VOL. 12 (1953) 



ACETYL COENZYME A SYNTHESIS 



147 



Reversibility. The conversion of the energy-rich bond of acetyl CoA into the energy- 

 rich pyrophosphoryl bond of ATP was demonstrated by using acetyl phosphate and 

 phosphotransacetylase as acetyl feeder system with catalytic amounts of CoA. The ex- 

 periment of Table V shows that the addition of pyrophosphate and AMP is necessary 

 for this reaction. 



Furthermore, the reversibility of the reaction can be shown spectrophotometrically 

 using the mercaptoester absorption at 232 |U.M as described by Stadtman^^ for determina- 

 tion of acetyl CoA. As shown in Fig. 5, on mixing the enzyme, ATP, CoA and acetate, 



0/25 



r 



8 0100- 

 a: 



i 0.075 



0050 



0025 



31 uM PP 



0.1 35i 



0.100 



9 3 M PP 

 * _ - — 



;' 0075 



/ Initial concentration 

 I Ac - 1.93 /.M/ml 



' ATP-016 fiM/mlaoSO 

 CoA -015 ,nM/ml 



0.025 



20 40 



60 80 

 minutes 



60 80 

 minutes 



the absorption reaches steady values in 

 about 10 minutes. If at this time pyro- 

 phosphate is added, a rather rapid de- 

 crease of the acetyl CoA absorption is 

 observed, the degree of which depends 

 on the pyrophosphate concentration. 

 The reason for the slow secondary de- 

 cline in the absorption of acetyl CoA 

 with higher pyrophosphate concentra- 

 tion is not understood at the present 

 time. 



Attempts to clarify the finer mecha- 

 nism of the reaction. In a previous report 

 experiments were presented which 

 seemed to indicate a primary reaction 

 between pyrophosphate and acetyl CoA 

 and were interpreted as an indication 

 of the formation of a CoA-pyrophos- 

 phate. However, it has been difficult 

 to reproduce these experiments with 

 more highly purified acetyl CoA prepa- 

 rations. It is now suspected that the 

 disappearance of acetyl CoA under these 

 conditions may be explained through 

 contamination with small amounts of 

 AMP. If large amounts of pyrophos- 

 phate are added, very little AMP is 

 needed for the reverse reaction. At- 

 tempts also were made to obtain a reaction between ATP and CoA. Frequently in such 

 experiments a small disappearance of ATP may be observed without addition of acetate. 

 However, it was found very difficult to exclude a contamination with small amounts 

 of acetate, to which this system is quite sensitive. 



We conclude, therefore, that a separate reaction either between ATP and CoA or 

 between acetyl CoA and pyrophosphate has not been proven convincingly. Furthermore, 

 attempts to divide the system into two enzyme fractions have not given encouraging 

 results (see also Addendum) . 



Fig. 5. Reversibility of ATP-CoA-acetate reac- 

 tion by addition of pyrophosphate. Each cell 

 contained, per ml: 0.15 ^M CoA-SH (reduced 

 with potassium borohydride) ; 0.16 fxM ATP; 

 1.93 i^iM potassium acetate; 35 f^iM KF; 2 ^M 

 MgClj; 40 iiM tris (hydroxymethyl) amino- 

 methane buffer, pH 7.5; and o.ooi ml (i unit) 

 of yeast enzyme fraction 4. 



Left Figure : Solid circles = control cell to which 

 no pyrophosphate is added. Open circles = cell 

 which contains the same reactants as the control 

 tube except that 3.1 fxM potassium pyrophos- 

 phate buffer, pH 7.5, are added at the time in- 

 dicated by the arrow. Crosses = cells which 

 contain the same reactants as the control cell 

 except that 3.1 [,iM potassium pyrophosphate 



buffer, pH 7.5, are added at the start. 



Right Figure: Curves as above except that 9.3 



/fM of the pyrophosphate buffer, pH 7.5, were 



added to the open circle and cross curves rather 



than 3.1 [jtM. 



Addendum {added in proof) 



Further information on the finer mechanism was recently obtained by Jones, 

 References p. i4g. 



