©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Site-specific Isotope Fractionation in the Characterization of Biochemical Mechanisms
THE GLYCOLYTIC PATHWAY (*)

Ben Li ZhangYunianta , Maryvonne L. Martin

From the (1)Laboratoire de Résonance Magnétique Nucléaire et Réactivité Chimique, URA-CNRS 472, Université de Nantes, 2 rue de la Houssinière, 44072 Nantes Cedex 03 France

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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.


EXPERIMENTAL PROCEDURES

Materials and Fermentation

The commercial glucoses (Prolabo) were obtained from maize and potato. -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%.

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 (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.

The composition of the fermentation medium was 2 g liter NHCl, 3 g liter KHPO, 3 g liter NaCl, 60 mg liter MgCl, 6 g liter NaHPO, 120 mg liter NaSO, 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) .

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.

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

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 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.

On-line formulae not verified for accuracy

When I 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.

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).


RESULTS

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 CHDCHOH exhibits only slight variation, whereas the amount of the CHDCHOH 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) .

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 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.

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 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.

The redistribution coefficient a can be further interpreted by considering separately the enantiomeric monodeuterated isotopomers of ethanol:

On-line formulae not verified for accuracy

The parameters k and kconnect 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.

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 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.

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% CHD, 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.


DISCUSSION

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.

In the diluted conditions of the fermentation experiments, the a 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, a0.2, 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).

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 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).

Enantiomeric resolution of the monodeuterated methylenic isotopomers provides further mechanistic information because the parameter k (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.

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.

  
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))

The starting natural glucose used in these experiments has a maize origin. Its overall carbon isotope ratio has a value, 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

Fermentation has first been conducted with natural glucose characterized by an overall isotope content of the carbon-bound hydrogen positions (D/H) = 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

(D/H) 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.



FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


ACKNOWLEDGEMENTS

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.


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