²H‐NMR resolution of the methylenic isotopomers of ethanol applied to the study of stereospecific enzyme‐catalysed exchange

Abstract

We have shown that site-specific natural isotope fractionation of hydrogen studied by NMR (SNIF-NMR) is an important source of information on the mechanistic and environmental effects which govern the photosynthesis of sugars and their fermentation into ethanol. Three isotope ratios associated with the methyl, methylene, and hydroxyl sites of ethanol are determined in achiral media. In this study we show that complementary information about possible stereospecific mechanisms involving the methylenic hydrogens is also rendered accessible by 2H-NMR enantiomeric resolution. The synthesis of mandelate esters enables exchange between the pro-R site of ethanol and water to be investigated. Simultaneous access to the three site-specific isotope ratios of the ethyl group is obtained at isotopic dilutions close to the natural ones. Mediation of the exchange by the enzymic system alcohol dehydrogenase-alpha-lipoyldehydrogenase and by the yeast Saccharomyces cerevisiae are compared. The progress of the reaction can be followed quantitatively as a function of time and the occurrence of glycolytic metabolism of endogeneous materials by yeast can be substantiated in a one-pot experiment.

Natural Abundance Isotopic Fractionation in the Fermentation Reaction: Influence of the Fermentation Medium

“…These effects of the added salts can be explained by a decrease in the percentage of intramolecular transfers, in particular that which involves site 2 of glucose at the phosphoglucoisomerase step of the reaction (17,18). Since the methylenic hydrogens originate primarily from water at two different steps of the reaction, variations in the values of the kinetic isotope effects may be invoked, but modifications in the rate of exchange between the pro-R site of ethanol and water may also intervene (19,20).…”

Natural Abundance Isotopic Fractionation in the Fermentation Reaction: Influence of the Nature of the Yeast

“…In general, an increase in the (D/H) II value may be observed when ethanol is left in contact with the yeast for a long time after consumption of glucose and in cases of poorly alcohologenic yeasts. This deuterium enrichment can be explained by a stereospecific exchange of the pro-R-methylenic site of ethanol with water mediated by the enzymatic couple alcohol dehydrogenase-diaphorase (15,16). Yeasts with low alcohologenic potential are thought to excrete ethanol from their cells more slowly than the Saccharomyces species.…”

Non-equivalence of Hydrogen Transfer from Glucose to the pro-R and pro-S Methylene Positions of Ethanol during Fermentation by Leuconostoc mesenteroides Quantified by 2H NMR at Natural Abundance

“…18. To an ethanol sample of 3.5-5.0 ml (56 -80 mmol) was added 20 ml of dry benzene in a 50-ml round-bottomed flask.…”

“…Deuterium Exchange in the Reduced Nucleotide Pools-Deuterium exchange between NAD(P) 2 H and water can potentially occur due to the action of flavin/diaphorase (see (18) To test whether such an exchange occurs in Ln. mesenteroides, a fermentation was carried out with natural abundance glucose and water until the glucose was used up.…”