The mechanism of acetoacetate synthesis.

The observation of a coenayme A (CoA)-catalyzed acetoacetate synthesis from acetate and adenosinetriphosphate in pigeon liver extracts (1) grouped this reaction with the larger family of acetylation reactions. Furthermore it furnished partial confirmation of an earlier calculation (2) showing that 16,000 calories are required for the condensation of two acetates to acetoacetate. Thus, one energy-rich phosphate bond could carry sufficient energy for such a condensation. A further and more direct support for these calculations was furnished by Stadtman and Barker (3) who found in extracts of Cbstridium kluyveri a phosphoroclastic split of acetoacetate, with the formation of acetyl phosphate and acetate. This observation suggested that in the reverse condensation reaction an activated carboxyl group (viz., acetyl phosphate) may react with the methyl group of “passive” acetate. Since the total energy required for the condensation could be furnished by the acetyl phosphate, it seemed plausible that the methyl group of the second acetate may not require activation. However, some doubt about the passive r81e of the second acetate arose when it was found that, if coupled with the appropriate animal acceptor enzymes, bacterial phosphotransacetylase can activate acetyl phosphate to serve as an acetyl donor in the synthesis of both citrate (4-6) and acetoacetate (5) and for the acetylation of sulfanilamide (7). It thus appears that the same donor system can deliver methyl-activated as well as carboxyl-activated acetate. By use of labeled acetyl phosphate and acetate, it seemed possible to decide whether, for the synthesis of acetoacetate, carboxyl activation was sufficient or whether a methyl activation was required also. The isotope experiments reported here somewhat unexpectedly showed that both 2-carbon molecules that condense to form