198 A. L. LEHNINGER, G. D. GREVILLE VOL. 12 (1953) 



ing evidence for the reaction sequence postulated in this paper. This dehydrogenase 

 is probably identical with that reported here and it appears quite probable from our 

 work therefore that the ^-hydroxybutyryl-CoA participating in the reaction catalyzed 

 contains the (i-isomer. 



That acetoacetyl-CoA is the probable oxidation product of d-BOH in the extracts 

 we have studied was indicated by the finding that on the addition of oxalacetate to a 

 system composed of mitochondrial extract, ATP, excess CoA, Mg++, d-BOH, and DPN+, 

 citrate was formed. Omission of any component caused complete or almost complete loss 

 of the capacity to form citrate. Citrate was not formed from free acetoacetate under the 

 same circumstances. See Table VIII. The formation of citrate from acetoacetyl-CoA 

 occurs in two steps by already well-known reactions^^-^^: 



Acetoacetyl-CoA + CoA ^ 2 acetyl-CoA (6) 



Acetyl-CoA -|- oxalacetate ^ citrate -f CoA (7) 



Since the "condensing enzyme" catalyzing reaction (7) requires acetyl-CoA, either 

 acetyl-CoA or a precursor of it must be formed in the oxidation of d-BOH. It is of course 



TABLE VIII 



FORMATION OF CITRATE FROM d-BOU 



Systems contained 0.4 ml extract (3-C-7), o.i M KCl, 0.05 M "tris" buffer pH 8.0, o.oi M 

 cysteine, 0.025 -^ ^-BOH or acetoacetate, o.oi M oxalacetate, 0.005 ^^ ATP, 0.005 M MgClj, and 

 o.ooi M CoA. Total volume, 2.0 ml. Time, 60 minutes; temp. 22° C. 



c- , , . CI Citrate formed 



Substrate System micromoles 



most likely that acetoacetyl-CoA is formed directly by dehydrogenation of (/-^-hydroxy- 

 butyryl-CoA and is spht to acetyl-CoA by reaction (6). The fact that "free" acetoacetate 

 does not form citrate excludes it as a direct intermediate and also excludes the presence 

 in the extracts used of the enzyme catalyzing the reaction 



Acetoacetate -f CoA ^^^ acetoacetyl-CoA (8) 



Other properties of the rf-BOH dehydrogenase system were examined. It was found, 

 using extracts containing no detectable /-/S-hydroxybutyric dehydrogenase activity, that 

 higher ^-hydroxy acids caused the reduction of DPN+ at about the same rate as did 

 ^-BOH, namely (//-^-hydroxyoctanoate and (^//-^-hydroxynonanoate. The optical isomers 

 of these acids were not available for direct test, but it appeared probable that only the 

 t?-isomers underwent dehydrogenation since both ATP and CoA were absolute require- 

 ments for the reduction of DPN+. Triphosphopyridine nucleotide was not detectably 

 reduced when it was substituted for DPN ' in the d-BOH system. 



The reduction of DPN + by d-BOH in the presence of ATP. Co.\, and Mg++ was not 

 inhibited by 0.005 M NaF and inhibited only 22% by 0.02 .1/ XaF. The presence of 

 orthophosphate did not increase the inhiI)ition. It is therefore probable that reactions 



References p. 202. 



