Substrate Stereochemistry of the Acetyl‐CoA Acetyltransferase Reaction

A specimen of symmetrically tritiated 3S‐3‐hydroxy[2‐3H2]butyryl‐CoA was prepared from acetoacetyl‐CoA by incubation in tritiated water, followed by enzymic reduction using 3S‐specific 3‐hydroxyacyl‐CoA dehydrogenase. In addition, a specimen of dideuterated, racemic 3RS‐3‐hydroxy‐[2‐2H2]butyryl‐CoA was prepared chemically from 3RS‐3‐hydroxy[2‐2H2]butyric acid.These acyl‐CoA derivatives were incubated respectively with deuterium oxide and tritiated water in presence of enoyl‐CoA hydratase. Stereospecifically tritiated compounds, respectively 2S,3S‐3‐hydroxy[2‐2H1,3H1]butyryl‐CoA and 2R,3S‐3‐hydroxy[2‐2H1,3H1]butyryl‐CoA (plus 3R‐diastereomer) were formed and isolated.3S‐3‐Hydroxy[2‐3H2]butyryl‐CoA and the 3S‐[2‐3H2,3‐14C]‐labelled material were also prepared as described in the preceding paper. The latter substrate was used to establish conditions in which little loss of tritium would occur (found: about 7%) on cleavage to acetyl‐CoA in the presence of 3‐hydroxyacyl‐CoA dehydrogenase and acetyl‐CoA acetyltransferase. Little loss of tritium indicates a small degree of racemization of the stereospecifically 2‐tritiated 3‐hydroxyacyl‐CoA derivatives at the level of transiently formed acetoacetyl‐CoA and acetyl‐CoA. This ensures high optical purity of the chiral acetates generated from the stereospecifically tritiated hydroxybutyryl‐CoA specimens listed in paragraph (2).These two acyl‐CoA derivatives and the symmetrically tritiated 3S‐3‐hydroxy[2‐3H2]butyryl‐CoA listed in paragraph (3) were converted to the acetates enzymically in the sequence hydroxy‐butyryl‐CoA → acetoacetyl‐CoA → acetyl‐CoA → acetate. The isolated acetates were assayed for chirality by conversion to the malates and fumarates as usual.The malate formed from 3S‐3‐hydroxy[2‐3H2]butyryl‐CoA on incubation with fumarase lost nearly 50%, that derived from 2R3S‐3‐hydroxy[2‐2H1,3H1]butyryl‐CoA (plus 3R‐diastereomer) retained about 26% and that derived from 2S,3S‐3‐hydroxy[2‐2H1,3H1]butyryl‐CoA retained about 78% of its total tritium content.It was concluded that the detachment of acetyl‐CoA from acetoacetyl‐CoA on acetyl‐CoA acetyltransferase occurs with inversion of configuration at the methylene group which becomes methyl.