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 Mg2P2O7 in the absence of arsenate and 0·6136 gram of Mg2P2O7 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.
CHAPTER VI. CARBOXYLASE.
An observation of remarkable interest, which promises to throw light on several important features of the biochemistry of yeast, was made in 1911, and has since then formed the subject of detailed investigation by Neuberg and a number of co-workers.
It was found that yeast had the power of rapidly decomposing a large number of hydroxy-and keto-acids [Neuberg and Hildesheimer, [1911]; Neuberg and Tir, [1911]; see also Karczag, [1912, 1], [2]]. The most important among these are pyruvic acid, CH3·CO·COOH, and a considerable number of other aliphatic a-keto-acids which are decomposed with evolution of carbon dioxide and formation of the corresponding aldehyde:—
R·CO·COOH = R·CHO + CO2.
The reaction is produced by all races of brewer's yeast which have been tried, as well as by active yeast preparations and extracts and by wine yeasts [Neuberg and Karczag, [1911, 4]; Neuberg and Kerb, [1912, 2]]. The phenomenon can readily be exhibited as a lecture experiment by shaking up 2 g. of pressed yeast with 12 c.c. of 1 per cent. pyruvic acid, placing the mixture in a Schrötter's fermentation tube, closing the open limb by means of a rubber stopper carrying a long glass tube and plunging the whole in water of 38–40°. Comparison tubes of yeast and water and yeast and 1 per cent. glucose may be started at the same time, and it is then seen that glucose and pyruvic acid are fermented at approximately the same rate [Neuberg and Karczag, [1911, 1]]. If English top yeast be used it is well to take 0·5 per cent. pyruvic acid solution and to saturate the liquids with carbon dioxide before commencing the experiment. The production of acetaldehyde can be readily demonstrated by distilling the mixture at the close of fermentation and testing for the aldehyde either by Rimini's reaction (a blue coloration with diethylamine and sodium nitroprusside) or by means of p-nitrophenylhydrazine which precipitates the hydrazone, melting at 128·5° [Neuberg and Karczag, [1911, 2], [3]]. [p082]
As the result of quantitative experiments it has been shown that 80 per cent. of the theoretical amount of acetaldehyde can be recovered. The salts of the acids are also attacked, the carbonate of the metal, which may be strongly alkaline, being formed. Thus taking the case of pyruvic acid, the salts are decomposed according to the following equation:—
2 CH3·CO·COOK + H2O = 2 CH3·CHO + K2CO3 + CO2.
Under these conditions a considerable portion of the aldehyde undergoes condensation to aldol [Neuberg, [1912]]:—
2 CH3·CHO = CH3·CH(OH)·CH2·CHO.
This change appears to be due entirely to the alkali and not to an enzyme since the aldol obtained yields inactive β-hydroxybutyric acid on oxidation [Neuberg and Karczag, [1911, 3]; Neuberg, [1912]]. The various preparations derived from yeast which are capable of producing alcoholic fermentation also effect the decomposition of pyruvic acid in the same manner as living yeast. They are, however, more sensitive to the acidity of the pyruvic acid, and it is therefore advisable to employ a salt of the acid in presence of excess of a weak acid, such as boric or arsenious acid, which decomposes the carbonate formed but has no inhibiting action on the enzyme [Harden, [1913]; Neuberg and Rosenthal, [1913]].
As already mentioned the action is exerted on α-ketonic acids as a class and proceeds with great readiness with oxalacetic acid, COOH·CH2·CO·COOH, all the three forms of which are decomposed, with α-ketoglutaric acid, and with α-ketobutyric acid. Hydroxypyruvic acid CH2(OH)·CO·COOH is slowly decomposed yielding glycolaldehyde, CH2(OH)·CHO, and this condenses to a sugar [Neuberg and Kerb, [1912, 3]; [1913, 1]]. Positive results have also been obtained with diketobutyric, phenylpyruvic, p-hydroxyphenylpyruvic, phenylglyoxylic and acetonedicarboxylic acids [Neuberg and Karczag, [1911, 5]].