Principles of General Physiology

We see, therefore, that the catalyst, acid, must act on both the hydrolytic and synthetic components of the reactions. That this is actually the case has been shown by experiment ; indeed, acid is commonly used both for hydrolysis and for synthesis of esters and other compounds. Another point, which is illustrated in the example chosen, is that it is not necessary that the two men should be equally excited by the same cause. If the one ran faster than the other, they would meet at a different equilibrium point. What is important to notice is that if they meet anywhere except at either extreme end, they must both have been accelerated. The conclusion to be drawn is that, if any slowly progressing reversible reaction is acted on by a catalyst, and the equilibrium position found to be anywhere except at nearly complete hydrolysis or synthesis, both of these processes must have been accelerated by the catalyst. In theory, therefore, every catalyst is capable of both hydrolytic and synthetic action and, instead of requiring special proof that an enzyme is a synthetic agent, proof must be demanded of any contrary statement. In the case of the ester acted on by lipase, if the hydrolytic reaction alone were accelerated, the synthetic reaction, going on at its own extremely slow pace, would not have proceeded to any perceptible extent before the hydrolytic one was complete. The equilibrium point would inevitably be close to that of complete hydrolysis, instead of being somewhere near one-third of the distance from the synthetic end.

This point of view is particularly insisted on by van't Hoff (1901, p. 211), and it is interesting to note that, at the time of his death, he was engaged in researches on enzymes with regard to their synthetic action (Cohen, 1912, pp. 575-576). He had already made - important advances in the elucidation of glucoside formation (1910), but, unfortunately, never reached the third part of his programme, the processes in the living organism. There is a pathetic interest attached to these latest researches of the great investigator, in that, as Ostwald says (1912, 1, p. 515), they were paid for with parts of his life itself. A portrait of van't Hoff in 1899 has been given in Fig. 24.

There are, in practice, many cases where the equilibrium position is so near complete change in one direction that it is held by some workers in this field to show absence of any reverse reaction whatever. Such a case is the action of emulsin on salicin ; even under such conditions of concentration that synthetic action would be most favoured, there appears, to a hasty observer, to be complete hydrolysis. Closer investigation shows, however, that the reaction is not quite complete. Both Visser (1905) and Bourquelot and Bridel (1913) found this incompleteness to be the fact and what is of importance is that both sets of independent experiments gave the same equilibrium point.

This fact obviously means that, even when accelerated by a catalyst, the synthetic reaction is very slow, compared with the hydrolytic one, when emulsin acts on salicin, or its components. The explanation was given by van't Hoft (1910), who pointed out that salicin is the glucoside of a tertiary alcohol, that is, of an alcohol which contains a carbon atom united directly to three other carbon atoms and with its third valency united to hydroxyl (see Bunge Plimmer, 1907, pp. 71-73). The group may be illustrated thus : —

Now, according to the work of Menschutkin (1879) on esterification, tertiary alcohols are very difficult to esterify, and van't Hoff shows that the same statement applies to the formation of glucosides. A primary alcohol, which contains the group CH9OH, on the contrary, is easily esterified or made into a glucoside. The alcohols of our first experiments, iso amyl and ethyl alcohols and glycerol, are all primary, so that the synthetic action is easy and the equilibrium position is a considerable distance from both ends. The synthetic reaction, in those cases where the equilibrium point is close to that of complete hydrolysis, is one that is of inherent chemical difficulty, and the facts given above with respect to the equilibrium position are thus to be accounted for. We shall see later, however, that even a small amount of synthesis is of considerable importance under such conditions that the product is removed as fast as it is formed.

A point of significance, in view of the mode of action of catalysts, is that they exercise a powerful action even when present in very minute amounts. This is not surprising when we remember that they usually reappear at the end of the reaction in an unaltered state, and are therefore ready for further work. We have already seen that the position of equilibrium under a particular kind of catalyst is not affected by the amount of this catalyst present. What is observed is that the time taken to attain equilibrium is shorter with the larger concentration of catalyst. This fact is a useful criterion in deciding whether, in a particular case, we have to deal with a catalytic process or with one in which the constituents enter into combination in molecular proportions. In the latter case, of course, the amount of product will depend on the amount added of the reagent whose nature we desire to test. Although it is not a matter for surprise that the final effect of a catalyst should be independent of its amount, it is striking to note how very minute are the quantities which are able to produce considerable results.

For instance, Erode (1901, p. 289) found that the reaction between hydrogen peroxide and hydriodic acid was appreciably accelerated by the presence of 1 gram molecule of molybdic acid in 31,000,000 litres. Again, a preparation of invertase, which probably consisted only to a small percentage of the active catalytic agent, was found by O'Sullivan and Tompson (1890) to hydrolyse 200,000 times its weight of cane-sugar. These facts will serve t« impress ujmn the reader the point that the amount of chemical eneri/y which a catalyst is capable <,f supplying to .a reaction is negligible, and the assumption cannot be used to explain any of the phenomena. This will be referred to again.

Heterogeneous Systems. — We shall find presently that the particular catalysts of especial interest to us are in the colloidal state. We have, indeed, seen already, in our first typical examples, that lipase and emulsin act in liquids in which they are completely insoluble. It is therefore necessary to consider briefly the mechanism of reactions in systems of more than one phase. The theory of these reactions is due chiefly to Nernst (1904). They may be said to take place in three stages. Suppose that the catalyst is present in the form of solid particles, and that the other components, which are to be brought into reaction, are in true solution. In order that they shall be influenced by the catalyst, it is obviously necessary that they shall diffuse to it, since it is not uniformly distributed throughout the system. The rate of diffusion is the first factor. If these solutes lower surface energy, as practically all solutes do, they will next be concentrated by adsorption at the interface between the catalyst and the solution. Adsorption is the second stage. The third stage is the chemical reaction proper. It is clear that the increased concentration on the surface will, in itself, hasten the reaction by mass action, and this was, in fact, the explanation suggested by Faraday (1839, 1, p. 184) for the effect of platinum in causing combination of oxygen and hydrogen. Whether all cases of heterogeneous catalysis can be explained on these lines is doubtful. In certain cases of catalysis in homogeneous systems, as we shall see later, there is an intermediate compound formed between the catalyst and the reagents ; but it is certain that this does not apply to all cases ; indeed, it appears to be exceptional.

Faraday's views on the possibility of the close approximation of oxygen and hydrogen on the surface of platinum being sufficient to cause their molecules to enter into combination led to a long discussion with De la Rive, who held that there is an intermediate formation of some oxide of platinum. With our knowledge of Faraday's wonderful insight into the mechanism of natural phenomena, we may well be inclined to think that he was most likely on the right side in this case. Kohlrausch remarked, " Er riecht die Wahrheit," "he smells the truth" (see Tyndall's "Faraday as a Discoverer," 1870, p. 55).

In the discussion of this question,' it is to be remembered that there is reason to belirvithat it is during the actual process of condensation itself that the molecular stresses result in unusual chemical activity (see Hardy's note to the paper by Drury, 1914, p. 175). In all heterogeneous reactions, the rate of the reaction, as measured, is naturally that of the slowest member of the series. Adsorption is a rapid process, when the substances are in contact, so that the rate of the reaction will be either that of diffusion or of the chemical component of the reaction. When the catalyst is in colloidal solution, the length of the way to be passed over by diffusion is very short, since the active substance is almost uniformly distributed ; so that the chemical reaction itself, unless of great rapidity, controls the rate of the whole

process. When a metal in mass is immersed in acid, the diffusion process is slower than the chemical reaction. When one substance is adsorbed on the surface of another, it does not follow of necessity that any chemical reaction will occur. Aniline on the surface of mercury in Lewis' experiments (1910, 3) may be given in illustration. When chemical reaction does occur, the rate at which it proceeds is obviously controlled by the amount adsorbed at any given moment, so that an exponential relation between the concentration and the velocity is to be expected.

The mode of action of catalysts, with especial reference to enzymes, will be discussed later. We have seen that certain substances, extracted from animals and plants, act in a catalytic way similar to that in which an inorganic compound, such as acid, does. These substances are known as "enzymes " or "ferments." In the discussion of their properties, certain names will have to be used, so that the terminology of the subject must first be referred to. The choice of correct words is really more than a mere matter of convenience. If the word used has a meaning, is connotative, it should tell us something about the thing named, although it frequently happens that the original meaning becomes changed as knowledge increases. As often pointed out, the progress of a science depends much on the language used in the description of its phenomena. It is a mistake, however, to be hasty in inventing new names ; more care must be exercised in attaining certainty that the new name is required to describe phenomena of a new kind, inadequately provided for by names already in use. Numerous names, at one time thought necessary, have disappeared.

As various substances were extracted from organisms, and the similarity of the action of these substances to that of alcoholic fermentation became obvious, it was natural to call them "fei'ments." And when Cagniard de Latour (1838) showed that alcoholic fermentation was due to a living organism, substances such as the "diastase," precipitated by Payen and Persoz (1833) from extracts of malt, were distinguished as " soluble," " unorganised," or " unformed " ferments from " living," "organised," or "formed" ferments. In process of time some confusion was caused by this double use of "ferment," so that Kiihne (1878, p. 293) thought it well to introduce a new name for the soluble, or unorganised ferments. The passage is sufficiently interesting to be translated here :—

"The latter designations (formed and unformed ferments) have not gained general acceptance, since on the one hand it was objected that chemical bodies, like ptyalin, pepsin, etc., could not be called ferments, since the name was already given to yeast cells and other organisms (Briicke) ; while, on the other hand, it was said that yeast cells could not be called ferment, because then all organisms, including man, would have to be so designated (Hoppe Seyler). Without stopping to inquire further why the name excited so much opposition, I have taken the opportunity to suggest a new one, and I give the name enzymes to some of the better known substances, called by many ' unformed ferments.' This name is not intended to imply any particular hypothesis, it merely states that cv typi) (in yeast) something occurs that exerts this or that activity, which is supposed to belong to the class called fermentative. The name is not, however, intended to be limited to the invertin of yeast, but it is intended to imply that more complex organisms, from which the enzymes, pepsin, trypsin, etc., can be obtained, are not so fundamentally different from the unicellular organisms as some people would have us believe."

On account of the important work done by Kiihne in the elucidation of the action of enzymes, I introduce his portrait in Fig. 83. The name " enzyme " has come into general use, although " ferment " is still to be met with as synonymous with it ; while the application of this name " ferment " to living organisms has dropped out of use. Enzymes may be shortly denned as the catalysts produced by living organisms. If we grant that the substances known by the name are a special kind of catalysts, which we have still to show, it is clear that the introduction of a name for them is merely a matter of convenience. At the same time, the majority of them have

certain incidental properties which distinguish them from the majority of inorganic catalystsj but this is all that we are justified in asserting. It is interesting to note that Von Wittich (1872) had already come to the conclusion that pepsin merely accelerates the action of hydrochloric acid on fibrin, but it is not quite dear whether he intended to make the statement that enzyme actions in general are catalytic actions, although it appears to be so.

A name is often wanted for the substance on which an enzyme acts. No satisfactory one has been suggested. " Hydrolyte " excludes all processes except hydrolysis, " zymolyte " applies only to enzymes and excludes other catalysts, a suggestion of difference which is to be deprecated. " Substrate " is in very general use ; it is rather an awkward word in English, but will be used in the following pages. To distinguish the different enzymes, Duclaux suggested adding the termination " ase " to the name of the substrate, thus "lactase" is the enzyme which acts on lactose. Certain old names, such as " pepsin " and " trypsin," are, however, still in use. The termination — " lytic " — has been used for a class of enzymes acting on a group of substances; a proteolytuenzyme is one that acts on the proteins in general, and includes pepsin and trypsiri. Armstrong (1890) has justly pointed out that "proteolytic," in analogy with " electrolytic," should mean decomposition by means of protein, not decomposition of protein itself. To avoid this misuse, the termination " -clastic " was suggested, and I will attempt to make consistent use of it.

A recent unfortunate introduction of " ese " as a termination for an enzyme which acts synthetically only has been shown to be unwarranted (Bayliss, 1913, 1) and need not concern us further. (Photograph of the Tablet in the Institute of Physiology, Heidelberg. ) We have seen that Berzelius himself placed the substances which we now call enzymes in the class of catalysts. Apart from theoretical interest, it is of some practical importance to know what kind of properties may be expected to be shown by a substance supposed to be an enzyme.

The only really essential property of a catalyst is that it changes the rate of a reaction, including the starting of one which does not appear to proceed of itself, and without entering as a constituent into the final chemical equilibrium. There are certain other properties usually present, but not essential, although they are sometimes of assistance in deciding the nature of particular cases. We will now proceed to inquire how far enzymes satisfy the above conditions.

It may be useful, first of all, to mention a few typical enzymes, remembering that there are new ones continually being discovered. How far many of these new ones are really such, and not new capabilities of old enzymes, may sometimes be a matter of doubt. In the following list, the name of the appropriate substrate is placed in brackets after that of the enzyme : amylase (starch), maltase (maltose and a-glucosides), emulsin (/3-glucosides), pepsin (proteins in acid medium), trypsin (proteins in alkaline medium), urease (urea), arginase (arginine), lipase (esters), peroxidase (organic peroxides, including that of hydrogen), and so on. We may also divide enzymes into classes according to the nature of the chemical change accelerated. The majority add or remove the elements of water, and may be called hydrolysiny from the one aspect of their activity. All of those mentioned above, with the exception of peroxidase, belong to this large class. Those that cause activation of oxygen or of hydrogen, bringing about oxidations and reductions, will be dealt with in Chapter XX. There is another class which appear simply to break up a complex molecule, although it is probable that this is done by a series of changes, involving oxidation, reduction, and hydrolysis, as in the case of the zymase system of yeast, converting glucose into alcohol and carbon dioxide —

Whether an enzyme merely accelerates a spontaneous reaction it is impossible to state as a general rule. But there are certainly some reactions which proceed slowly by themselves and are accelerated by enzymes : the esters in water may be mentioned. In other cases, the change, rapid under the action of an enzyme, is, at ordinary temperatures, too slow to be detected, although it can be made to proceed at a measurable rate by raising the temperature ; the hydrolysis of cane-sugar and of salicin by water are cases in point. When a reaction can be made obvious by heat, it is justifiable to conclude that it is not entirely absent at ordinary temperatures.

In such cases of solutions in water, the question arises as to whether the spontaneous change might be due to the catalytic action of the small quantity of hydrogen and hydroxyl ions always present. Consideration will show, however, that, even when a reaction is proceeding under the influence of one catalyst, if it is further accelerated by another substance, this second is no less an additional catalyst ; except in those cases where the second acts by increasing the activity of the first, and is inactive alone.

A more important point is the question of the relation of the enzyme to the fatal products. In some cases the enzyme has been recovered at the end of the reaction unchanged, as the acid in ester reactions. In other cases it disappears, partially or entirely. This disappearance, however, is found to be due to the instability of the enzyme itself. That it does not form a component of the final equilibrium is shown by the numerous experiments in which it has been found that the total amount of change is independent of the amount of enzyme added, which would be impossible in the other case. A series of curves illustrating this fact will be found in my monograph (1913, 2), which shows also how the rate of the change depends on the amount of the catalyst. Another case, using the synthetic aspect of the action of emulsin, is given in Fig. 84 below.

If the enzyme formed a component of the final equilibrium, the position of this equilibrium would be altered by mass action if more enzyme were added after its attainment. This, in point of fact, does not happen (Bayliss, 1913, 1, p. 246). Certain views as to the attainment of what has been called a "false equilibrium," in which the final result appears to be in proportion to the concentration of the enzyme, will be found discussed in a paper by myself (1913, 1). It will suffice to say here that careful examination of the experimental facts shows that they do not compel us to make an assumption of this kind, and are, for the most part, to be accounted for by destruction of the enzyme before it

has had time to carry the reaction as far as the equilibrium position. Naturally, the nicmenzyme is present at first, the faster the reaction proceeds ; and, moreover, it will be longer before the whole of the enzyme has disappeared. The fact that the position of equilibrium is found to be the same whether we start from the system consisting only of substrate or only of products, is again of considerable importance as regards the proof that we are dealing

with a true equilibrium in a reversible reaction (see Figs. 80 and 81, page 300 above). We see then that enzymes are, beyond doubt, typical catalysts in the comparatively simple cases hitherto considered. Since, however, many of the reactions taking place in the living organism under the influence of enzymes are of a complex chemical nature, not as yet completely understood, it is not to be wondered at that we meet with phenomena which seem, at first sight, to be difficult to reconcile with the hypothesis of catalysis in reversible systems. We shall presently meet with further evidence that, in cases of enzyme action where we have all the factors under control, the reactions obey all the laws they would be expected to do on the hypothesis mentioned. It appears to me that we are hereby justified in holding that the more complex cases, such, for example, as those where proteins are concerned, will be found to require no assumptions contrary to the laws obeyed in the simpler cases. In these heterogeneous, colloidal

FIG. 84. RATE OF SYNTHESIS OF ULYCEROL-GLCCOSIDE WITH DIFFERENT CONCENTKA- TIONS OF EMULSIN. — The ratios are as 1 :4 : 12, the uppermost curve having the lowest concentration. All arrive finally at the same position of equilibrium. systems, the facts brought forward in the preceding chapters are sufficient to indicate what innumerable possibilities of modification are present, in the way of surface action, electric charge, and so on. It is the work of the future to investigate the intervention of these factors in the course of the chemical reactions brought about by the various individual enzymes.

Certain incidental properties common to inorganic catalysts and enzymes serve to strengthen our position. The fact that very minute quantities are active has been mentioned already : how minute the really active substance is in the case of enzymes we do not know, owing to the difficulty of preparing them in a chemically pure state from the complex mixtures in which they are found. In the definition of enzymes, we sometimes find the qualifications introduced that they are colloidal, specific catalysts destroyed by heat. It is true that the substances which we separate as active enzymes are practically all in the colloidal state, but we are not absolutely certain that this state is necessary to their activity in all cases. As to #pecifc nature, the circumstance that a particular catalyst acts on a limited class of substrates is by no means peculiar to those produced by living organisms ; some inorganic catalysts are very specific, as, for example, tungstic acid is a powerful catalyst for the oxidation of hydriodic acid by hydrogen peroxide, but not for its oxidation by persulphates or by bromic acid. On the other hand, some enzymes are not particularly specific, emulsin acts on the whole series of

/3-glucosides, which are almost innumerablein number. Some investigators seem to l>e prepared to postulate a separate enzyme for each glucoside. This question requires more detailed discussion in a later page. As to the action of heat, the sensibility of enzymes varies considerably, according to the conditions present. As a rule, they are coagulated or precipitated by heat, but in some cases the enzymes seem to be merely carried down by adsorption or changed in their physical state, reversibly. In practice, however, this property is frequently useful in deciding the nature of a particular agent. If the action is stopped by moderate heat, say up to that of boiling water, it is almost certainly due to an enzyme or to the action of living protoplasm, using this latter name for the present as a cloak for ignorance. To distinguish an enzyme action from it, use is made of antiseptics, which have a more powerful action on what we call " vital activity" than on that of enzymes. But, again, the distinction is one of degree only, some enzymes are very sensitive to antiseptics, others not; the difference probably depends on complexity of structure. Invertase is comparatively insensitive, zymase is very sensitive. Meyer hof (1913 and 1914) finds the inhibiting effect of indifferent narcotics on enzymes to be completely reversible, and interprets it as being due to the driving off of adsorbed substrate by the more strongly adsorbed narcotic. We shall see later that preliminary adsorption is a phase in the action of enzymes.

We may take it, then, that enzymes are a special class of catalysts ; this being so, their function is to alter the rate of reactions. The factors involved in the velocity with which a reaction takes place have been incidentally dealt with to some extent, but it may be well to spend a little more time on the question, as it affects catalytic action especially. When one large molecule is undergoing the process of being divided up into smaller ones, spontaneously, or under the action of a catalyst, there is no difficulty in seeing that the number of molecules split -in a given time is proportional to those present.