalase breaks down hydrogen peroxide and nothing else; whereas inorganic catalysts, such as iron filings and manganese dioxide, may break down hydrogen peroxide but will also catalyze numerous other reactions.
What accounts for the remarkable specificity of enzymes? Lunge’s and Langmuir’s theories about the behavior of a catalyst as a middleman suggested an answer. Suppose we consider that an enzyme forms a temporary combination with the substrate—the substance whose reaction it catalyzes. The form, or configuration, of the particular enzyme may therefore play a highly important role. Plainly, each enzyme must present a very complicated surface, for it has a number of different side chains sticking out of the peptide backbone. Some of these side chains have a negative charge; some, positive; some, no charge. Some are bulky; some, small. One can imagine that each enzyme may have a surface that just fits a particular substrate. In other words, it fits the substrate as a key fits a lock. Therefore, it will combine readily with that substance, but only clumsily or not at all with others. Hence, the high specificity of enzymes: each has a surface made to order, so to speak, for combining with a particular compound. That being the case, no wonder that proteins are built of so many different units and are constructed by living tissue in such great variety.
This theory of enzyme action was first suggested by the work of an English physiologist, William Maddock Bayliss, working with a digestive enzyme named trypsin. In 1913, the theory was used by the German chemist, Leonor Michaelis, and his assistant, Maud Lenora Menten, to work out the Michaelis-Menten equation which described the manner in which enzymes carry out their functions, and robbed these catalysts of much of their mystery.
This lock-and-key view of enzyme action was borne out also by the discovery that the presence of a substance similar in structure to a given substrate will slow down or inhibit the substrate’s enzyme-catalyzed reaction. The best known case involves an enzyme called succinic acid dehydrogenase, which catalyzes the removal of two hydrogen atoms from succinic acid. That reaction will not proceed in the presence of a substance called malonic acid, which is very similar to succinic acid. The structures of succinic acid and malonic acid are:
The only difference between these two molecules is that succinic acid has one more CH2 group at the left. Presumably the malonic acid, because of its structural similarity to succinic acid, can attach itself to the surface of the enzyme. Once it has pre-empted the spot on the surface to which the succinic acid would attach itself, it remains jammed there, so to speak, and the enzyme is out of action. The malonic acid “poisons” the enzyme, so far as its normal function is concerned. This sort of action is called competitive inhibition.
The most positive evidence in favor of the enzyme-substrate-complex theory has come from spectrographic analysis. Presumably, if an enzyme combines with its substrate, there should be a change in the absorption spectrum: the combination’s absorption of light should be different from that of the enzyme or the substrate alone. In 1936, the British biochemists David Keilin and Thaddeus Mann detected a change of color in a solution of the enzyme peroxidase after its substrate, hydrogen peroxide, was added. The American biophysicist Britton Chance made a spectral analysis and found that there were two progressive changes in the absorption pattern, one following the other. He attributed the first change in pattern to the formation of the enzyme-substrate complex at a certain rate, and the second to the decline of this combination as the reaction was completed. In 1964, the Japanese biochemist Kunio Yagi announced the isolation of an enzyme-substrate complex, made up of a loose union of the enzyme n-amino acid oxidase and its substrate alanine.
Now the question arises: Is the entire enzyme molecule necessary for catalysis, or would some part of it be sufficient? This is an important question from a practical as well as a theoretical standpoint. Enzymes are in wide use today; they have been put
to work in the manufacture of drugs, citric acid, and many other chemicals. If the entire enzyme molecule is not essential and some small fragment of it would do the job, perhaps this active portion could be synthesized, so that the processes would not have to depend on the use of living cells, such as yeasts, molds, and bacteria.
Some promising advances toward this goal have been made. For instance,
Northrop found that when a few acetyl groups (CH3CO) were added to the side chains of the amino acid tyrosine in the pepsin molecule, the enzyme lost some of its activity. There was no loss, however, when acetyl groups were added to the lysine side chains in pepsin. Tyrosine, therefore, must contribute to pepsin’s activity, while lysine obviously does not. This was the first indication that an enzyme might possess portions not essential to its activity.
Recently the active region of another digestive enzyme was pinpointed with more precision. This enzyme is chymotrypsin. The pancreas first secretes it in an inactive form called chymotrypsinogen. This inactive molecule is converted into the active one by the splitting of a single peptide link (accomplished by the digestive enzyme trypsin): that is, it looks as if the uncovering of a single amino acid endows chymotrypsin with its activity. Now it turns out that the attachment of a molecule known as DFP (diisopropylfluorophosphate) to chymotrypsin stops the enzyme’s activity. Presumably, the DFP attaches itself to the key amino acid. Thanks to its tagging by DFP, that amino acid had been identified as serine. In fact, DFP has also been found to attach itself to serine in other digestive enzymes. In each case, the serine is in the same position in a sequence of four amino acids: glycine-aspartic acid-serine-glycine.
