Enzymic Generation of Chiral Acetates. A Quantitative Evaluation of Their Configurational Assay

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Abstract
1 R-Acetate was generated enzymically from R-acetate in the sequence acetate → malate → oxaloacetate → acetate, and S-acetate likewise from S-acetate. It was concluded that the formation of malate on malate synthase involves the operation of a normal isotopic effect combined with inversion of configuration. The malate synthase kH/K2H, was determined as 3.7 ± 0.5 by a method which yields results independently of the stereochemical purity of the chiral acetates used initially. 2 R-Acctatc was also generated from R-acetate in the sequence acetate → citrate → malate → oxaloacctate → acetate, and S-acetate likewise from S-acetate. The conclusion is the same as given above, but refers to the formation of citrate on the re-synthase. 3 2S,3R-[2−2H1.3−2H1,3H1]Malate and 2S,3S-[2−2H1, 3−2H1]malate were prepared from 2S[2.3−2H3]malate by treatment with fumarase in tritiated water and normal water, respectively. It was assumed that these malate specimens were pure with respect to chirality as generated by isotopic labelling. 4 These two malate specimens were partially converted (about 9%) to acetates in conditions where no racemization at the level of transiently formed oxaloacetate occurred. That no racemization took place was demonstrated experimentally. Oxidative enzymic hydrolysis of 2S,3R[2−2H1,3−2H1,3H1]malate in normal water and of 2S,3S-[2−2H1,3−2H1]malate in tritiated water produced S-[2 H1, 3H1]acetate and R-[2H1,3H1]acetate, respectively. 5 The isolated R-[2H1, 3H1]acetate and S-[2H1. 3H1]acetate on configurational analysis yielded malates which in the presence of fumarase retained 79.7 ± 0.7% and 20.3 ± 0.9 respectively, of their total tritium content. The symmetric deviation from the 50% value found with [3H1]acetate strenghtens the conclusion that stereochemically pure chiral acetates were analyzed. The malate svnthase kH/k2H was determined from the data of this study as 3.9 ± 0.2. 6 The average of the values given under paragraphs 1 and 5 for the isotopic discrimination on malate synthase corresponds to kH, k2H= 3.8 ± 0.1. It was concluded that the configurational analysis of stereochemically pure R-[2H1,3H1]acetate and S-[2H1, 3H1]acetate yields malates which in the presence of fumarase retain 79 ± 2% and 21 ± 2%, respectively, of their total tritium content. Hence, a deviation of 29 ± 2% from the 50% value represents the actual amplitude of the configurational assay. 7 Outlines are given for an enzymic generation of chiral acetates in preparative scale.