From the
For a given biochemical transformation, such as the fermentation
reaction, the redistribution coefficients, which relate the natural
site-specific isotope contents in end products to those of their
precursors, are a source of mechanistic information. These coefficients
characterize the traceability of specific hydrogens in the products
(ethanol and water) to their parent hydrogens in the starting materials
(glucose and water). In conditions of complete transformation, they
also enable intermolecular exchanges with the water medium to be
estimated. Thus it is directly confirmed that hydrogens 1, 2, 6, and 6`
of glucose are strongly connected to the methyl site I of ethanol
obtained by fermentation by Saccharomyces cerevisiae. However,
whereas hydrogens 6 and 6` are transferred to a great extent, transfer
is only partial for hydrogen 2, and it is even less for hydrogen 1.
Because the two moieties of glucose corresponding to carbons
1-2-3 and 4-5-6 are scrambled by the aldolase and
triose-phosphate isomerase reactions, additional exchange of hydrogens
at positions 1 and 2 must have occurred before these steps. The value
of the coefficient that relates site 2 of glucose to site I of ethanol
in particular can be used to quantify the contribution of
intermolecular exchange occurring in the course of the transfer from
site 2 of glucose 6-phosphate to site 1 of fructose 6-phosphate
mediated by phosphoglucoisomerase. The average hydrogen isotope effects
associated with the transfer of hydrogen from the water pool to the
methyl or methylene site of ethanol are estimated. In contrast to
conventional experiments carried out in strongly deuterium-enriched
media where metabolic switching may occur, the NMR investigation of
site-specific natural isotope fractionation, which operates at tracer
isotopic abundance, faithfully describes the unperturbed metabolic
pathways.
By studying site-specific natural isotope fractionation by
nuclear magnetic resonance(1) , end products can be powerful
sources of information on the chemical or biochemical mechanisms that
have governed their elaboration(2) . In particular, the natural
abundance of isotopic distribution in fermentation ethanol has been
shown to reflect the physiological and environmental conditions of the
photosynthesis of its carbohydrate precursors(3) . This aptitude
of the ethanol probe to infer properties of sugars is based on the
traceability of the various isotopomeric species determined in the end
fermentation medium to those contained in the starting sugar and
water(4) .
Much interest has been devoted to the study of
glucose fermentation and most steps of the glycolysis have been
investigated independently using the appropriate
enzymes(5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16) .
The behavior of the carbon atoms is simple because C-1 and C-6 of
glucose become the methyl carbons of two ethanol molecules, whereas C-2
and C-5 give the methylenic carbons. However, the problem of the
hydrogen relationships is much more complex, because both
intramolecular and intermolecular hydrogen transfers may occur, and the
isotope fractionation effects depend on the relative rates and
reversibility of several reaction steps. Most investigations of the
glycolytic pathways have involved labeling experiments using either
tritium or deuterium, and a number of kinetic isotope effects have been
estimated for isolated enzymatic steps (9-14). However, the final
isotope fractionations resulting from a whole fermentation experiment
are expected to depend not only on the overall yield of the
transformation but also on the extent of hydrogen exchange with the
water medium and on the associated isotope effects.
Mechanistic
information on hydrogen transfer during fermentation by Saccharomyces cerevisiae has been obtained by comparing mirror
experiments in which fully protonated or deuterated glucose or mannose
are fermented in heavy or light water, respectively(5) .
However, these results are not necessarily transferable to fermentation
in natural isotopic conditions because, in fully deuterated media, the
existence of a large isotope effect on one branch of a metabolic
pathway may possibly modify the isotopic flux through a competing
branch with a different isotopic preference. Such a phenomenon is known
to alter, for instance, the proportion of intra- and intermolecular
transfers in the interconversion of D-glucose 6-phosphate and D-fructose 6-phosphate catalyzed by yeast
phosphoglucoisomerase(10, 11) .
From a mechanistic
point of view, the investigation of site-specific natural isotope
fractionation by NMR at natural, or close to natural, deuterium
abundance offer a means of simultaneously comparing the behavior of the
different monodeuterated isotopomers in media that ensure competitive
kinetic conditions. It is therefore possible to develop a strategy that
will enable a complex mechanistic pathway to be characterized
isotopically by investigating only the starting and end products of the
transformation. To this aim, model experiments have been carried out to
elucidate the fate of the individual carbohydrate atoms and to estimate
both the extent of hydrogen exchange with the water medium and the
resulting influence of the kinetic and thermodynamic isotope effects
affecting different steps of the biochemical transformation.
The commercial glucoses (Prolabo) were obtained from maize
and potato.
Glucose samples slightly enriched at
specific positions were prepared by adding small quantities of labeled
glucose (6-24 mg) to 60 g of the reference corn glucose dissolved
in water. Increases in the hydrogen isotope ratio of a given site (i) of glucose by values
The composition of the fermentation
medium was 2 g liter
On-line formulae not verified for accuracy
The overall hydrogen isotope ratio of glucose and that of water
were measured by means of a VG SIRA 9 isotope ratio mass spectrometer.
In the case of glucose, the deuterium content of the carbon-bound
positions was obtained by synthetizing the pentanitrate derivative
(19). The reduction of water into hydrogen gas in the spectrometer was
catalyzed by zinc (BDH AnalaR Zinc coarse powder) at 550 °C. The
standard deviation of the deuterium analyses was 0.5 ppm for glucose
and 0.2 ppm for water.
The site-specific natural isotope fractionation was studied
by NMR(2) . For a given molecular site (i) the hydrogen
isotope ratio at tracer conditions of the deuterium isotope is defined
as,
On-line formulae not verified for accuracy
where N
On-line formulae not verified for accuracy
When I
The isotope
ratios of the methyl and methylene positions of ethanol can be
accurately determined on samples obtained from distillation of the
fermentation medium, provided that the results are corrected for
liquid-vapor fractionation effects associated with incomplete yield
(17).
On-line formulae not verified for accuracy
In a fermentation experiment, j = 1 to n denotes a given site of the fermentation products, ethanol and
water, and i = 1 to m specifies the considered
site in the starting materials, glucose and water. In matrix notation
the set of n linear equations (4) is represented by,
On-line formulae not verified for accuracy where D
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy N is the number of experiments, S(D/H) is the standard deviation on the
estimation of the isotope ratio, and F is the Fischer test at
a 95% confidence level.
Taking into account the high level of sugar
dilution, the a
The redistribution coefficient a
On-line formulae not verified for accuracy The parameters k
Complex
scrambling of hydrogens may occur at several reversible steps of the
transformation (Fig. 2), and sites j = I and II of ethanol and W
Intermolecular exchange with the
water medium may affect either directly or indirectly several hydrogens
ending at ethanol in the course of the glycolytic pathway (Fig. 2). In this respect fermentation of glucose in fully
deuterated water has been shown (5) to introduce a large amount
of deuterium into ethanol because the following proportions of
deuterated fragments have been determined: 26.6% CH
In
the diluted conditions of the fermentation experiments, the a
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy This result corroborates in particular the observation of deuterium
enrichment at positions 6 and 6` of glucose from C
Enantiomeric resolution of the monodeuterated
methylenic isotopomers provides further mechanistic information because
the parameter k
It
should be emphasized that the present results, obtained at or close to
natural isotopic abundance, are safely interpretable in terms of
hydrogen connectivity existing in unperturbed biosyntheses. Different
mechanistic behavior may occur in experiments involving strong isotopic
labeling. In this respect, the role of intermolecular exchange in the
glycolytic pathway seems to be significantly enhanced in fermentation
reactions performed in deuterated water, for instance(5) ,
because, as discussed above, up to 70% of the methyl hydrogens of
ethanol are then found to originate from water, whereas this percentage
is about 50% in natural media. The present approach also benefits from
the possibility of simultaneously determining the fate of several
isotopomeric species.
In addition, the described method, which
elucidates overall mechanistic relationships between specific molecular
positions of starting and end products, provides general quantitative
criteria for ultimately estimating parameters of disappeared precursors
such as sugars from natural abundance isotopic parameters easily
measured in end products such as ethanol.
The starting
natural glucose used in these experiments has a maize origin. Its
overall carbon isotope ratio has a value,
Fermentation has first been conducted with
natural glucose characterized by an overall isotope content of the
carbon-bound hydrogen positions (D/H)
(D/H)
We are grateful to M. Trierweiler and F. Mabon for
collaboration in the determinations of site-specific natural isotope
fractionation by NMR, to Prof. N. Naulet for help in the mass
spectrometry determinations, and to Prof. G. J. Martin for fruitful
discussions.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
Materials and Fermentation
-D-Glucose-1-d,
-D-glucose-2-d, and
-D-glucose-6,6`-d
were purchased
from Aldrich. Their isotopomeric purity was checked by
H
NMR. It was higher than 98%.
(D/H)
= 50, 100,
150, and 400 ppm were thus produced. The fermentation experiments were
conducted in Nantes tap water characterized by a (D/H)
ratio
of 150 ppm. Aqueous media slightly enriched in deuterium (180-211
ppm) were prepared by adding small amounts of deuterated water to
Nantes tap water. Experiments were also performed with strongly
depleted water (Eurisotop) characterized by a (D/H)
value
close to 0 ppm. In this case, however, the isotope ratio of the
starting medium reached 20.5 ppm as a result of exchange with the
hydroxyl sites of glucose.
NH
Cl, 3 g
liter
KH
PO
, 3 g
liter
NaCl, 60 mg liter
MgCl
, 6 g liter
Na
HPO
, 120 mg liter
Na
SO
, and 171.4 g liter
glucose. The yeast S. cerevisiae was used in all
experiments. The fermentation temperature was 25 ± 1 °C, the
duration was 7 days, and the yield in ethanol was higher than 95%.
Ethanol was extracted by distillation in controlled
conditions(17) .
Spectrometric Techniques
Mass Spectrometry
The overall carbon-13 contents
of the glucose and ethanol samples were measured by isotope ratio mass
spectrometry using a Finnigan Delta E spectrometer coupled with a Carlo
Erba NA 1500 elemental analyzer. The carbon-13 isotope content of a
compound (P) is expressed in percent/thousand on the relative
-scale (Equation 1), which refers the isotope ratio of the sample
to that of an international standard Pee Dee Belemnite (PDB).
The precision of the determination is usually better than 0.3
%(18) .
Nuclear Magnetic Resonance
The deuterium NMR
spectra were recorded at 61.4 MHz with a Bruker AM400 spectrometer
equipped with a fluorine locking device. The following experimental
conditions were adopted in most determinations of site-specific natural
isotope fractionation by NMR: broad band proton decoupling, a frequency
window of 1200 Hz, memory size of 16 K, and exponential multiplication
corresponding to a line broadening of 1 Hz. Usually 200-800 scans
were accumulated. The quantitative evaluation of the monodeuterated
isotopomers was performed by using a new curve-fitting algorithm based
on a complex least squares treatment (20) of the H
NMR signal. This analysis involves automatic integrated management of
all the experimental parameters, including the phases of the individual
resonances. The repeatability of the NMR determinations was better than
0.2 ppm.
Methods
is the number of
isotopomers monodeuterated at site i, N
is the number of the fully protonated species, and P
is the stoichiometric number of
hydrogens at position i. This parameter can be determined by
referring the deuterium content in site i to that of a
reference substance (tetramethylurea) with a known isotope ratio (D/H = 136 ppm) and added to the sample in an
accurately measured quantity. In the case of ethanol, a relative
parameter (R) directly accessible from the intensities of the
H NMR signals has also been defined. This parameter would
represent the deuterium content in the methylenic site (II) in
a situation where the methyl site (I) is arbitrarily given the
statistical value 3.
and I
are taken as signal heights instead of signal areas, a
higher accuracy is reached, but the R parameter has only an
empirical status. R is equal to
2(D/H)
/(D/H)
when I
and I
are expressed in terms of signal areas.
Site-specific Fractionation Factors of the
Glycolysis
Several series of fermentation experiments were
performed on reference glucose samples, characterized by their natural
overall carbon and hydrogen isotope ratios, and, on the same samples,
slightly enriched in a given molecular position by the addition of a
very small quantity of specifically labeled glucose (Tables I, II, and
III). Deuterium enrichments, ranging from 0 to 400 ppm, at positions 1,
2, 6, and 6` of glucose have been considered. Fermentations were also
conducted either in Nantes tap water or in water with slightly
increased deuterium contents that still fulfills tracer conditions. The
tight connection between the methylene site of ethanol and water is
illustrated in Fig. 1A, which shows that fermentation in
deuterium-depleted water leads to ethanol with a strong reduction of
the (D/H) value. Fermentation
by S. cerevisiae was performed in standardized conditions
ensuring a high level of transformation of glucose into ethanol
(
95%). Consequently, the experimental results accommodate only
restricted repercussions (<5%) of isotopic spread through other
metabolites.
Figure 1:
H NMR spectra of ethanol
obtained from fermentation of glucose resulting from hydrolysis of
maize starch. A, glucose has been fermented in water strongly
depleted in deuterium (
0 ppm). Due to exchange with the hydroxyl
sites of glucose in particular, the starting aqueous medium was
characterized by an isotope ratio of (D/H)
= 20.5 ppm. The measured isotope content in the methylene site
of ethanol, (D/H), is only 22.2 ppm in these
conditions. B, the starting glucose has been slightly enriched
at positions 6 and 6` by adding a small amount of glucose specifically
labeled at these positions (
(D/H)
= 150 ppm). Fermentation was carried out in Nantes tap
water
((D/H)
= 150 ppm). Due to isotope effect on the deuterium chemical
shift, the isotopomers of ethanol bideuterated on the methyl site (I) are observed separately (see expanded spectrum). In both
cases tetramethylurea (TMU) with a known hydrogen isotope
ratio (136 ppm) was used as a reference for determining the
site-specific deuterium contents.
When the deuterium content at position 6 of glucose is
slightly varied (0-400 ppm) by adding small amounts of glucose
6,6`-d, ethanol isotopomers bideuterated at the
methyl position are separately observed on the
H NMR
spectrum due to a chemical shift isotope effect of about 0.02 ppm (Fig. 1B). Quantitative analysis of the signals as a
function of the increase in the deuterium content of glucose at sites 6
and 6` proves that the amount of monodeuterated isotopomers
CH
DCH
OH exhibits only slight variation, whereas
the amount of the CHD
CH
OH species regularly
increases.
Isotopic Relationships between End and Starting
Products
At natural or close to natural abundance of deuterium,
the site-specific isotope ratios of the end products (Q) can
be considered as linear combinations of the isotope parameters of the
starting materials (S)(4) . and D
are the column vectors of the deuterium contents in the m starting and n end molecular positions and A is the (m, n) matrix of the redistribution coefficients, a
, which summarizes the isotopic
transfers occurring in the global fermentation reaction (Fig. 2).
Figure 2:
Glycolytic pathway. The fate of the
hydrogens is illustrated in the case of hydrogen H-1
(H), H-2 (
H), and H-6 pro-R (
H) of glucose. Steps where hydrogens from
water are introduced and steps with possible intermolecular exchanges
are mentioned. Hydrogen transfer to and from NADH are also indicated.
The numbering of the carbon atoms follows that of glucose. G6P, glucose 6-phosphate; F6P, fructose 6-phosphate; F1,6P, fructose 1,6-diphosphate; DHAP, dihydroxy
acetone phosphate; G3P, glyceraldehyde 3-phosphate; 1,3 di
PG, 1,3-diphosphoglycerate; 2PG, 2-phosphoglycerate; PEPy, phosphoenol pyruvate; Py,
pyruvate.
The monovariable experiments described in Tables I, II, and III
enable the a coefficients, which relate
sites j = I and II of ethanol to
sites i = 1, 2, 6, and 6` of maize glucose and to starting water W
to be separately determined.
parameter relating the
isotope ratio of end water, (D/H)
, to
that of any site i of glucose is not accessible with
sufficient accuracy. It can be predicted that, in the hypothesis of
complete transfer of hydrogen i of glucose to water for
instance, an increase of (D/H)
by 400 ppm would increase the value of (D/H)
by
only 2.2 ppm.
can be further interpreted by considering separately the
enantiomeric monodeuterated isotopomers of ethanol:
and k
connect the methylenic sites pro-S and pro-R of ethanol, respectively, to the starting water
medium. These parameters, the average value of which is a
0.73 (Equation 10), can be
selectively determined by enantiomeric resolution of the monodeuterated
isotopomers in the
H NMR spectrum of ethanol. An
appropriate diastereotopy of the methylenic hydrogens can be achieved
by reacting ethanol with L(-)-mandelic acid(21) .
Thus the enantiomeric ratio II-R/II-S = 1.37 was determined from the spectrum of mandelate
synthetized from ethanol obtained by fermentation of maize glucose in
Nantes tap water.
Estimation of Intermolecular Exchanges
The
isotopic redistribution parameters (a)
provide information on the mechanistic paths followed by hydrogens in
the course of multiple reaction steps. Because the overall
transformation of glucose is nearly complete and the yield in ethanol
is very high, kinetic isotope effects associated with the individual
enzymatic steps are not expected to result in final significant isotope
fractionation. The observed fractionation effects are therefore mainly
due to exchange with the water medium. From a general point of view,
the redistribution coefficients (a
) are
representative of the origin of the hydrogens on one hand and of the
extent of intermolecular transfer from water on the other hand.
Because, at low sugar concentrations, water may be approximately
considered as providing an open pool of hydrogens, the a
parameters involving water provide
information on the magnitude of kinetic or thermodynamic isotope
effects accompanying the intermolecular hydrogen transfers.
of
the end water medium may be more or less strongly connected to sites i = 1 to 6,6` of glucose and W
of the starting water. If a given hydrogen atom of glucose (i) is protected from any kind of exchange with the medium in
the course of the reaction pathway and ends at position j of
the products, the value of the a
factor
can be predicted. Because one glucose molecule produces two ethanol
molecules, the theoretical values are a
= for the methyl site and a
= for the methylene site in conditions of complete
transformation. Conversely, experimental values of a
equal to these theoretical values
would prove that one of the hydrogens of site j originates
exclusively from the considered site i of glucose and that all
proton transfers possibly undergone by i have a purely
intramolecular character or, at least, are preserved from any exchange
with the aqueous medium. Ignoring the effect of incomplete yield, the
deviations observed with respect to the predicted values are therefore
indicative of hydrogen exchange.
D, 33.7%
CHD
, and 37.9% CD
on one hand and 11.2% CHD and
88.8% CD
on the other hand. 69% of the hydrogen atoms found
in the methyl sites therefore originate from water, and this proportion
reaches 94% for the methylene site. This behavior does not necessarily
reflect the hydrogen transfers occurring in natu-ral conditions. At
tracer concentrations of deuterium, the individual fate of the glucose
hydrogens can be inferred from Equations 6-11.
Hydrogen Connections and Exchanges Associated with the
Methyl Site of Ethanol
A tight connection between positions i = 1 and 6,6` of glucose and the methyl site j = I of ethanol is proved by the high values of the
redistribution coefficients a (Equation 6) and a
(Equation 8). The a
value in particular is not far from the theoretical value 2/6
= 0.33 corresponding to a total transfer of hydrogens 6 and 6`
of glucose to the methyl group of pyruvate and subsequently to ethanol.
This behavior excludes the hypothesis of large exchanges resulting
either from reversibility at the step of the pyruvate kinase reaction
or from pyruvate enolization, because significant exchange with water
would then decrease the sensitivity of (D/H)
to (D/H)
to the benefit of that to (D/H)
.
Similarly, the ethanol site I is strongly connected to site 1 of
glucose because the a
coefficient reaches a value of 0.12 (Equation 6) compared with
the theoretical one, . If we admit, as substantiated below, that the
triose-phosphate interconversion mediated by triose-phosphate isomerase
is relatively fast(5, 22) , scrambling of the two
moieties issued from carbons 1-2-3 and 4-5-6 of glucose
confers subsequent identical fates to the hydrogens pertaining to
carbons 1 and 6 on one hand and 2 and 4 on the other hand. Any
difference in the redistribution coefficients a
and a
on one hand and a
and a
on the other hand is
therefore expected to result from reaction steps prior to aldolization
and triose-phosphate isomerization. In this respect, the occurrence of
exchange with water involving hydrogens at positions 1 and 2 of glucose
6-phosphate in the presence of both phosphoglucoisomerase and
phosphomannoisomerase has been unambiguously
proved(9, 16) . Thus hydrogen at position 2 of glucose
6-phosphate is first transferred by phosphoglucoisomerase to carbon 1
of fructose 6-phosphate at the pro-R position. However, this
transfer may be only partly intramolecular. Because the hydrogen
previously at C-2 of glucose, which later occupies the pro-R position 3 of glyceraldehyde 3-phosphate and then the methyl
position of pyruvate, ends at the methyl group of ethanol, the
redistribution coefficient a
may quantify the proportion of hydrogens at position C-2 of
glucose which is intramolecularly transferred by phosphoglucoisomerase,
provided that no further intermolecular exchanges occur in the
following reaction steps. When comparing the redistribution
coefficients a
and a
, it is observed that a
reaches only 70% of the a
value (Equations 6 and 7).
This result confirms that the transfer from C-2 of glucose 6-phosphate
to C-1 of fructose 6-phosphate is only partly intramolecular, as
concluded from tritium labeling in the phosphoglucoisomerase
reaction(10) . The present approach provides an easy and
reliable way for appraising the influence of experimental factors such
as the temperature and composition of the medium (nature and
concentration of salts, in particular inorganic phosphate) on the
relative contributions of the intra- and intermolecular hydrogen
transfers. Thus the redistribution coefficient a
is a useful criterium for
characterizing the technological conditions of the glycolysis.
coefficient, which relates site I of
ethanol to the starting water medium (Equation 9), represents the sum
of the isotopic contributions of water at different steps of the
reaction. The main contribution occurs at the pyruvate kinase reaction,
where phosphoenolpyruvate acquires one hydrogen to give pyruvate. In
the absence of any isotope effect, this reaction would contribute a
value of 2/6 to the a
parameter. In
addition, a
integrates all medium
effects resulting from exchanges possibly intervening in the course of
the reaction paths that link sites 1, 2, 6, and 6` of glucose to site I
of ethanol. In particular, taking into account the intermolecular
contributions discussed above, a value of the order of 3/6 = 0.5
is predicted for a
. By comparing this
value to the experimental one, a
0.2, a mean value of about 2.5 is estimated
for the average kinetic isotope effect accompanying proton transfers
from water. The constant term of Equation 9 is also mechanistically
meaningful, because it represents the average contribution of sites 1,
2, 6, and 6` of glucose to the methyl site of ethanol. Its relatively
high value further confirms the high level of purely intramolecular
transfer of these hydrogens. It is interesting to note that this term
depends on the nature of the fermented sugar. It is significantly
higher for glucose obtained from maize (81 ppm) than for potato glucose
(62 ppm).
plants
as compared with glucose from C
plants(23) .
Hydrogen Connections and Exchanges Associated with the
Methylene Site of Ethanol
The redistribution coefficients a involving the methylene site j = II of ethanol and sites i = 1,
2, 6, and 6` of glucose are close to zero (Tables I and II), thus
confirming the absence of direct connection between these sites i and j (Fig. 2). From a mechanistic point of view, a
relation between hydrogen at position 4 of glucose and the pro-R methylenic site of ethanol via NADPH formed in the oxidation step
of glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate could be
considered (Fig. 2). Unfortunately, the a
coefficient is not
accessible from the present experiments. However, the percentage of
hydrogen transferred from glucose to the methylene site of ethanol is
also reflected in the constant term of the linear Equation 10. Because
the aldehydic hydrogen, which is abstracted to produce NADPH, has been
previously exchanged with water at the aldolase reaction step in half
of the glyceraldehyde 3-phosphate molecules, the maximum theoretical
contribution originating from glucose is a
= 0.25. A (D/H)
value of 150 ppm, for instance,
would then lead to a constant term of 37.5 ppm. In addition, several
hydrogens of glucose contribute indirectly because they are transferred
to the water medium, which is subsequently used for introducing
methylenic hydrogens. Nevertheless, due to the diluted level of the
fermentation substrate, this deuterium enrichment of the water medium,
(and therefore such an indirect contribution of glucose) is restricted
to a few parts/million. When corrected approximately for this effect,
the experimental value of the constant term does not exceed about 4
ppm. This low value excludes the possibility of a close connection
between sites j = II and i =
4. In this respect, it should be emphasized that the fermentation
experiments carried out in highly depleted water provide a direct
unambiguous proof of the primarily aqueous origin of the methylenic
hydrogens (Fig. 1A). Loss into water of both hydrogens
originating from C-3 and C-4 of glucose is in fact expected if the
reversible aldolase and triose-phosphate isomerase reactions are
relatively fast with respect to the oxidation step of glyceraldehyde
3-phosphate (Fig. 2). Moreover, in the presence of the yeast S. cerevisiae, a break in the connection between NADPH and
ethanol might result from an exchange of the pro-R methylenic
hydrogen of ethanol with water, mediated by the enzyme
-lipoyl
dehydrogenase(21, 24) . However this exchange phenomenon
being relatively slow and requiring active yeast, our results are more
likely explained by a substantial scrambling of the two moieties of
fructose diphosphate resulting from the reactions catalyzed by
triose-phosphate isomerase and aldolase. In such conditions a water
origin is conferred to the aldehydic hydrogens of most glyceraldehyde
3-phosphate molecules, which formerly pertained either to carbon 3 or
to carbon 4 of glucose. In this context, the redistribution coefficient a
(Equation 10) provides a limit for
the average value of the kinetic isotope effects associated with
hydrogen transfers from water to the methylenic sites of ethanol (k
/k
1.4).
(Equation 12), associated
with the pro-S site of ethanol, is mainly governed by the
kinetic isotope effect for stereospecific hydrogen transfer from water
to site 2 of pyruvate in the pyruvate decarboxylase reaction step,
whereas k
, which relates the pro-R site of ethanol to water through the NADPH intermediate, conveys
kinetic isotope effects associated with the stereospecific hydrogen
transfer from water to site 3 of fructose diphosphate. It is concluded
that the isotopic effects involved in these different kinds of
stereospecific reactions are rather similar for the pro-R and
pro-S positions because the experimental ratio between the R
and S isotopomers of ethanol is not very different from unity.
Table: Influence of slight increases
((D/H)
) in the hydrogen isotope ratio at
position i = 1 or 2 of glucose, on the isotopic parameters of
the methyl ((D/H)
) and methylene
((D/H)
) sites of ethanol, and of water of the
fermented medium ((D/H)
)
C =
-11.1‰ which is in agreement with a C
metabolic pathway of the plant precursor. The overall isotope
ratio of its carbon-bound (nonexchangeable) hydrogens measured by
isotope ratio mass spectrometry on the pentanitrate derivative has a
value (D/H)
= 144.1 ppm (where
GNE stands for glucose nonexchangeable sites). The isotope ratio of
Nantes tap water used in all fermentations is (D/H)
= 150
ppm. The relative parameter R defined in Equation 3 is
calculated from signal heights. It characterizes the evolution of the
deuterium content in the methylene site with respect to that of the
methyl site, which is arbitrarily given the statistical value 3.
Table: Influence of the isotope content at
positions 6 and 6` of glucose on the site-specific isotope ratios of
fermentation ethanol
= 144.1 ppm. In the other experiments, small amounts
of glucose bideuterated at positions 6 and 6` have been added in order
to increase the site-specific isotope ratio by the value
(D/H)
. The
H NMR signals of the isotopomers of ethanol mono- and
bideuterated at the methyl position (I) can be quantified separately
(Fig. 1B). Due to signal overlap, the repeatability of the
determinations is degraded in this case (1.5 ppm). (D/H)
and (D/H)
are the
isotope ratios of the methylenic position of ethanol and of water of
the fermented medium. ND, not determined.
Table: Site-specific hydrogen isotope ratio of
ethanol resulting from the fermentation of glucose in Nantes tap water
characterized by an isotope ratio (D/H) = 150 ppm
and in water either depleted or slightly enriched in deuterium
is the overall isotope ratio
of the carbon-bound hydrogens of the starting glucose. The potato
glucose used in these experiments is characterized by a
C value equal to -24.5‰. This value is
in agreement with a C
metabolic pathway of the plant
precursor. (D/H)
and (D/H)
are the isotope ratios of the
methyl and methylene positions of ethanol, respectively, and (D/H)
is that of
water of the fermented medium. R is the relative parameter
defined in Equation 3. It is computed from signal heights.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.