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, CH{3}·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 + CO{2}.
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:--
2CH{3}·CO·COOK + H{2}O = 2CH{3}·CHO + K{2}CO{3} + CO{2}.
Under these conditions a considerable portion of the aldehyde
undergoes condensation to aldol [Neuberg, 1912]:--
2CH{3}·CHO = CH{3}·CH(OH)·CH{2}·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·CH{2}·CO·COOH, all the three forms of which are decomposed, with
α-ketoglutaric acid, and with α-ketobutyric acid. Hydroxypyruvic
acid CH{2}(OH)·CO·COOH is slowly decomposed yielding glycolaldehyde,
CH{2}(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].
RELATION OF CARBOXYLASE TO ALCOHOLIC FERMENTATION.
With regard to the relation of carboxylase to the process of alcoholic
fermentation, nothing definite is yet known. As Neuberg points out
[see Neuberg and Kerb, 1913, 1] the universal presence of the enzyme
in yeasts capable of producing alcoholic fermentation, and the
extreme readiness with which the fermentation of pyruvic acid takes
place create a [p083] strong presumption that the decomposition of
pyruvic acid actually forms a stage in the process of the alcoholic
fermentation of the sugars. On the other hand Ehrlich's alcoholic
fermentation of the amino-acids (p. 87) provides another function
for carboxylase--that of decomposing the α-ketonic acids produced
by the deaminisation of the amino-acids. It must be remembered in
this connection that carboxylase is not specific in its action, but
catalyses the decomposition not only of pyruvic acid but also of
a large number of other α-ketonic acids, including many of those
which correspond to the amino-acids of proteins and are doubtless
formed in the characteristic decomposition of these amino-acids by
yeast. Carboxylase undoubtedly effects one stage in the production of
alcohols from amino-acids, whether it is also the agent by which one
stage in the alcoholic fermentation of sugar is brought about still
remains to be proved.
A comparison of the conditions of action of carboxylase and zymase
has revealed several interesting points of difference. Neuberg and
Rosenthal [1913] have observed that the fermentation of pyruvic
acid by maceration extract commences much more rapidly than that
of glucose and interpret this to mean that in the fermentation of
glucose a long preliminary process occurs before sufficient pyruvic
acid has been produced to yield a perceptible amount of carbon
dioxide. The long delay (3 hours) which they sometimes observed in
the action of maceration juice on glucose is however by no means
invariable (see p. 46), but in any case indicates that the sugar
fermentation can be affected by conditions which are without influence
on the pyruvic fermentation. A similar conclusion is to be drawn
from the fact that the pyruvic acid fermentation is less affected
by antiseptics than the glucose fermentation [Neuberg and Karczag,
1911, 4; Neuberg and Rosenthal, 1913], chloroform sufficient to stop
the glucose fermentation brought about by yeast or dried yeast being
usually without effect on the fermentation of the pyruvates either
alone or in presence of boric or arsenious acid. A more important
difference is that carboxylase decomposes pyruvic acid in the absence
of the co-enzyme which is necessary for the fermentation of glucose
[Harden, 1913; Neuberg and Rosenthal, 1913]. This can be demonstrated
experimentally by washing dried yeast or zymin with water (see p.
63) until it is no longer capable of decomposing glucose (Harden),
or by allowing maceration extract to autolyse or dialyse until it is
free from co-enzyme (Neuberg and Rosenthal). The zymase of maceration
extract is moreover inactivated in 10 minutes at 50-51°, whereas after
this treatment the carboxylase is still active. [p084]
The only conclusion that can be legitimately drawn from these highly
interesting facts is that if the decomposition of pyruvic acid
actually be a stage in the alcoholic fermentation of glucose the
soluble co-enzyme is required for some change precedent to this,
so that in its absence the production of pyruvic acid cannot be
effected. [p085]