VOL. 12 (1953) 



ENZYMES OF FATTY ACID METABOLISM 



305 



gel, fractionation with ethanol in the presence of Zn ions, and refractionation with 

 ammonium sulfate. 



Fig. 5 shows the appearance ot the enol band of S-acetoacetyl thioesters when 

 succinyl Co A is incubated with acetoacetate and transferase at pH 8.1, indicating the 

 formation of S-acetoacetyl CoA. Within the range 290 to 330 m/u, this band corresponds 

 closely with the corresponding band ot S-acetoacetyl-N-acetyl thioethanolamine. It may 

 also be seen that most of the absorption in this region disappears after the further 

 addition of CoA-SH and thiolase since the equilibrium position of the thiolase reaction 

 favors the formation of acetyl CoA. The forward course and the reversal of the transferase 



06 



0.5 



0.4 

 0.3 

 0.2 

 0.1 



INITIAL 



AFTER TRANSFERASE 



AFTER THIOLASE 



A. 



270 280 



340 



290 300 310 320 

 WAVE LENGTH {m/<) 

 Fig. 5. Spectral changes accompanying the enzy- 

 matic synthesis and breakdown of S-acetoacetyl 

 CoA at pH 8.1. Volume, 1.5 ml; d = 0.5 cm; 

 temp. 25°. — O — O — S-SuccinylCoA('^^o.i fiM). 

 — — % — After establishment of equilibrium 

 on addition of acetoacetate (50 fiM) and trans- 

 ferase (60 jug of protein) . — /\ — /\ — After further 

 addition of CoA-SH (0.15 fiM) and thiolase (90 

 /ig of protein). Acetoacetate and CoA-SH added 

 to both blank and experimental cells. MgClg (8.0 

 /.iM) present in reaction mixture. 



»- 



SUCCINATE 



i 



12 16 

 MINUTES 



Fig. 6. Optical demonstration of transferase and 

 thiolase activities. Volume, 1.5 ml; d — 0.5 cm; 

 pH, 8.1; temp, 25°. Upper curve: Transferase 

 (17 fig of protein) added at zero time to a mix- 

 ture of succinyl CoA {r~^ o.ii /liM) and acetoace- 

 tate (50 fiM) ; succinate (40 /nM) added at the 

 arrow. Lower curve : Transferase ( 1 7 //g of protein) 

 added at zero time to a mixture of succinyl CoA 

 {'-^ 0.06 fiM) and acetoacetate (50 /nM) ; CoA-SH 

 (0.15 /uM) and thiolase (2.7 fig of protein) added 

 at the arrow. Other details as in Fig. 5. 



reaction as followed at 305 m/x are shown in Fig. 6. The upper curve shows the increase 

 in absorption at 305 m/x on adding transferase to a mixture of succinyl CoA and aceto- 

 acetate and the reversal of the reaction by succinate after equilibrium was established. 

 The equilibrium constant of the transferase reaction (/v^^ = (Succinate) (acetoacetyl-S- 

 CoA)/Succinyl-S-CoA) (acetoacetate)) is about lO^^ at pH 8.1. 



The acetoacetyl CoA formed by the reaction between succinyl CoA and aceto- 

 acetate, in the presence of transferase, was isolated as a crude alcohol-insoluble barium 

 salt and further purified by paper chromatography^^. In ethanol-acetate its Rp is 0.52 

 at 24°, while that of acetoacetate is 0.75. Like acetyl CoA*^>^', acetoacetyl CoA gives, a 

 positive sulfhydryl reaction with nitroprusside only after alkaline hydrolysis. 



Some insight into the mechanism of action of the transferase has been gained by 

 experiments with methylene labelled ^^C-succinate^^^. When ^^C-succinate and succinyl- 

 CoA are incubated with transferase a rapid exchange of free- and thioester-bound 

 succinate occurs. This suggests the possibility that succinyl CoA, or acetoacetyl CoA, 

 reacts with the enzyme to form a CoA-enzyme compound which retains the energy of the 



References p. 313 1 314. 



