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

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 k H /K 2H , 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 −2 H 1 .3 −2 H 1 , 3 H 1 ]Malate and 2S,3S‐[2 −2 H 1 , 3 −2 H 1 ]malate were prepared from 2S[2.3 −2 H 3 ]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 −2 H 1 ,3 −2 H 1 , 3 H 1 ]malate in normal water and of 2S,3S‐[2 −2 H 1 ,3 −2 H 1 ]malate in tritiated water produced S‐[ 2 H1, 3 H 1 ]acetate and R‐[ 2 H 1 , 3 H 1 ]acetate, respectively. 5 The isolated R‐[ 2 H 1 , 3 H 1 ]acetate and S‐[ 2 H 1 . 3 H 1 ]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 [ 3 H 1 ]acetate strenghtens the conclusion that stereochemically pure chiral acetates were analyzed. The malate svnthase k H /k 2H 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 k H , k 2H = 3.8 ± 0.1. It was concluded that the configurational analysis of stereochemically pure R‐[ 2 H 1 , 3 H 1 ]acetate and S‐[ 2 H 1 , 3 H 1 ]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.