BIOCONVERSION OF D-GLUCOSE IN HEAVY WATER
2059
methylene and methyl groups grow, this growth being
noticeably faster in the latter, therefore differences in the
relative isotope content in it and in the methylene group
are gradually leveled, decreasing from 2.23 in 5% heavy
water to 1.26 in concentrated heavy water (99%). The
deuterium distribution in ethanol for each experiment
was compared with the isotopomeric composition of
water in use, as calculated by the binomial distribution
equation [3].
Thus, it has been found that in the case of the
D-glucose bioconversion in deuterowater solutions the
observed leveling of differences in relative deuterium
contents in methyl and methylene groups of ethanol
coincides with the appearance of significant amounts of
the d2 isotopomeric form in water and manifests itself the
more strongly, the higher is its content, reaching a maxi-
mal value in concentrated heavy water. Moreover, it has
been found that the distribution curves of heavy water
isotopomers d0, d1, and d2 are reproduced practically
completely also for ethanol, d0, d1, and d2–5, respectively
(Fig. 1). A negligible deviation is connected with the
fact that, as a heavy water concentration increases, the
fraction of the alcohol molecules [1,2-2H2]-СН3СН2ОН,
which are the d1 isotopomer by fragments and d2, by the
gross-content, grows. As they are referred completely to
the isotope group d2–5, the content of the first isotopomers
is lower and of the second higher compared to the cor-
responding isotopomers in water. It is evident that the
observed analogy in the distribution of isotopomers in
water and in alcohol formed in it is connected with a spe-
cial feature of exchange reactions inside the substrate-
enzyme complex.
Fig. 1. Dependence of the contents A (%) of isotopomeric water
2
forms and ethanol on the concentration c (%) of Н2О in the
2
medium. (1) HOH, (2) HO2H, (3) HO2H, (4) d0-EtOH, (5)
d1-EtOH, (6) d2-d5-EtOH.
As deuterium is present in isotope-dilute water in the form
of НО2Н molecules, it is obvious that the intermolecular
interchanging in the hexose stage occurs preferentially
due to the protium atom because of the isotopic effect.
Thus, under these conditions the deuterium atom of wa-
ter participates in the formation of the ethanol molecule
mainly in the second stage as a result of enolpyruvic acid
conversions. This conclusion is supported by the fragment
ratio of deuterium in the methylene and methyl groups,
(2.23–2.07) : 1, respectively, that practically corresponds
to the number of vacant places at the molecule kitting-up
from enolpyruvic acid up to ethanol: two for the methy-
lene group and one for the methyl group.
The general view of the 13С NMR spectrum of the
methyl group of ethanol obtained by the glucose frag-
mentation in 99% heavy water is presented in Fig. 2.
When the heavy water concentration is increased the
isotopic equilibrium in it is displaced aside the d2 isoto-
pomers. In their presence the possibility of the isotope
alternation in the first stage is limited, therefore deuterium
atoms are preferentially involved in the intermolecular
exchange of hexoses, thereby providing its additional
entering in the methyl group of ethanol proportionally to
the content of these molecules in water. Thus, the total
content of deuterium in the methyl group starts to grow
noticeably faster than in the methylene group.
The interpretation of the results of the present work was
fulfilled within the limits of the Embden–Mayerhoff–
Parnas standard scheme, according to which hydrogen
atoms of water inside the substrate-enzyme complex
can be brought into fructose as a result of the isotope
substitution at the С2 carbon atom [4] or in final stages of
the conversion of enolpyruvic acid to ethanol (reactions
a, b, and c) [5]:
RUSSIAN JOURNAL OF APPLIED CHEMISTRY Vol. 83 No. 11 2010