CHAPTER V.

ACTION OF SOME INHIBITING AND ACCELERATING AGENTS ON THE ENZYMES OF YEAST-JUICE.

One of the most interesting and at the same time most difficult problems concerning enzyme action in general is the nature of the inhibiting or accelerating effect produced by many substances upon the rate or total result of the chemical process set up in presence of the enzyme. Inhibition, it is usually supposed, involves either the decomposition of the enzyme, in which case it is irreversible, its removal from the sphere of action by some change in its mode of solution, or the formation of an inactive or less active compound between the enzyme and the inhibiting agent. This compound it may sometimes be possible to decompose, with the result that the activity of the enzyme is restored. A striking example of this, to which allusion has already been made, is the effect of hydrocyanic acid on alcoholic fermentation (p. 37).

Acceleration of enzyme action can in some cases be ascribed to the fact that the accelerating substance possesses an assignable chemical function in the reaction, so that an increase in the concentration of this substance causes an increase in the rate of the reaction. As we have seen in Chapter III, this is the explanation of the accelerating effect of phosphates on fermentation by yeast-juice. In many other cases, however, no such chemical function can be traced, as, for example, in the effect of neutral salts on the hydrolytic action of invertase, or the effect of the addition of the co-enzyme to zymase, and it is necessary to fall back on some assumption, such as that the accelerating agent acts by increasing the effective concentration of the enzyme or by combining either with the enzyme or the substrate, forming a compound which undergoes the reaction more readily.

The interest in the following examples of inhibition and acceleration of fermentation by yeast-juice lies not only in their relation to these general problems but also, and perhaps chiefly, in their bearing on the specific problem of the nature and mode of action of the various agents concerned in the production of alcohol and carbon dioxide from sugar in the yeast-cell. [p071]

I. INFLUENCE OF CONCENTRATION OF PHOSPHATE ON THE COURSE OF FERMENTATION.

Prominent among these instances of inhibition and acceleration are the phenomena attendant on the addition of excess of phosphate to yeast-juice.

When a phosphate is added to a fermenting mixture of a sugar and yeast-juice, the effect varies with the concentration of the phosphate and the sugar and with the particular specimen of yeast-juice employed. With low concentrations of phosphate in presence of excess of glucose the acceleration produced is so transient that no accurate measurements of rate can be made. As soon as the amount of phosphate added is sufficiently large, it is found that the rate of evolution of carbon dioxide very rapidly increases from five to ten times, and then quickly falls approximately to its original value.

As the concentration of phosphate is still further increased, it is first observed that the maximum velocity, which is still attained almost immediately after the addition of the phosphate, is maintained for a certain period before the fall commences, and then, as the increase in concentration of phosphate proceeds, that the maximum is only gradually attained after the addition, the period required for this increasing with the concentration of the phosphate. Moreover, with still higher concentrations, the maximum rate attained is less than that reached with lower concentrations, and further, the rate falls off more slowly. The concentration of phosphate which produces the highest rate, which may be termed the optimum concentration, varies very considerably with different specimens of yeast-juice [Harden and Young, 1908, 1].

All these points are illustrated by the accompanying curves (Fig. 7) which show the rate of evolution per five minutes plotted against the time for four solutions in which the initial concentrations of phosphate were (A) 0·033, (B) 0·067, (C) 0·1, and (D) 0·133 molar, the volumes of 0·3 molar phosphate being 5, 10, 15, and 20 c.c. in each case added to 25 c.c. of yeast-juice, and made up to 45 c.c, each solution containing 4·5 grams of glucose. The time of addition is taken as zero, the rate before addition being constant, as shown in the curves.

It will be observed that 5 and 10 c.c. (A and B) give the same maximum, whilst 15 c.c. (C) produce a much lower maximum, and 20 c.c. (D) a still lower one, the rate at which the velocity diminishes after the attainment of the maximum being correspondingly slow in these last two cases. By calculating the amount of phosphate which has disappeared as such from the amount of carbon dioxide evolved, [p072] it is found that the maximum does not occur at the same concentration of free phosphate in each case.

These results suggest that the phosphate is capable of forming two or more different unstable associations with the fermenting complex. One of these, formed with low concentrations of the phosphate, has the composition most favourable for the decomposition of sugar, whilst the others, formed with higher concentrations of phosphate, contain more of the latter, probably associated in such a way with the fermenting complex as to render the latter partially or wholly incapable of effecting the decomposition of the sugar molecule. As the fermentation proceeds slowly in the presence of excess of phosphate, the concentration of the latter is reduced by conversion into hexosephosphate, and a re-distribution of phosphate occurs, resulting in the gradual change of the less active into the more active association of phosphate with fermenting complex, and a consequent rise in the rate of fermentation.

In those cases in which the maximum rate corresponding to the optimum concentration of phosphate is never attained, some secondary cause may be supposed to intervene, such as a permanent change in a portion of the fermenting complex, accumulation of the products of the reaction, etc.

It is also possible as suggested by Buchner for the analogous case of arsenite (p. 78) that the addition of increasing amounts of phosphate causes a progressive but reversible change in the mode of dispersion [p073] of the colloidal enzyme, and that this has the secondary effect of altering the rate of fermentation. No decisive evidence is as yet available upon the subject.

The results obtained by Euler and Johansson [1913] to which reference has already been made indicate that in presence of a moderate excess of phosphate esterification is more rapid than production of carbon dioxide. No explanation of this phenomenon has yet been given, but it might obviously be due either to the production of some phosphorus compound which subsequently takes part in the production both of hexosediphosphate and of carbon dioxide, or, less probably, to the entire independence of the two changes--esterification of phosphate and production of carbon dioxide--which might then be differently affected by the presence of excess of phosphate and therefore take place at different rates.

II. REACTION OF FRUCTOSE WITH PHOSPHATES IN PRESENCE OF YEAST-JUICE.

Although, as has been pointed out (p. 42), glucose, mannose, and fructose all react with phosphate in a similar manner in presence of yeast-juice, there are nevertheless certain quantitative differences between the behaviour of glucose and mannose on the one hand, and fructose on the other, which appear to be of considerable importance. Fructose differs from the other two fermentable hexoses in two particulars: (1) the optimum concentration of phosphate is much greater; (2) the maximum rate of fermentation attainable is much higher [Harden and Young, 1908, 2; 1909].

These points are clearly illustrated by the following results, which all refer to 10 c.c. of yeast-juice, and show that the optimum concentration of phosphate for the fermentation of fructose is from 1·5 to 10 times that of glucose, and that the maximum rate of fermentation for fructose in presence of phosphate is 2 to 6 times that of glucose.

───────┬─────────┬──────────────────────┬────────────────────── Sugar │ Total │ Optimum Volume of │ Maximum Rate in Cubic in │ Volume. │ 0·6 Molar Phosphate │ Centimetres of CO{2} Grams.│ │ in c.c. │ per Five Minutes │ │ Glucose. │ Fructose. │ Glucose. │ Fructose. ───────┼─────────┼──────────┼───────────┼──────────┼─────────── 2 │ 35 │ 2 │ 5 │ 7·5 │ 32·2 4 50 1 10 5·4 28·4 1·6 23 2 5 8 17 1 25 1·75 5 5·2 25·9 2 25 5 7·5 16·2 31·2 2 20 2 3·5 7·9 22·6 2 22·5 0·75 2 3·4 22·2 ───────────────────────────────────────────────────────────────

[p074]

It is interesting to note that the two high rates, 32·2 and 31·2 c.c. per five minutes, are equal to about half the rate obtainable with an amount of living yeast corresponding to 10 c.c. of yeast-juice, assuming that about 16 to 20 grams of yeast are required to yield this volume of juice, and that this amount of yeast would give about 56 to 70 c.c. of carbon dioxide per five minutes at 25°, which has been found experimentally to be about the rate obtainable with the top yeast employed for these experiments.

III. EFFECT OF THE ADDITION OF FRUCTOSE ON THE FERMENTATION OF GLUCOSE OR MANNOSE IN PRESENCE OF A LARGE EXCESS OF PHOSPHATE.

When the maximum rate of fermentation of glucose or mannose by yeast-juice in presence of phosphate is greatly lowered by the addition of a large excess of phosphate, the addition of a relatively small amount of fructose (as little as 2·5 per cent. of the weight of the glucose) causes rapid fermentation to occur. This induced activity is not due solely to the selective fermentation of the added fructose, since the amount of gas evolved may be greatly in excess of that obtainable from the quantity added.

Another way of expressing the same thing is to say that the optimum concentration of phosphate (p. 71) is greatly raised when 2·5 per cent. of fructose is added to glucose, and that consequently the rate of fermentation rises. The effect is extremely striking, since a mixture of glucose and yeast-juice fermenting in the presence of a large excess of phosphate at the rate of less than 1 c.c. of carbon dioxide in five minutes may be made to ferment at six to eight times this rate by the addition of only 0·05 gram of fructose (2·5 per cent. of the glucose present), and to continue until the total gas evolved is at least five to six times as great as that obtainable from the added fructose, the concentration of the phosphate being the whole time at such a height as would limit the fermentation of glucose alone to its original value.

The effect is not produced when the concentration of the phosphate is so high that the rate of fermentation of fructose is itself greatly lowered.

This remarkable inductive effect is specific to fructose and is not produced when glucose is added to mannose or fructose, or by mannose when added to glucose or fructose, under the proper conditions of concentration of phosphate in each case.

This interesting property of fructose, taken in connection with the [p075] facts that this sugar in presence of phosphate is much more rapidly fermented than glucose or mannose, and that the optimum concentration of phosphate for fructose is much higher than for glucose or mannose, appears to indicate that fructose when added to yeast-juice does not merely act as a substance to be fermented, but in addition, bears some specific relation to the fermenting complex.

All the phenomena observed are, indeed, consistent with the supposition that fructose actually forms a permanent part of the fermenting complex, and that, when the concentration of this sugar in the yeast-juice is increased, a greater quantity of the complex is formed. As the result of this increase in the concentration of the active catalytic agent, the yeast-juice would be capable of bringing about the reaction with sugar in presence of phosphate at a higher rate, and at the same time the optimum concentration of phosphate would become greater, exactly as is observed. The question whether, as suggested above, fructose actually forms part of the fermenting complex, and the further questions, whether, if so, it is an essential constituent, or whether it can be replaced by glucose or mannose with formation of a less active complex, remain at present undecided, and cannot profitably be more fully discussed until further information is available.

It must, moreover, be remembered that different samples of yeast-juice vary to a considerable extent in their relative behaviour to glucose and fructose, so that the phenomena under discussion may be expected to vary with the nature and past history of the yeast employed.

IV. EFFECT OF ARSENATES ON THE FERMENTATION OF SUGARS BY YEAST-JUICE AND ZYMIN.

The close analogy which exists between the chemical functions of phosphorus and arsenic lends some interest to the examination of the action of sodium arsenate upon a mixture of yeast-juice and sugar, and experiments reveal the fact that arsenates produce a very considerable acceleration in the rate of fermentation of such a mixture [Harden and Young, 1906, 3; 1911, 1]. The phenomena observed, however, differ markedly from those which accompany the action of phosphate.

The acceleration produced is of the same order of magnitude as that obtained with phosphate, but it is maintained without alteration for a considerable period, so that there is no equivalence between the amount of arsenate added and the extra amount of fermentation effected. Further, no organic arsenic compound corresponding in composition with the hexosephosphates appears to be formed.

Increase of concentration of arsenate produces a rapid inhibition of [p076] fermentation, probably due to some secondary effect on the fermenting complex, possibly to be interpreted as the formation of compounds incapable of combining with sugar and hence unable to carry on the process of fermentation. An optimum concentration of arsenate therefore exists just as of phosphate, at which the maximum rate is observed, and this optimum concentration and the corresponding rate vary with different samples of juice and are less for glucose than for fructose. The rate of fermentation by zymin is relatively less increased than that by yeast-juice.

Owing to the fact that the rate is permanently maintained the addition of a suitable amount of arsenate increases the total fermentation produced to a much greater extent than phosphate.

The nature of these effects may be gathered from the result of a few typical experiments. In one case the rate of fermentation of glucose by yeast-juice was raised by the presence of 0·03 molar arsenate from 2 to 23 c.c. per five minutes, and the total evolved in ninety-five minutes from 51 to 459 c.c. The accelerating effect on 20 c.c. of juice, of as little as 0·005 c.c. of 0·3 molar arsenate, containing 0·11 mgrm. of arsenic, can be distinctly observed, but the maximum effect is usually produced by about 1 to 3 c.c., the concentration being therefore 0·015 to 0·045 molar. Greater concentrations than this produce a less degree of acceleration accompanied by a shorter duration of fermentation, as shown by the following numbers which refer to 20 c.c. of yeast-juice in a total volume of 40 c.c. containing 10 per cent. of glucose:--

─────────────┬──────────────────┬──────────────── C.cs. of │ Molar │ Maximum 0·3 Molar │ Concentration of │ Rate of Arsenate │ Arsenate. │ Fermentation. in 40 c.c. │ │ ─────────────┴──────────────────┴──────────────── 0 0 3·5 0·005 0·0000375 6·3 0·01 0·000075 8 0·02 0·00015 14·2 0·04 0·0003 19·9 0·1 0·00075 29·7 0·2 0·0015 35 ───────────────────────────────────────────────── 0·5 0·00375 34·9 1·0 0·0075 29·5 2·0 0·015 23·2 5·0 0·0375 14·5 10·0 0·075 8·7 15·0 0·1125 5·3 20 0·15 3·2 ─────────────────────────────────────────────────

The contrast between glucose and fructose in their relations to [p077] arsenate are well exhibited in the following table, in which the rates of fermentation produced by arsenate in presence of excess of glucose and fructose respectively are given:--

──────────────────────────────────────────────────── Rate. Concentration of Arsenate. ─────────────────────── Glucose. Fructose. ──────────────────────────────────────────────────── 0·0075 molar 12·1 26·6 0·0225 (opt. for glucose) 13·4 -- 0·0525 (opt. for fructose) -- 45·8 0·1125 5·1 39 ────────────────────────────────────────────────────

Here the optimum concentration for fructose is more than twice that for glucose, whilst the maximum rate of fermentation obtainable with fructose is between three and four times the maximum given by glucose.

V. EFFECT OF ARSENITES ON THE FERMENTATION PRODUCED BY YEAST-JUICE.

Effects somewhat similar to those produced by arsenates were observed by Buchner [Buchner and Rapp, 1897; 1898, 1, 2, 3; 1899, 2; Buchner, E. and H., and Hahn, 1903, pp. 184-205] when potassium arsenite was added to yeast-juice. This substance, the action of which on yeast had been adduced by Schwann as a proof of the vegetable nature of this organism, was employed by Buchner on account of its poisonous effect on vegetable cells as an antiseptic and as a means of testing for the protoplasmic nature of the agent present in yeast-juice. Its effect on the fermentation was, however, found to be irregular, and at the same time it did not act as an efficient antiseptic in the concentrations which could be employed. Even 2 per cent. of arsenious oxide, added as the potassium salt, had in many cases a decided effect in diminishing the total fermentation obtained with cane sugar, and this effect increased with the concentration. A number of irregularities were also observed which cannot here be discussed. It was further found that in some cases 2 per cent. of arsenious oxide inhibited the fermentation of glucose but not of saccharose, or of a mixture of glucose and fructose, whilst its effect on fructose alone was of an intermediate character.

The important observation was also made by Buchner that the addition of a suitable quantity of arsenite as a rule caused a greatly increased fermentation during the first sixteen hours even in experiments in which the total fermentation was diminished. By examining the effect of arsenite on fermentation in a similar manner to that of arsenate, Harden and Young [1911, 1] have found that a close analogy exists [Pg 078] between the effects and modes of action of these substances, but that arsenite produces a much smaller acceleration than arsenate. An optimum concentration of arsenite exists, just as in the case of arsenate, which produces a maximum rate of fermentation. Further increase in concentration leads to inhibition, and in no case is there any indication of the production of an exactly equivalent amount of fermentation as in the case of phosphate. In various experiments with dialysed, evaporated, and diluted yeast-juice in which 2 per cent. of arsenious oxide was found by Buchner to inhibit fermentation, it is probable that, owing to the small amount of fermenting complex left, this amount of arsenious oxide was considerably in excess of the optimum concentration, although Buchner ascribes the effect to the removal of some of the protective colloids of the juice, owing to the prolonged treatment to which it had been subjected.

The extent of the action of arsenite appears from the following results. In one case a rate of 1·7 c.c. was increased to 7 c.c. by 0·06 molar arsenite. In another experiment it was found that the optimum concentration was 0·04 molar arsenite, the addition of which increased the rate three-fold. As in the case of arsenate the optimum concentration and the corresponding maximum rate of fermentation are considerably greater for fructose than for glucose. The relative rates produced by the addition of equivalent amounts of arsenate and arsenite (1 c.c. of 0·3 molar solution in each case to 20 c.c. of yeast-juice) were 27·5 and 3·1, the original rate of the juice being 1·7. In general the optimum concentration of arsenite is considerably greater than that of arsenate.

The inhibiting effects of higher concentrations of arsenite and arsenate also present close analogies, but this most interesting aspect of the question has not yet been sufficiently examined to repay detailed discussion. Buchner [Buchner, E. and H., and Hahn, 1903, pp. 199-205] has suggested that the inhibition is due primarily to some change in the colloidal condition of the enzyme and has shown that certain colloidal substances appear to protect it, as does also sugar. The possibility is also present that inactive combinations of some sort are formed between the fermenting complex and the inhibiting agent, in the manner suggested to account for the inhibiting effect of excess of phosphate (p. 72). It seems most probable that the effect is a complex one, in which many factors participate.

NATURE OF THE ACCELERATION PRODUCED BY ARSENATE AND ARSENITE.

In explanation of the remarkable accelerating action of arsenates and arsenites two obvious possibilities present themselves. In the [p079] first place the arsenic compound may actually replace phosphate in the reaction characteristic of alcoholic fermentation, the resulting arsenic analogue of the hexosephosphate being so unstable that it undergoes immediate hydrolysis, and is therefore only present in extremely small concentration at any period of the fermentation and cannot be isolated. In the second place it is possible that the arsenic compound may accelerate the action of the hexosephosphatase of the juice, and thus by increasing the rate of circulation of the phosphate produce the permanent rise of rate. With this effect may possibly be associated a direct acceleration of the action of the fermenting complex.

The experimental decision between these alternative explanations is rendered possible by the use of a mixture of enzyme and co-enzyme free from phosphate and hexosephosphate. As has already been described (p. 55) a mixture of boiled yeast-juice, which has been treated with lead acetate, glucose or fructose, and washed zymin can be prepared which scarcely undergoes any fermentation unless phosphate be added. If now arsenates or arsenites can replace phosphate, they should be capable of setting up fermentation in such a mixture. Experiment shows that they do not possess this power. For fermentation to proceed phosphate must be present and it cannot be replaced either by arsenate or arsenite [Harden and Young, 1911, 1].

The effect of these salts on the action of the hexosephosphatase can also be ascertained by a modification of the foregoing experiment. If a hexosephosphate be made the sole source of phosphate in such a mixture as that described above, in which it must be remembered abundance of sugar is present, the rate at which fermentation can proceed will be controlled by the rate at which the hexosephosphate is decomposed with formation of phosphate. Experiment shows that in the presence of added arsenate or arsenite the rate of fermentation is largely increased, so that the effect of these salts must be to increase the rate of liberation of phosphate, or in other words, to accelerate the hydrolytic action of the hexosephosphatase.

This conclusion is even more strikingly confirmed by a comparison of the direct action of yeast-juice on hexosephosphate in presence and in absence of arsenate, as measured by the actual production of free phosphate. In a particular experiment this gave rise to 0·0707 gram of Mg{2}P{2}O{7} in the absence of arsenate and 0·6136 gram of Mg{2}P{2}O{7} in the presence of arsenate.

The results obtained with arsenite are throughout very similar to those given by arsenate, but are not quite so striking. It may therefore be affirmed with some confidence that the chief action of arsenates [p080] and arsenites in accelerating the rate of fermentation of sugars by yeast-juice or zymin, consists in an acceleration of the rate at which phosphate is produced from the hexosephosphate by the action of the hexosephosphatase.

It has further been found that arsenates, and to a less degree arsenites, also produce an acceleration of the rate of autofermentation of yeast-juice and of the rate at which glycogen is fermented. This turns out to be due in all probability to an increase in the activity of the glycogenase by the action of which the sugar is supplied which is the direct subject of fermentation. Thus in one case an initial rate of fermentation of glycogen of 1·9 c.c. per five minutes was increased by 0·05 molar arsenate to 9·7 and the amount of carbon dioxide evolved in two hours from 38 to 158 c.c. Even this enhanced production of glucose from glycogen, however, is not nearly sufficient for the complete utilisation of the phosphate also being liberated by the action on the hexosephosphatase, for the addition of an excess of sugar produces a much higher rate, in this case 36 c.c. per five minutes. The effect of arsenate on the rate of action of the glycogenase seems therefore to be much smaller than on that of the hexosephosphatase.

No other substances have yet been found which share these interesting properties with arsenates and arsenites, and no advance has been made towards an understanding of the mechanism of the accelerating action of these salts on the specific enzymes which are affected by them. [p081]