2 C6H12O6 + 2 R2HPO4 = 2 CO2 + 2 C2H6O + 2 H2O + C6H10O4(PO4R2)2,
render it in the highest degree probable that two molecules of the sugar are concerned. The most reasonable interpretation of this equation appears to be that in the presence of phosphate and of the complicated machinery of enzyme and co-enzyme two molecules of the hexose, or possibly of the enolic form, are each decomposed primarily into two groups.
Of the four groups thus produced, two go to form alcohol and carbon dioxide and the other two are synthesised to a new chain of six carbon atoms, which forms the carbohydrate residue of the hexosephosphate. The introduction of the phosphoric acid groups may possibly occur before the rupture of the original molecules, and may even be the determining factor of this rupture, or again this introduction may take place during or after the formation of the new carbon [p118] chain. Sufficient information is not yet available for the exact formulation of a scheme for this reaction. Such a scheme, it may be noted, would not necessarily be inconsistent with the views of Wohl and of Buchner as to the way in which the carbon chain of a hexose is broken in the process of fermentation, but would interpret differently the subsequent changes which are undergone by the simpler groups which are the result of this rupture. The reaction might thus proceed without the formation of definite intermediate products, whilst opportunity would be afforded for the production of a small quantity of by-products such as formaldehyde, glycerol, lactic acid, acetic acid, etc., by secondary reactions.
A symmetrical scheme can readily be constructed for such a change, but much further information is required before any decisive conclusion can be drawn as to the precise course of the reaction which actually occurs in alcoholic fermentation.
CHAPTER IX. THE MECHANISM OF FERMENTATION.
The analysis of the process of alcoholic fermentation by yeast-juice and other preparations from yeast which has been carried out in the preceding chapters has shown that the phenomenon is one of a very complex character. The principal substances directly concerned in the change appear to be the enzyme and co-enzyme of the juice, a second enzyme, hexosephosphatase, and, in addition, sugar, phosphate, and the hexosephosphate formed from these. During autofermentation two other factors are involved, the complex carbohydrates of the juice, including glycogen and dextrins, and the diastatic ferment by which these are converted into fermentable sugars. It is also possible that the supply of free phosphate is partially provided by the action of proteoclastic ferments on phosphoproteins. Under special circumstances the rate at which fermentation proceeds may be controlled by the available amount of any one of these numerous substances.
When the juice from well-washed yeast is incubated, the phenomenon of autofermentation is observed. The juice contains an abundant supply of enzyme, co-enzyme, and phosphate or hexosephosphate, and in this case the controlling factor is usually the supply of sugar, which is conditioned by the concentration of the diastatic enzyme or of the complex carbohydrates as the case may be. When this is the case the measured rate of fermentation is the rate at which sugar is being produced in the juice, this being the slowest of the various reactions which are proceeding under these circumstances. If sugar be now added, an entirely different state of affairs is set up. As soon as any accumulated phosphate has been converted into hexosephosphate, the normal rate of fermentation which is usually higher than that of autofermentation is attained, and, provided that excess of sugar be present, fermentation continues for a considerable period at a slowly diminishing rate and finally ceases. During the first part of this fermentation the rate is controlled entirely by the supply of free phosphate, and this depends mainly on the concentration of the hexosephosphatase and of the hexosephosphate, and only in a secondary degree on the decomposition [p120] of other phosphorus compounds by other enzymes and on the concentration of the sugar. The amount of hexosephosphate in yeast-juice is usually such that an increase in its concentration does not greatly affect the rate of fermentation, and hence the measured rate during this period represents the rate at which hexosephosphate is being decomposed, and this in its turn depends on the concentration of hexosephosphatase, which is therefore the controlling factor. As fermentation proceeds, the concentration of both enzyme and co-enzyme steadily diminishes, as already explained, probably owing to the action of other enzymes, so that at an advanced stage of the fermentation, the controlling factor may be the concentration of either of these, or the product of the two concentrations (see p. [122]). The hexosephosphatase appears invariably to outlast the enzyme and co-enzyme. The condition at any moment could be determined experimentally if it were possible to add enzyme, co-enzyme and hexosephosphatase at will and so ascertain which of these produced an acceleration of the rate.
Unfortunately this can at present be only very imperfectly accomplished, owing to the impossibility of separating these substances from each other and from accompanying matter which interferes with the interpretation of the result.
A third condition can also be established by adding to the fermenting mixture of the juice and sugar a solution of phosphate. The supply of phosphate is now almost independent of the action of the hexosephosphatase, and the measured rate represents the rate at which reaction (1), p. [51], can occur between sugar and phosphate in the presence of the fermenting complex consisting of enzyme and co-enzyme. This change is controlled, so long as sugar and phosphate are present in the proper amounts, by the concentration of the fermenting complex or possibly of either the enzyme or the co-enzyme. If only a single addition of a small quantity of phosphate be made, the rate falls as soon as the whole of this has been converted into hexosephosphate and the reaction then passes into the stage just considered, in which the rate is controlled by the production of free phosphate.