The rapid diminution in the amount of free phosphate during stage 1 corresponds with the occurrence of reaction (1). During the whole period of fermentation the enzymic hydrolysis of the hexosephosphate is proceeding according to equation (2). Up to the end of stage 2 the phosphate thus produced enters into reaction, according to equation (1), with the sugar which is present in excess and is thus reconverted into hexosephosphate, so that as long as alcoholic fermentation is proceeding freely, no accumulation of free phosphate can occur.
As soon as alcoholic fermentation ceases, however, it is no longer possible for the phosphate to pass back into hexosephosphate, and hence it accumulates in the free state.
A similar hydrolysis of hexosephosphate and accumulation of phosphate occur when a solution of hexosephosphate is treated with yeast-juice which has been deprived of the power of fermentation by dialysis, or with zymin freed from co-enzyme by washing (p. [63]).
The actual rate of fermentation observed in any particular case in presence of excess of sugar, enzyme, and co-enzyme must on this view depend on the supply of phosphate which is available.
In presence of an adequate amount of phosphate, as well as of sugar, the highest rate attained represents the maximum velocity at which reaction (1) can proceed in that sample of yeast-juice or zymin, and this high rate is characteristic of the initial period of accelerated fermentation which follows the addition of a suitable quantity of phosphate. By the simple expedient of renewing the supply of phosphate as rapidly as it is converted into hexosephosphate, this high rate can be maintained for a considerable time [Harden and Young, [1908, 1]]. In this way, for example, an average rate of evolution of carbon dioxide of 15 c.c. in five minutes was maintained for an hour and a [p054] quarter, whereas the normal rate in the absence of added phosphate was 3 c.c.
As soon as all the free phosphate has entered into the reaction, however, the supply of phosphate depends in the main on the rate at which the resulting hexosephosphate is decomposed, and the rate of fermentation now attained is conditioned by the rate at which reaction (2) proceeds, and this evidently depends on the existing concentration of the hydrolytic enzyme, which may be provisionally termed hexosephosphatase.
The rates attained during the initial period of rapid fermentation and the subsequent period of slow fermentation are thus seen to represent the velocities of two entirely different chemical reactions.
These considerations also explain why it is the extra carbon dioxide evolved during the initial period, and not the total carbon dioxide, which is equivalent to the added phosphate. As the production of phosphate is proceeding throughout the whole period at a rate which is equivalent to the normal rate of fermentation, it is obviously necessary to deduct the corresponding amount of carbon dioxide from the total evolved in order to ascertain the amount equivalent to the added phosphate.
An explanation is also afforded of the fact that a considerable increase in the concentration of hexosephosphate does not materially increase the normal rate of fermentation. This is probably due to the circumstance that, in accordance with the general behaviour of enzymes in presence of excess of the fermentable substance, the hexosephosphatase hydrolyses approximately equal amounts of hexosephosphate in equal times whatever the concentration of the latter may be, above a certain limit.
According to the experiments of Euler and Johansson [[1913]] the hydrolytic activity of the hexosephosphatase is greatly diminished by the presence of toluene.