BUTYRIC AC1D-BUTANOL FERMENTATIONS 45 



Now the above reaction was catalyzed by a relatively crude 

 pigeon liver extract, and there was no certainty that it rep- 

 resented a single enzymatic step. Another possibility 24,43 

 was a two-step reaction sequence in which one enzyme 

 catalyzed the reversible transfer of an acetyl group from 

 one molecule of acetyl-SCoA to another to give acetoacetyl- 

 SCoA plus free coenzyme A (reaction 25) , and another 

 enzyme hydrolyzed acetoacetyl-SCoA to acetoacetate (reac- 

 tion 26) . 



2CH 3 COSCoA ^^= 



CH3COCH2COSC0A + HSCoA (25) 



CH3COCH2COSC0A + H 2 — > 



CH3COCH2COOH + HSCoA (26) 



The postulated formation of acetoacetyl-SCoA was consist- 

 ent not only with all the available information concerning 

 the pigeon liver acetoacetate-forming system but also with 

 certain observations made on the CI. kluyveri system. For 

 example, at that time the necessity of the activation of 

 butyrate prior to its oxidation was known and the activated 

 compound was suspected to be butyryl-SCoA. The oxida- 

 tion of butyryl-SCoA would be expected to give acetoacetyl- 

 SCoA, and the reverse reaction would also be anticipated. 

 More direct evidence for the reversible formation of 

 acetoacetyl-SCoA from acetyl-SCoA was first obtained by 

 Lynen et al., 44 - 45 with soluble enzymes from sheep liver. 

 This was made possible by the discovery that thioesters of 

 acetoacetate such as S-acetoacetyl-N-acetyl-thioethanolamine 

 and acetoacetyl-SCoA show a strong ultraviolet absorption 

 peak at 303 m^ in alkaline solution. By means of this opti- 

 cal property the formation and decomposition of acetoacetyl- 

 SCoA according to reaction 25 were demonstrated. The 

 enzyme catalyzing this reaction, called acetoacetyl thiolase 



