VOL. 12 (1953) ENZYMIC OXIDATION OF ^-HYDROXYBUTYRATE I95 



by /-BOH (in the absence or presence of ATP, CoA, and Mg++) is, at the most, less than 

 5% of the rate of reduction ot DPN+ by rf-BOH under the conditions employed. 



TABLE VI 



REDUCTION OF DPN+ BY d-BOH IN EXTRACTS ESSENTIALLY FREE OF /-BOH DEHYDROGENASE 



Test conditions exactly as in Table IV, with the exception that the extracts used were prepared 

 from acetone-powdered KCl-washed liver residue (see "Experimental Details"), o.io ml of each 



extract in total volume of i.o ml, as in Table IV. 



The experiments in Table VI are not consistent with the view that the Z-/8-hydroxy- 

 butyric dehydrogenase is involved in the oxidation of the i-isomer, following an enzymic 

 racemization. Such a hypothesis would require that the rate of oxidation of d-BOH could 

 not exceed the maximal rate of oxidation of /-BOH, measured under identical conditions. 

 It is evident from the data that the rate of oxidation of the i-isomer is at least twenty- 

 fold greater than the rate of oxidation of the /-isomer. It may be concluded that the 

 /-specific dehydrogenase is not involved in the oxidation of the ^-isomer observed. 



As a working hypothesis it was proposed that i-BOH reduces DPN+ in these 

 extracts through a sequence of two reactions: 



(/-^-hydroxybutyrate -4- CoA ^^-^ ^-jS-hydroxybutyryl-CoA (3) 



i-/3-hydroxybutyryl-CoA -f DPN+ ^ acetoacetyl-CoA -f DPNH + H+ (4) 



Such a mechanism would account for the requirement of ATP and CoA in the reduction 

 of DPN+ by d-BOH. Experimental evidence supporting this mechanism follows. 



That the ^/-isomer undergoes reaction (3) was indicated by the finding that the 

 hydroxamic acid derivative of BOH is formed when d-BOH is incubated with the ex- 

 tracts in the presence of ATP, CoA, Mg++, and hydroxylamine as trapping agent (Table 

 VII). It is seen from the data that the formation of the hydroxamic acid derivative is 

 absolutely dependent on the presence ot ATP and CoA and is maximal in the presence 

 of Mg++. The hydroxamic acid derivative formed from ^-BOH was established as that 

 of |8-hydroxybutyric acid by paper chromatography. The hydroxamic acid formed in 

 the test system was separated and chromatographed on paper by the procedure outlined 

 by Stadtman and Barker^^. The Rp of the hydroxamic acid derivative on Whatman 

 No. 43 paper, using a butanol-water system, was found to be 0.35. An authentic specimen 

 of ^-hydroxybutyrohydroxamic acid, prepared from the pure ethyl ester, showed an 

 References p. 202. 



