Asimov's New Guide to Science

by Isaac Asimov

  It turns out that a peptide consisting of those four amino acids alone will not display catalytic activity. In some way, the rest of the enzyme molecule plays a role, too. We can think of the four-acid sequence—the active center—as analogous to the cutting edge of a knife, which is useless without a handle.

  Nor need the active center, or cutting edge, necessarily exist all in one piece in the amino-acid chain. Consider the enzyme ribonuclease. Now that the exact order of its 124 amino acids is known, it has become possible to devise methods for deliberately altering this or that amino acid in the chain and noting the effect of the change on the enzyme’s action. It was discovered that three amino acids, in particular, are necessary for action, but that they are widely separated. They are a histidine in position 12, a lysine in position 41, and another histidine in position 119.

  This separation, of course, exists only in the chain viewed as a long string. In the working molecule, the chain is coiled into a specific three-dimensional configuration, held in place by four cystine molecules, stretching across the loops. In such a molecule, the three necessary amino acids are brought together into a close-knit unit.

  The matter of an active center was made even more specific in the case of lysozyme, an enzyme found in many places, including tears and nasal mucus. It brings about the dissolution of bacterial cells by catalyzing the breakdown of key bonds in some of the substances that make up the bacterial cell wall. It is as though it causes the wall to crack and the cell contents to leak away.

  Lysozyme was the first enzyme whose structure was completely analyzed (in 1965) in three dimensions. Once this was done, it could be shown that the molecule of the bacterial cell wall that is subject to lysozyme’s action fits neatly along a cleft in the enzyme structure. The key bond was found to lie between an oxygen atom in the side chain of glutamic acid (position 35) and another oxygen atom in the side chain of aspartic acid (position 52). The two positions were brought together by the folding of the amino-acid chain with just enough separation that the molecule to be attacked could fit in between. The chemical reaction necessary for breaking the bond could easily take place under those circumstances—and it is in this fashion that lysozyme is specifically organized to do its work.

  Then, too, it happens sometimes that the cutting edge of the enzyme molecule is not a group of amino acids at all but an atom combination of an entirely different nature. A few such cases will be mentioned later in the book.

  We cannot tamper with the cutting edge, but could we modify the handle without impairing the usefulness of the tool? The handle has its purposes, of course. It would seem that the enzyme in its natural state is “jiggly” and can take up several different shapes without much strain. When the substrate adds on to the active site, the enzyme adjusts itself to the shape of the substrate, thanks to the “give” of the non-active portion of the molecule so that the fit becomes tight and the catalytic action is highly efficient. Substrates of slightly different shape might not take advantage of the “give” quite as well and will not be affected—might, indeed, inhibit the enzyme.

  Still, the enzyme might be simplified, perhaps, at the cost of the loss of some efficiency, but not all. The existence of different varieties of such protein as insulin, for instance, encourages us to believe that simplification might be possible. Insulin is a hormone, not an enzyme, but its function is highly specific. At a certain position in the G-chain of insulin there is a three-amino-acid sequence that differs in different animals: in cattle, it is alanine-serine-valine; in swine, threonine-serine-isoleucine; in sheep, alanine-glycine-valine; in horses, threonine-glycine-isoleucine; and so on. Yet any of these insulins can be substituted for any other and still perform the same function.

  What is more, a protein molecule can sometimes be cut down drastically without any serious effect on its activity (as the handle of a knife or an ax might be shortened without much loss in effectiveness). A case in point is the hormone called ACTH (adrenocorticotropic hormone). This is a peptide chain made up of thirty-nine amino acids, whose order has now been fully determined. Up to fifteen of the amino acids have been removed from the C-terminal end without destroying the hormone’s activity. On the other hand, the removal of one or two amino acids from the N-terminal end (the cutting edge, so to speak) kills activity at once.

  The same sort of thing has been done to an enzyme called papain, from the fruit and sap of the papaya tree. Its enzymatic action is similar to that of pepsin. Removal of the pepsin molecule’s 180 amino acids from the N-terminal end does not reduce its activity to any detectable extent.

  So it is at least conceivable that enzymes may yet be simplified to the point where they will fall within the region of practical mass synthesis. Synthetic enzymes, in the form of fairly simple organic compounds, may then be made on a large scale for various purposes. This would be a form of chemical miniaturization.

  Metabolism

  An organism, such as the human body, is a chemical plant of great diversity. It breathes in oxygen and drinks water. It takes in as food carbohydrates, fats, proteins, minerals, and other raw materials. It eliminates various indigestible materials plus bacteria and the products of the putrefaction they bring about.

  It also excretes carbon dioxide via the lungs, gives up water by way of both the lungs and the sweat glands, and excretes urine, which carries off a number of compounds in solution, the chief of these being urea. These chemical reactions determine the body’s metabolism.

  By examining the raw materials that enter the body and the waste products that leave it, we can tell a few things about what goes on within the body. For instance, since protein supplies most of the nitrogen entering the body, we know that urea (NH2CONH2) must be a product of the metabolism of proteins. But between protein and urea lies a long, devious, complicated road. Each enzyme of the body catalyzes only a specific small reaction, rearranging perhaps no more than two or three atoms. Every major conversion in the body involves a multitude of steps and many enzymes. Even an apparently simple organism such as the tiny bacterium must make use of many thousands of separate enzymes and reactions.

  All this may seem needlessly complex, but it is the very essence of life. The vast complex of reactions in tissues can be controlled delicately by increasing or decreasing the production of appropriate enzymes. The enzymes control body chemistry as the intricate movements of fingers on the strings control the playing of a violin; and without this intricacy, the body could not perform its manifold functions.

  To trace the course of the myriads of reactions that make up the body’s metabolism is to follow the outline of life. The attempt to follow it in detail, to make sense of the intermeshing of countless reactions all taking place at once, may indeed seem a formidable and even hopeless undertaking. Formidable it is, but not hopeless.

  THE CONVERSION OF SUGAR TO ETHYL ALCOHOL

  The chemists’ study of metabolism began modestly with an effort to find out how yeast cells convert sugar to ethyl alcohol. In 1905, two British chemists,

  Arthur Harden and William John Young, suggested that this process involves the formation of sugars bearing phosphate groups. Harden and Young were the first to note that phosphorus plays an important role in metabolism (and phosphorus has been looming larger ever since). Harden and Young even found in living tissue a sugar-phosphate ester consisting of the sugar fructose with two phosphate groups (PO3H2) attached. This fructose diphosphate (still sometimes known as Harden-Young ester) was the first metabolic intermediate to be identified definitely—the first compound, that is, recognized to be formed momentarily, in the process of passing from the compounds as taken into the body to the compounds eliminated by it. Harden and Young had thus founded the study of intermediary metabolism, which concentrates on the nature of such intermediates and the reactions involving them. For this work and for further work on the enzymes involved in the conversion of sugar to alcohol by yeast (see chapter 15), Harden shared the Nobel Prize in chemistry in 1929.

  Wh
at began by involving only the yeast cell became of far broader importance when the German chemist Otto Fritz Meyerhof demonstrated in 1918 that animal cells, such as those of muscle, break down sugar in much the same way as yeast does. The chief difference is that in animal cells the breakdown does not proceed so far in this particular route of metabolism. Instead of converting the six-carbon glucose molecule all the way down to the two-carbon ethyl alcohol (CH3CH2OH), they break it down only as far as the three-carbon lactic acid (CH3CHOHCOOH).

  Meyerhof’s work made clear for the first time a general principle that has since become commonly accepted: with only minor differences, metabolism follows the same routes in all creatures, from the simplest—to the most complex. For his studies on the lactic acid in muscle, Meyerhof shared the Nobel Prize in physiology and medicine in 1922 with the English physiologist Archibald Vivian Hill. The latter had tackled muscle from the standpoint of its heat production and had come to conclusions quite similar to those obtained from Meyerhofs chemical attack.

  The details of the individual steps involved in the transition from sugar to lactic acid were evolved between 1937 and 1941 by Carl Ferdinand Cori and his wife Gerty Theresa Cori, working at Washington University in St. Louis. They used tissue extracts and purified enzymes to bring about changes in various sugar-phosphate esters, then put all the changes together like a jigsaw puzzle. The scheme of step-by-step changes that they presented has stood with little modification to this day, and the Caris were awarded a share in the Nobel Prize in physiology and medicine in 1947.

  In the path from sugar to lactic acid, a certain amount of energy is produced and is utilized by the cells. The yeast cell lives on it when it is fermenting sugar, and so, when necessary, does the muscle cell. It is important to remember that this energy is obtained without the use of oxygen from the air. Thus, a muscle is capable of working even when it must expend more energy than can be replaced by reactions involving the oxygen brought to it at a relatively slow rate by the blood. As the lactic acid accumulates, however, the muscle grows weary, and eventually it must rest until oxygen breaks up the lactic acid.

  METABOLIC ENERGY

  Next comes the question: In what form is the energy from the sugar-tolactic—acid breakdown supplied to the cells, and how do they use it? The German-born American chemist Fritz Albert Lipmann found an answer in researches beginning in 1941. He showed that certain phosphate compounds formed in the course of carbohydrate metabolism store unusual amounts of energy in the bond that connects the phosphate group to the rest of the molecule. This high-energy phosphate bond is transferred to energy carriers present in all cells. The best known of these carriers is adenosine triphosphate (ATP). The ATP molecule and certain similar compounds represent the small currency of the body’s energy. They store the energy in neat, conveniently sized, readily negotiable packets. When the phosphate bond is hydrolyzed off, the energy is available to be converted into chemical energy for the building of proteins from amino acids, or into electrical energy for the transmission of a nerve impulse, or into kinetic energy via the contraction of muscle, and so on. Although the quantity of ATP in the body is small at any one time, there is always enough (while life persists), for as fast as the ATP molecules are used up, new ones are formed.

  For his key discovery, Lipmann shared the Nobel Prize in physiology and medicine in 1953.

  The mammalian body cannot convert lactic acid to ethyl alcohol (as yeast can); instead, by another route of metabolism, the body bypasses ethyl alcohol and breaks down lactic acid all the way to carbon dioxide (CO2) and water. In so doing, it consumes oxygen and produces a great deal more energy than is produced by the non-oxygen-requiring conversion of glucose to lactic acid.

  The fact that consumption of oxygen is involved offers a convenient means of tracing a metabolic process—that is, finding out what intermediate products are created along the route. Let us say that at a given step in a sequence of reactions a certain substance (for example, succinic acid) is suspected to be the intermediate substrate. We can mix this acid with living tissue (or in many cases with a single enzyme) and measure the rate at which the mixture consumes oxygen. If it shows a rapid uptake of oxygen, we can be confident that this particular substance can indeed further the process.

  The German biochemist Otto Heinrich Warburg devised the key instrument used to measure the rate of uptake of oxygen. Called the Warburg manometer, it consists of a small flask (where the substrate and the tissue or enzyme are mixed) connected to one end of a thin U-tube, whose other end is open. A colored Huid fills the lower part of the V. As the mixture of enzyme and substrate absorbs oxygen from the air in the flask, a slight vacuum is created there, and the colored liquid in the V-tube rises on the side of the V connected to the flask. The rate at which the liquid rises can be used to calculate the rate of oxygen uptake (figure 12.4).

  Figure 12.4. Warburg manometer.

  Warburg’s experiments on the uptake of oxygen by tissues won him the Nobel Prize in physiology and medicine in 1931.

  Warburg and another German biochemist, Heinrich Wieland, identified the reactions that yield energy during the breakdown of lactic acid. In the course of the series of reactions, pairs of hydrogen atoms are removed from intermediate substances by means of enzymes called dehydrogenases. These hydrogen atoms then combine with oxygen, with the catalytic help of enzymes called cytochromes. In the late 1920s, Warburg and Wieland argued strenuously over which of these reactions is the important one, Warburg contending that it is the uptake of oxygen, and Wieland that it is the removal of hydrogen. Eventually, David Keilin showed that both steps are essential.

  The German biochemist Hans Adolf Krebs went on to work out the complete sequence of reactions and intermediate products from lactic acid to carbon dioxide and water. This is called the Krebs cycle, or the citric-acid cycle, citric acid being one of the key products formed along the way. For this achievement, completed in 1940, Krebs received a share in the Nobel Prize in physiology and medicine in 1953 (with Lipmann).

  The Krebs cycle produces the lion’s share of energy for those organisms that make use of molecular oxygen in respiration (which means all organisms except a few types of anaerobic bacteria that depend for energy on chemical reactions not involving oxygen). At different points in the Krebs cycle, a compound will lose two hydrogen atoms, which are eventually combined with oxygen to form water. This “eventually” hides a good deal of detail. The two hydrogen atoms are passed from one variety of cytochrome molecule to another, until the final one, cytochrome oxidase, passes it on to molecular oxygen. Along the line of cytochromes, molecules of ATP are formed and the body is supplied with its chemical “small change” of energy. All told, for every turn of the Krebs cycle, eighteen molecules of ATP are formed. The entire process, because it involves oxygen and the piling up of phosphate groups to form the ATP, is called oxidative phosphorylation and is a key reaction of living tissue. Any serious interference with it (as when one swallows potassium cyanide) brings death in minutes.

  All the substances and all the enzymes that take part in oxidative phosphorylation are contained in tiny granules within the cytoplasm. These were first detected in 1898 by the German biologist C. Benda, who did not at that time, of course, understand their importance. He called them mitochondria (“threads of cartilage,” which he wrongly thought they were), and the name stuck.

  The average mitochondrion is football-shaped, about 1/10,000 of an inch long and 1/25,000 of an inch thick. An average cell might contain anywhere from several hundred to a thousand mitochondria. Very large cells may contain a couple of hundred thousand, while anaerobic bacteria contain none. After the Second World War, electron-microscopic investigation showed the mitochondrion to have a complex structure of its own, for all its tiny size. The mitochondrion has a double membrane, the outer one smooth and the inner one elaborately wrinkled to present a large surface. Along the inner surface of the mitochondrion are several thousand tiny structures called elementary particles. I
t is these that seem to represent the actual sites of oxidative phosphorylation.

  THE METABOLISM OF FATS

  Meanwhile biochemists also made headway in solving the metabolism of fats. It was known that the fat molecules are carbon chains, that they can be hydrolyzed to fatty acids (most commonly sixteen or eighteen carbon atoms long), and that the molecules are broken down two carbons at a time. In 1947, Fritz Lipmann discovered a rather complex compound, which plays a part in acetylation—that is, transfer of a two-carbon fragment from one compound to another. He called the compound coenzyme A (the A standing for “acetylation”). Three years later, the German biochemist Feodor Lynen found coenzyme A to be deeply involved in the breakdown of fats. Once it attaches itself to a fatty acid, there follows a series of four steps which end in lopping off the two carbons at the end of the chain to which the coenzyme A is attached. Then another coenzyme A molecule attaches itself to what is left of the fatty acid, chops off two more atoms, and so on. This is called the fatty-acid oxidation cycle. This and other work won Lynen a share in the 1964 Nobel Prize in physiology and medicine.

  The breakdown of proteins obviously must be, in general, more complicated than that of carbohydrates or fats, because some twenty different amino acids are involved. In some cases it turns out to be rather simple: one minor change in an amino acid may convert it into a compound that can enter the citric-acid cycle (as the two-carbon fragments from fatty acids can). But mainly amino acids are decomposed by complex routes.

  We can now go back to the conversion of protein into urea—the question that I considered in the section on enzymes. This conversion happens to be comparatively simple.

  A group of atoms that is essentially the urea molecule forms part of a side chain of the amino acid arginine. This group can be chopped off by an enzyme called arginase, and it leaves behind a kind of truncated amino acid, called ornithine. In 1932, Krebs and a co-worker, K. Henseleit, while studying the formation of urea by rat-liver tissue, discovered that when they added arginine to the tissue, it produced a flood of urea—much more urea, in fact, than the splitting of every molecule of arginine they had added could have produced. Krebs and Henseleit decided that the arginine molecules must be acting as agents that produce urea over and over again. In other words, after an arginine molecule has its urea combination chopped off by arginase, the ornithine that is left picks up amine groups from other amino acids (plus carbon dioxide from the body) and forms arginine again. So the arginine molecule is repeatedly split, re-formed, split again, and so on, each time yielding a molecule of urea. This is called the urea cycle, the ornithine cycle, or the Krebs-Henseleit cycle.