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



would be expected if reaction (5) is reversible, the data reported are probably not suffi- 

 cient to establish reversibility of reaction beyond any doubt (4). However with the 

 appearance of the work of Lynen et al.^^, further work on this question appeared re- 

 dundant. 



DISCUSSION 



The work reported in this communication demonstrates that ^/-/3-hydroxybutyrate 

 undergoes biological oxidation, and clarifies the mechanisms involved. It also indicates 

 that ^/-/3-hydroxybutyrate, and possibly higher (i-^-hydroxy acids may be normal meta- 

 bolic intermediates. This suggestion derives from the very probable reversibility of 

 reaction (4) for which evidence has been presented by us and by Lynen and his colleagues. 

 It appears quite probable on the basis of current knowledge, that the oxidation of fattv 

 acids follows the pattern : 



RCH2CH2CH2COOH + CoA -^^^ RCH0CH2CH2COC0A (11) 



RCH2CH2CH2COC0A > RCHoCH = CHCOCoA + 2 £ (12) 



RCHgCH = CHCOCoA + HgO > RCH2CHCH2CC0A (13) 



I i: 



OH O 



RCH2CHOHCH2CC0A + DPN+ > RCH2CCH2COC0A + DPNH + H+ (14) 



II il 



O O 



followed by "thioclastic" cleavage^^ of the ^-keto acid-CoA derivative to yield acetyl- 



CoA and a shortened fatty acid CoA derivative. Our work suggests that the ^-hydroxy- 



acid participating as the CoA complex in these reactions may be in the dextrorotatory 



form. 



It may be pointed out that longer chain dextrorotatory /3-hydroxy acids have re- 

 cently been isolated as hydrolysis products of certain complex lipids of micro-organisms^". 



The two different and specific mechanisms for dehydrogenation of the two isomers 

 described in this report, taken together with the enzymic reactions of acetoacetate 

 summarized by Stern et al.^^, also provide an enzymic mechanism for biological race- 

 mization of /3-hydroxybutyrate. This mechanism may be outlined briefly as follows: 

 ^^BOH, as its CoA derivative, may be oxidized to acetoacetyl-CoA, as already described. 

 A. deacylase exists, capable of hydrolyzing the latter, with the formation of free aceto- 

 acetate, which may then be reduced reversibly to Z-BOH by the /-j8-hydroxybutyric 

 dehydrogenase. The reverse transformation may take place by intervention of the enzyme 

 catalyzing the formation of acetoacetyl-CoA at the expense of ATP. Although this en- 

 zyme apparently has only limited activity in the liver, it is quite active in other tissues^^. 

 It appears possible that the general reaction pattern involved here may underlie other 

 instances of biological racemization which are dependent on the "sparking" phenom- 

 enon"-''^. Preliminary experiments by one of us (A.L.L.) suggest such a pattern to be 

 involved in the enzymic oxidation of the stereoisomers of lactic acid. 



In the work on intact mitochondria reported here it was observed that /-BOH gave 

 rise to small but significant amounts of citrate. Although the liver appears not to attack 

 free acetoacetate readily^'' ^'''^'', it is possible that the citrate formed from Z-BOH derives 

 from acetoacetate, the major oxidation ])roduct of l-BOH. On the other hand, it is 

 conceivable that /-^-hydroxybutyryl-CoA, which has been shown to be formed by 



References p. 202. 



