DIFFERENTIATION OF DIASTEREOMERIC SALTS OF HYDROXYETHYL(THIAMIN) AND ANALYSIS OF STEREOCHEMICAL REQUIREMENTS IN PYRUVATE DECARBOXYLASE

Pyruvate undergoes enzyme-catalyzed decarboxylation through formation of an adduct with thiamin diphosphate. The adduct is chiral and loss of carbon dioxide produces a second chiral intermediate, 2-(1-hydroxyethyl)thiamin diphosphate (HETDP). It has been shown previously that 2-(1-hydroxyethyl)thiamin (HET) can be resolved as a salt of (+)- or (-)-2,3-dibenzoyltartaric acid. The resulting optically active samples of HET have been converted to HETDP and used for enzyme studies. Both (+) and (-) HETDP were converted by the apoenzyme of pyruvate decarboxylase. This apparent lack of stereospecificity prompted us to establish independently the extent of the resolution accomplished by fractional crystallization. The diastereomeric dibenzoyltartrate salts have now been shown to be distinguishable by 1H NMR spectroscopy of solutions in methanol-d4. Calculated spectra permit an estimation of the relative concentrations of the enantiomers of HET in any sample under these conditions. On the basis of this information, it is concluded that the enzyme can react with both enantiomers of HETDP, suggesting that the active site of wheat germ pyruvate decarboxylase can accept the interchange of the hydrogen and methyl groups of the C2α position of HETDP. © 1990.