An enzymatic method for the determination of enantiomeric composition and absolute configuration of deuterated or tritiated succinic acid.

The distribution of hydrogen isotope between pro-R and pro-S positions of succinic acid has been determined by comparison of its isotopic content before and after incubation with isocitrate lyase. This enzyme, in the presence of glyoxylate, exchanges exclusively the pro-S protons of succinate with water (M. Sprecher, R. Berger, and D. B. Sprinson (1964) J. Biol. Chem. 239, 4268-4271). With [1-14C,2(R,S)-3H]succinate as substrate, the exchange was easily followed by the decrease of 3H/14C ratio (dried aliquots), which accounted for the high isotopic effect of this reaction. The final ratio was within +/- 5% of the theoretical one. The evolution of the exchange of deuterated succinate added with [1-14C,2(R,S)-3H]succinate acid was again followed by 3H/14C ratio. The deuterium content of [2,3-2H2]succinic acid, [2-2H2]succinic acid (derived from L-[4-2H2]glutamic acid by oxidation) and of the corresponding succinates isolated after incubation with isocitrate lyase was determined by gas chromatography-mass spectroscopy of their dimethylester under NH4+ chemical ionization. This method provides the basis for a quantitative measurement of the distribution of hydrogen isotopes in unsymmetrically 2-labeled succinate or 4-labeled glutamate.