Buchner and Klatte [[1908]] made use of yeast-juice rendered free from co-enzyme by incubation with sugar solution to examine the nature of the agent by which the co-enzyme is destroyed. This agent is certainly an enzyme, since boiled yeast-juice can be preserved with unimpaired powers for a considerable length of time, and suspicion fell naturally, in the first instance, on the endotryptase of the yeast cell. Direct experiment showed, however, that yeast-juice, which, when fresh, rapidly destroyed the co-enzyme of boiled juice, lost this power on preservation, but retained its proteoclastic properties without diminution, so that the tryptic enzyme could not be the one concerned. The direct action of commercial trypsin on boiled yeast-juice also yielded [p068] a negative result, although this cannot strictly be regarded as an indication of the effect of the specific proteoclastic enzymes of yeast-juice. On the other hand, it was found that when boiled juice was treated for some time with an emulsion containing the lipase of castor oil seeds, the co-enzyme was completely destroyed. This is a result of great importance, inasmuch as it probably indicates that the co-enzyme is chemically allied to the class of substances hydrolysable by lipase, i.e. to the fats and other esters.
Further, observations by Buchner and Haehn [[1909]] have shown that digestion with potassium carbonate solution containing 2·5 grams per 100 c.c. also brings about the destruction of the co-enzyme, and that this is also slowly accomplished by the repeated boiling of the juice. The co-enzyme is also destroyed both by acid and alkaline hydrolysis, and when the solution is evaporated to dryness and the residue ignited.
Beyond this general indication nothing is known of the chemical nature of the co-enzyme. The intimate relation of phosphoric acid to the process of fermentation renders it not impossible that the co-enzyme may contain this group, but there is no definite evidence for such a belief. Purely negative results have been obtained with all the substances of known composition which have yet been tested, among these being soluble phosphates, hexosephosphates and a number of oxidisable and reducible substances, such as quinol, p-phenylenediamine, methylene blue, peptone beef broth, etc. (Harden and Young; Harden and Norris [[1914]]; see also Euler and Bäckström [[1912]]), glycero-phosphates (Buchner and Klatte).
The precise function of the co-enzyme is even more obscure than its chemical nature. The system of reacting substances consisting of fermentable material, enzyme and co-enzyme, bears, however, an obvious superficial resemblance to many of the systems required for the accomplishment of chemical changes in the animal or vegetable organism. Such a triad of substances is, for example, requisite for the process by which the red blood corpuscles of an animal are broken up by the serum of a different animal into the blood of which the red corpuscles of the first animal have been injected. This effect is only produced when two substances are present, the amboceptor or immune body and the complement. The analogy may be carried to a further stage since the amboceptor is, like the co-enzyme, more thermostable than the complement, which therefore corresponds with the enzyme. Immune serum can, in fact, be freed from complement by being heated at 57–60° for half an hour, whereas the amboceptor is unaffected by this treatment. On the other hand, the complement and amboceptor do not [p069] appear to act like enzymes but rather like ordinary chemical reagents, remaining in combination even after the blood corpuscle has been broken up, whereas the enzyme and co-enzyme of yeast-juice are again liberated when the reaction between sugar and phosphate has been completed.
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]