VOL. 12 (1953) 



ENZYMES OF FATTY ACID METABOLISM 



309 



has been found***^ to be 5.2-102 ^^ pjj ^ q -pj^g gj^. 

 zyme does not react with free acetoacetate or with 

 ethyl acetoacetate ; it also fails to react with S-aceto- 

 acetyl glutathione. With acetoacetyl-S-CoA the reac- 

 tion is much faster than with the thioethanolamine 

 derivative. This is undoubtedly due to the much 

 higher affinity of the enzyme for the natural 

 compound. In tact, in kinetic studies with S-aceto- 

 acetyl-N-acetyl thioethanolamine it was not pos- 

 sible to reach saturation of the enzyme with the 

 analogue^^. 



In the presence of thiolase, ^-keto reductase, 

 and DPNH, the latter is oxidized on addition of 

 acetyl-S-CoA; DPN+, j8-hydroxybutyryl-S-CoA and 

 HS-CoA are the reaction products^. This occurs 

 according to the reactions below : 



a. 



e 



CO 



-1- 



? 



2 4 6 8 10 12 

 MINUTES 



Fig. g. Optical /?-keto reductase 

 test. Pyrophosphate buffer pH 7.4, 

 50 ixM; DPNH, 0.13 fxH ; S-aceto- 

 acetyl-N-acetyl thioethanolamine, 

 5.0 jxM . Volume, 2.0 ml; d = 1.0 

 cm; temp. 25°. 



O 



2CH3— C— S— CoA: 

 O 



HS— CoA + CH3— CO— CH 2— O— S— Co A (thiolase) 



O 



CH3— CO— CHj— C— S— C0A + DPNH + H+ ^ CH3— CHOH— CH,— C— S— C0A + DPN+ (reductase) 



O 



Sum : 2 CH3— C— S— Co A + DPNH + Hh 



O 



II 

 CH3— CHOH— CH2—C—S— CoA + HS— CoA + DPN+ 



The formation of HS-CoA can be followed through the appearance of sulfhydryl 

 groups. /?-hydroxybutyryl-S-CoA was extracted from the acidified reaction mixture with 

 /j-cresol and converted into the corresponding hydroxamic acid by reaction with 



hydroxylamine. The j3-hydroxybutyrohydrox- 

 amic acid was identified by paper chro- 

 matography {Rp in aqueous butanol, 0.29). 

 On incubation of the natural j8-hydroxy- 

 butyryl-S-CoA with DPN+ and purified ^- 

 ketoreductase at pH 9.05, the reduction of 

 DPN+, followed at 340 m/x, is accompanied by 

 the formation of acetoacetyl-S-CoA as shown 

 by the increase in optical density at 303 mju,. 

 As previously mentioned both j3-hydroxy- 

 butyryl- and acetoacetyl-S-CoA have recently 

 become available synthetically. The course 

 of the j3-ketoreductase reaction with these 

 two compounds*^^ is shown in Fig. 10. 



Lehninger AND Greville*^ have recent- 

 ly reported the interesting observation that 

 liver contains two different /D-ketoreduc- 

 tases. One of them catalyzes the reversible 

 oxidation of free /-hydroxybutyrate by DPN+, 

 the other catalyzes the reversible oxidation 



Fig. 10. Optical experiments with /S-keto 

 reductase. Pyrophosphate bufter pH 6.58, 

 100 (iM; DPN+, 5.o/iAf; S-;3-hydroxybutyryl 

 CoA, 2.0 [xM. Volume, 2.0 ml; A, 366 raij,; 

 d = i.o cm; temp. 21°. /3-keto reductase 

 (1.5 mg of protein) added at the first arrow. 

 S-acetoacetyl CoA (0.43 fiM) added at the 

 second arrow. 



References p. 313J314. 



