VOL. 12 (1953) ENZYMES OF FATTY ACID MET.\BOLISM 307 



due to the formation ot a second thioester bond, since two molecules oi acetyl CoA are 

 formed per molecule of acetoacetyl CoA disappearing. In the presence of CoA-SH, oxal- 

 acetate, thiolase, and citrate condensing enzyme, acetoacetyl CoA yields two molecules 

 of citrate per molecule of sulfhydryl [i.e., per molecule of CoA-SH) appearing^^, accor- 

 ding to the following reactions: 



Acetoacetyl — S — CoA+HS — CoA ^ 2 acetyl — S — CoA (thiolase) 



2 Acetyl — S — C0A+2 oxalacetate-f-2H20 :^ 2 citrate + aHS — CoA (citrate condensing enzyme) 



Sum- Acetoacetyl — S — CoA + HS — CoA + 2 oxalacetate + aHgO ^ 2 citrate + 2 HS — CoA 



The equilibrium position of the thiolase reaction is very far toward cleavage^^-^^'"". 

 For this reason it was not feasible to use this reaction for the isolation of acetoacetyl 

 CoA. Determinations of the equilibrium constant (A'gq = (Acetyl-S-Co A) 2/ (Acetoacetyl 

 CoA) (CoA-SH)) by means of the optical method gave an approximate value of 5-10^ at 

 pH 8.1, and I • 10* at pH 9.0. 



The reversibility of the reaction can be demonstrated by the optical method as 

 previously reported^. The synthesis of acetoacetyl CoA from acetyl CoA can be followed 

 directly at alkaline pH (■ — ' 9.0) as a small increase in the optical density at 305 m/^ in 

 the presence of large amounts of acetyl CoA^^^, or indirectly through coupling with the 

 j8-keto reductase to effect the oxidation of reduced DPN^^. 



The purified heart enzyme is highly specific for acetoacetyl CoA. /3-Ketovaleryl CoA 

 reacts at 20% of the rate of acetoacetyl CoA, and /3-ketocaproyl- and ?socaproyl-CoA 

 react practically not at all. This is in contrast to the broader specificity of crude enzyme 

 fractions^^ and indicates that there must be other thiolases, acting on S-j8-ketoacyl CoA 

 derivatives of higher chain length. 



Under the conditions of the optical test, the best preparations of the heart enzyme 

 so far obtained catalyze the cleavage of 3000 to 4000 moles of acetoacetyl CoA per 

 minute per 100,000 g of enzyme at 25°. When coupled with j8-keto reductase about 30 

 times more acetoacetate condensing enzyme must be used in the back reaction to reach 

 the rates obtained in the direction of acetoacetyl CoA cleavage. 



/3-Ketothiolase has been found to be inhibited by sulfhydryl reagents such as 

 iodoacetic acid or arsenoxide'*^^. This indicates that the enzyme is an "SH enzyme" and 

 suggests the following mechanism of action : 



O 9 9 



!l , I II _ 



(a) R—CHj— CO— CHg— C— S— CoA + HS-Enzyme ^ R—CHg— C— S-Enzyme + CHg— C— S— CoA 



O O 



(b) R— CHg— C— S-Enzyme + HS— CoA ^ R— CHg— C— S— CoA + HS-Enzyme 



Such a mechanism is further supported by experiments'*^^ with CoA labelled with ^^S. 



On incubation of propionyl-S-CoA with ^^S-H-CoA, in the presence of purified heart 



thiolase, radioactive propionyl-S-CoA is formed indicating the occurrence ot the following 



reaction : 



Propionyl S — CoA+HS-enzyme :^ Propionyl-S-enzyme + HS- — CoA 



The above mechanism provides an explanation for the unequal isotope distribution 

 in acetoacetate observed during oxidation of isotopic tatty acids in liver^'''*^'^^'^^'^^. 

 For example, octanoic acid labelled with ^^C in the carboxyl group can yield acetoacetate 

 in which the ratio of the radioactivity in the carbonyl and carboxyl carbons is less than 

 unity. Some acetoacetate labelled exclusively in the carboxyl group must arise when 



References p. 313I314. 



