VOL. 12 (1953) ENZYMIC OXIDATION OF j8-HYDR0XYBUTYRATE I99 



{3) and (4) are not the fluoride-sensitive points in the conversion of d-BOH to citrate 

 observed in intact respiring mitochondria. It is of some interest in this connection that 

 Drysdale and Lardy^^ found no inhibitory effects of fluoride on the oxidation of 

 (presumably) butyryl-CoA to acetoacetyl-CoA although the oxidation of fatty acids in 

 intact mitochondria is inhibited by fluoride (c/.-^'"'^^). 



The reduction of DPN+ by d-BOH was strongly inhibited by pyrophosphate (85% 

 by 0.005 M pyrophosphate) but not by orthophosphate at concentrations up to 0.03 M. 

 This finding would be expected, on the basis of mass action effects, if the activation reac- 

 tion (3) is written in detail as follows : 



ATP + CoA + RCOOH ^ R— C— S— CoA + pyrophosphate + adenylate (9) 



II 

 O 



The activation of fatty acids apparently proceeds according to this general equation^^'^^. 



There was no striking experimental evidence of a competitive antagonism between 

 d- and /-BOH in the d-BOU. reaction; the addition of o.oi M /-BOH to a system acting 

 on o.oi M d-BOH had no detectable effect on the rate of reduction of DPN+. Much higher 

 molar ratios of /-BOH to ^-BOH were not tested. It should be pointed out, however, 

 that without knowledge of which reaction is rate-limiting in the sequence of reactions 

 (3) and (4) it cannot necessarily be concluded that competitive antagonism between 

 d- and /-BOH or their CoA derivatives does not take place in either of the reactions in 

 this sequence. 



The reduction of DPN+ by d-BOH in the presence of ATP and CoA was not severely 

 inhibited by hydroxylamine except at very high concentrations of the latter. For most 

 effective trapping as the hydroxamic acid it was necessary to use 0.5 M hydroxylamine. 

 In the presence of this concentration of hydroxylamine slight reduction of DPN+ 

 could still be observed. This finding is consistent with the relatively low inhibitory effects 

 of hydroxalamine on the fatty acid oxidase system in extracts of C. KluyverP'^ and in 

 mitochondrial extracts^*. It appears probable that the ability of hydroxylamine to react 

 non-enzymically with various acyl-CoA derivatives varies considerably. Acetyl-CoA 

 is known to be trapped effectively at much lower hydroxylamine concentrations than 

 the CoA derivatives of higher fatty acids (c/. 38) . 



Some evidence was obtained that the dehydrogenation reaction (4) is reversible. 

 When the extract is supplemented with excess acetate, ATP, CoA, Mg++, and DPNH, 

 the DPNH is slowly reoxidized as indicated by disappearance of the characteristic 

 absorption at 340 m^. The reaction requires the presence of acetate, ATP, CoA, and 

 Mg+"'-. The reoxidation of DPNH presumably occurs by the intervention of the following 

 known reactions : 



Acetate -\- CoA ^^-> Acetyl-CoA (10) 



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



Acetoacetyl-CoA + DPNH + H+ ^ |8-hydroxybutyryI-CoA + DPN+ (5) 



This experimental approach resembles that employed by Lynen et al.^^. 



It has been established that reaction (10) occurs in these extracts, by means of the 

 iiydroxylamine trapping reaction. Reaction (6), known to be reversible^^, is responsible 

 for formation of acetyl-CoA from acetoacetyl-CoA in the citrate synthesis experiments 

 just described and in citrate synthesis from fatty acids in mitochondrial extracts des- 

 cribed by others^^. Although the reoxidation of DPNH by acetate in the system described 



References p. 202. 



