304 F. LYNEN, S. OCHOA VOL. 12 (1953) 



enzyme preparation was active on a,j8-unsaturated and /3-hydroxy derivatives. The 

 formation of crotonyl- and ^-hydroxybutyryl CoA by liver enzymes under similar 



O 



— CHg— CH2— COOH + HS— CoA + ATP ^ — CHj— CH^— C— S— CoA + AMP + PP (_■) 



conditions has also been observed in other laboratories*^ •'^". The activation of higher 

 fatty acids, presumably C^^ to jg- by ATP and HS-CoA is catalyzed by yet another 

 enzyme discovered by Kornberg and Pricer^^-^^ in liver. Kornberg's enzyme 

 catalyzes the formation of S-stearyl CoA, AMP, and PP from stearic acid, HS-CoA and 

 ATP. 



CoA transferases. In CI. kluyveri extracts activation of fatty acids appears to occur 

 predominantly by transfer of CoA from acetyl-S-CoA. The first enzyme of the CoA 

 transferase type was discovered by Stadtman in extracts of CI. kluyveri and named 

 CoA transphorasg^''. The enzyme catalyzes the reversible transfer of CoA from acetyl- 

 CoA to propionate (Reaction 3). 



O O 



CH3— C— S— C0A + CH3— CHj— COOH ^ CH3— COOH + CH3— CH2— C— S— CoA (3) 



CI. kluyveri extracts also catalyze the transfer of CoA from acetyl CoA to butyrate, 

 vinyl acetate, and lactate^**^. This enzyme (enzymes) is (are) similar to the succinyl-CoA- 

 acetoacetate transferase of heart muscle but with different substrate specificity. 



The reversible transfer of CoA from succinyl CoA to acetoacetate was discovered 

 independently by Green and co workers-*- 2- and by Stern et al.^'^^. The enzyme 

 which, as already mentioned, is present in heart and probably in skeletal muscle and 

 kidney but not in liver, catalyzes Reaction 4. In the early stages of purification^^ the 



O 



H OOC— CH 2— CH 2— C— S~Co A + CH 3— CO— CH 2— COOH ;=i Q 



HOOC— CH2— CH 2— COOH + CH3— CO— CH 2— C— S— Co A ( \) 



enzyme assay was based on the rate of citric acid synthesis from succinyl CoA and aceto- 

 acetate in the presence of oxalacetate, an excess of thiolase, and crystalline citrate 

 condensing enzyme, as indicated by the reactions below: 



Succinyl — S — CoA -)- acetoacetate ^ succinate -)-acetoacetyl — S — CoA (transferase) 



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



2 Acetyl — S — C0A + 2 oxalacetate -f- 2 H„0 ^ 2 citrate + 2HS — CoA (citrate condensing enzyme) 



Sum: Succinyl — S — Co A + acetoacetate -|-HS — CoA + 2 oxalacetate + 2 HgO ^ Succinate + 



2 citrate + 2 HS — CoA 



A heated ammonium sulfate fraction from ox liver, free of transferase, was used as the 

 source of thiolase. After removal of thiolase, the transferase assay was based on the 

 increase in optical density at pH 8.1 and wavelength 305 m/x due to the formation of 

 acetoacetyl-S-CoA. Although magnesium ions are not required for the reaction, as 

 mentioned previously, Mg*"^ markedly augments tlie light absorption and was added to 

 the reaction mixture in order to increase the sensitivity of the assay. 



The enzyme has been isolated from pig heart and purified about 700-fold over the 

 initial phosphate extract^''^. The purification involved ammonium sulfate and acetone 

 fractionation, removal of inactive proteins by heat and liy adsorj^tion on Ca ])hosphate 



References p. 3131314- 



