\OL. 12 (1953) ENZYMIC OXIDATION OF ^-HYDROXYBUTYRATE 197 



for ATP and Co A. It would appear that each of these acids must also form CoA deriva- 

 tives prior to dchydrogenation. This question was tested by means of the hydroxyl- 

 amine trapping reaction and both substrates were found to form amounts of hydrox- 

 amic acid approximately equivalent to the formation from d-BOH. These enzymically 

 formed hydroxamic derivatives were isolated and chromatographed on paper, by the 

 ])rocedures used for the BOH derivatives, with the following results: product formed 

 from crotonate, Rp = 0.70, authentic crotonylhydroxamic acid, Rp — 0.70, product 

 formed from vinyl acetate, /^^j- = 0.74, authentic vinylacetylhydroxamic acidi?jr = 0.74, 

 It is evident, then, that both crotonate and vinyl acetate yield their corresponding CoA 

 derivatives in the trapping reaction. Since both cause reduction of DPN+ it appears un- 

 likely that /-BOH as such or as the CoA derivative can be an intermediate in the oxida- 

 tion of crotonyl-CoA or vinylacetyl-CoA, which presumably would first require hydration 

 to ^-hydroxybutyryl-CoA. These data indicate that the trapping reaction does in fact 

 trap the corresponding CoA derivative in each case and that apparently no significant 

 conversion of one of these acids to another, either as such or as the CoA derivative takes 

 place in the presence of hydroxylamine in the concentrations used (0.5 il/). Furthermore 

 no evidence could be found that either d- or /-BOH undergo enzymic dehydration to 

 form either vinyl acetate or crotonate, in the absence of ATP, CoA, and DPN+ in these 

 extracts. As a sensitive test, d- or /-BOH were incubated with the extract and then the 

 medium, after acidification, was extracted with ether; the extracted material was exami- 

 ned in the ultraviolet region for the characteristic absorptions of the unsaturated acids. 

 No conversion of free /-BOH or d-BOH to crotonate or vinyl acetate was detectable. 



These findings, taken together, therefore indicate quite strongly that the CoA 

 derivative formed from ^-BOH is probably the t/-j8-hydroxybutyryl-CoA. Direct proof 

 could only come from direct polarimetric analysis of a relatively large quantity of the 

 highly purified reaction product, an experimental development that is extremely un- 

 likely in the near future because of the relatively large amount of material required 

 and the relatively low specific rotation expected, or perhaps by use of a highly purified 

 sample of the /-specific ^-hydroxybutyric dehydrogenase as an enzymic test of the optical 

 properties of free /3-hydroxybutyrate liberated from the enzymically formed CoA 

 derivatives by alkaline hydrolysis. This dehydrogenase has unfortunately never been 

 purified to any significant extent. 



Very significant complementary evidence for the findings described in this paper 

 is the report of Lynen et al.^^ which recently appeared as our work was being completed. 

 These investigators isolated from sheep liver in apparently pure form a dehydrogenase 

 catalyzing the following reaction: 



Acetoacetyl-CoA + DPNH + H+ ^ ^-hydroxybutyryl-CoA + DPN+ (5) 



The reaction was followed in the forward direction as written by measuring the dis- 

 appearance of DPNH in the presence of enzymically formed acetoacetyl-CoA or, 

 alternatively, in the presence of synthetic S-acetoacetyl-N-acetyl-cysteamine, a "model" 

 of acetoacetyl-CoA. /3-hydroxybutyryl-CoA was identified as the reduction product by 

 the hydroxylamine reaction and paper chromatography of the hydroxamic acid. The 

 reaction was found to be reversible and approximations of the equilibrium constant 

 were made. The enzyme did not act on free acetoacetate or free jS-hydroxybutyrate. The 

 authors did not discuss the question of the stereochemical configuration or the rotation 

 of the ^-hydroxybutyryl-CoA formed. Their findings however furnished strong support- 

 References p. 202. 



