44 BACTERIAL FERMENTATIONS 



reaction. Later experiments by Bartsch 38 have shown that 

 CI. kluyveri extracts contain an enzyme called vinylacetyl 

 isomerase which converts vinylacetyl-SCoA to crotonyl-SCoA. 



CH 2 =CHCH 2 COSCoA — > CH 3 CH=CHCOSCoA (23) 



Vinylacetyl-SCoA Crotonyl-SCoA 



The latter compound may be the substance actually re- 

 duced, as it appears to be in animal systems. 39 - 40 



A key reaction in the synthesis of butyrate is the reversible 

 formation of the C 4 compound at the oxidation level of 

 acetoacetate from its C 2 precursor. The elucidation of this 

 reaction required a series of investigations most of which 

 were done with animal enzyme preparations. Studies 

 carried out in Lipmann's laboratory with a soluble multi- 

 enzyme preparation from pigeon liver showed that ATP, 

 coenzyme A, and acetate are required for acetoacetate syn- 

 thesis. 41 This suggested that a coenzyme A derivative of 

 acetate was a precursor of acetoacetate, but it was not 

 known whether one molecule of "active" acetate condensed 

 with a molecule of acetate to give acetoacetate directly or 

 whether more complicated reactions were involved. This 

 situation was clarified by Stadtman et al., 42 who studied 

 acetoacetate synthesis in a system containing acetyl phos- 

 phate, acetate, coenzyme A, phosphotransacetylase from CI. 

 kluyveri, and a pigeon liver extract. In separate experi- 

 ments, either the acetyl phosphate or the acetate was labeled 

 with C 14 and the incorporation of the isotope into aceto- 

 acetate was studied. The data showed that free acetate was 

 not used, but on the contrary both halves of the acetoacetate 

 molecule were derived from acetyl phosphate presumably 

 via acetyl-SCoA (reaction 24) . 



2CH 3 COSCoA + H 2 — > 



CH3COCH2COOH + 2HSCoA (24) 



