3o6 



F. LYNEN, S. OCHOA 



VOL. 12 (1953) 



thioester bond and can transfer CoA to the free acid, acetoacetic or succinic. The reaction 

 could be visuaHzed as involving a carboxyl group of the enzyme as indicated below : 



o 



1 



HOOC— CH 2— CH 2— C— S— Co A + Enzyme— COOH 



O 



Enzyme— C—S— CoA +CH3— CO— CH 2— COOH ^ 



Enzyme— COOH + CH3— CO— CH2—C—S— CoA 



HOOC— CH2—CH2— COOH + Enzyme— C—S— CoA 



O 



The best preparations of transferase so far obtained are free of thiolase and, under 

 the conditions of the optical assay, catalyze the formation of 250 moles of acetoacetyl- 

 CoA per minute per 100,000 g of enzyme at 25°. As assayed optically, the purified trans- 

 ferase catalyzes the transfer of CaA from succinyl CoA to acetoacetate, /3-ketovalerate, 

 ^-ketoisocaproate, and j8-ketocaproate in order of decreasing activity. jS-ketooctanoatc is 

 inactive. The enzyme catalyzes the transfer of CoA from acetoacetyl CoA to succinate but 

 not to j3 -hydroxy by turate, crotonate, butyrate or octanoate. 



^-keto thiolase. The enzyme catalyzing Reaction 5 has been partially purified from 

 sheep liver^ and more extensively from pig heart'''**. The enzyme has also been referred 

 to as the acetoacetate condensing enzyme®^. In the lower curve of Fig. 6, addition of 

 CoA-SH and thiolase to a mixture of succinyl CoA and acetoacetate previously incubated 

 with transferase is shown to cause a decrease in optical density- at 305 mfi due to cleavage 

 of the acetoacetyl CoA formed by the CoA transfer reaction. The assay used for the 

 purification of the pig heart enzyme was based on the decrease in optical density at 



O O 



CH3— CO— CH2— C— S— CoA + HS— CoA ; 



:CH3— C— S^CoA 



(5) 



-r .100 



E 



' .0T5 - 



E 

 10 



050 - 



-"I" 



.025 



pH 8.1 and wavelength 305 m/u, with acetoacetyl CoA and CoA-SH as substrates in the 



presence of Mg+-. Solutions of acetoacetyl CoA were prepared every few days by 



incubating synthetic succinyl CoA and acetoacetate with 



purified Co A-transf erase. When the reaction reached 



equilibrium, the pH of the mixture was brought to 5.5 



with acetic acid, the solution was heated to 75° for 2 



minutes to destroy the transferase, cooled, centrifuged 



and the supernatant adjusted to pH 8.0. AcetoacetylCoA 



was stable for several days if stored at -18° when not in 



use. 



The enzyme has been purified about 300 fold over 

 the initial phosphate extract through steps involving 

 ammonium sulfate and acetone fractionation, removal 

 of inactive proteins at pH 5.3, refractionation with am- 

 monium sulfate, and low temperature ethanol fraction- 

 ation in the presence of Zn ions. The time course of the 

 reaction in the optical test with varying concentrations 

 of purified pig heart enzyme is shown in Fig. 7. 



The thioclastic cleavage of acetoacetyl CoA to ace- 

 tyl CoA results not only in a decrease of light absorption 

 at 305 mix but also in a concomitant increase in the absorption in the 240 m/x region 



References p. 313I314. 



MINUTES 



Fig. 7. Optical thiolase test. 

 Tris ( hydroxymcthyl ) amino - 

 methane-HCI buflcr pH 8.1, 

 200 [xM; MgClg, 8.0 1.1M; re- 

 duced glutathione, 10. o [xM; 

 CoA-SH, 0.15 fiM; S-aceto- 

 acetylCoA,' — -0.03 ///I/. Volume, 

 1.5 ml; d = 0.5 cm; temp., 25°. 



