^•OL. 12 (1953) 



ACETYL COENZYME A SYNTHESIS 



145 



Identification of the reaction products. Chou had frequently observed that the amount 

 of phosphate Hberated was far below the amount of hydroxamic acid formed. Recently 

 these studies were resumed, particularly in 

 view of Lynen's identification of acetyl Co A 

 as the mercaptoester of CoA^^. Various ob- 

 servations then indicated that inorganic 

 pyrophosphate rather than orthophosphate 

 was being liberated. It was found that the 

 liberation of inorganic phosphate could be 

 almost completely suppressed by the addition 

 of fluoride. Under these conditions the easily 

 hydrolyzable phosphate did not change, but 

 a substance appeared which gave, with the 

 Fiske-Subbarow molybdate reagent, a slowly 

 developing colour similar to that observed 

 earlier by Sacks and Davenport with inor- 

 ganic pyrophosphate^^. In Table II an experi- 

 ment is presented with Co A as acetyl acceptor. 

 If fluoride is added, approximately equiva- 

 lent amounts of pyrophosphate appear, as 

 determined by the manganese precipitation 

 method of Kornberg^^ and also by a colori- 

 metric method utilizing the above-mentioned 

 colour increase with the molybdate reagent. 

 This colour reaction has been developed into the convenient colorimetric method for 

 determination of inorganic pyrophosphate, which will be described elsewhere. 



;o 



?0 30 40 



50 60 70 80 

 units CoA added 



Fig. 4. CoA-concentration curve. Each 

 tube contained, in 1 ml: 25 units CoA; 

 10 ^M ATP; 10 /^Mpotassiumacetate; 

 50 juM KF; 100 /J.M potassium phos- 

 phate buffer (pH 7.4); 200 /j.M 

 hydroxylamine neutralized with KOH 

 to pH 7.4; 10 /xM glutathione; 0.02 ml 

 (i unit) of yeast fraction i. Tubes were 

 incubated at 37° for 20 minutes at 

 which time 1.5 ml of the 10% FeClg 

 reagent were added. 



TABLE II 



EFFECT OF FLUORIDE ON PYROPHOSPHATE FORMATION 



I fiM CoA = 310 units. 



** Determined by colour increase. 



*** Determined by Mn precipitation method. 



Each vessel contained in i ml: 12 /^M ATP; 10 j^iM potassium 

 acetate; 10 fiM MgClgl 20 ^M HgS; 200 jxM tris(hydroxymethyl) 

 aminomethane buffer, pH 7.5 and 0.02 ml (20 units) of yeast enzyme 

 fraction 4. Vessels were incubated at 37° for 30 minutes. 



Without fluoride the excess of inorganic phosphate over the CoA-free blank corre- 

 sponds to about twice the amount of acetyl CoA formed. As shown by Kunitz^^, fluoride 

 inhibits strongly the pyrophosphatase which is present in our yeast fractions. For this 

 reason the presence of fluoride is necessary to preserve the pyrophosphate formed. 

 References p. i4g. 



