VOL. 12 (1953) BIOCHIMICA ET BIOPHYSICA ACTA I4I 



ACETYL COENZYME A SYNTHESIS THROUGH PYROPHOSPHORYL 

 SPLIT OF ADENOSINE TRIPHOSPHATE 



by 



MARY ELLEN JONES*, SIMON BLACK**. RUTH M. FLYNN, and FRITZ LIPMANN 



Biochemical Research Laboratory, Massachusetts General Hospital, 

 Department of Biological Chemistry, Harvard Medical School, Boston, Massachusetts (U.S.A.) 



Two different enzymic pathways exist for the activation of acetate. One pathway 

 is initiated by phosphorylation of acetate^ : 



acetate + ATP ^ acetyl phosphate + ADP*** (i) 



followed by acetyl transfer through transacetylase^- ^ to coenzyme A (CoA) : 



acetyl phosphate + CoA ^ acetyl CoA + phosphate (2) 



This relatively well understood mechanism is present in most micro-organisms*'^, but 

 not in animal tissues^. In tissues, yeast and probably also in some micro-organisms, 

 another enzymic pathway leads through a complex reaction between ATP, CoA and 

 acetate to acetyl CoA'>^. Inorganic pyrophosphate was recently found to be a product 

 of this reaction^' ^". It appears that a pyrophosphate split of ATP is involved in this 

 energy transfer, which is formulated as follows : 



ATP -f CoA + acetate ^ acetyl CoA -f AMP -f pyrophosphate (3) 



Earlier experiments seemed to support a primary reaction between ATP and CoA, 

 yielding presumably CoA-pyrophosphate. A more exhaustive study, however, has left 

 us undecided with regard to the finer mechanism of reaction^, which we assume to 

 represent a double transfer system of some kindi Some details of our experiments on 

 this problem are presented here. 



ENZYiME PREPARATIONS AND ASSAY SYSTEM 



Pigeon liver enzyme was prepared as described by Kaplan and Lipmann^^ and by Chou and 

 Lipmann12_ 



In most experiments yeast enzyme was used. Most brands of commercial Bakers' yeast were 

 found to be suitable; the best preparations were obtained with "National" brand yeast. 



The system extracts well from quick- frozen yeast; i part of yeast is mixed with i part of ether 

 and 1.5 parts of dry ice, and stirred well. After 30 minutes the liquid is poured off, the yeast spread 

 out in a thin layer on a cloth, and air is blown over it by a fan under a well-ventilated hood. An ad- 

 ditional 1.5 parts of dry ice are mixed with the yeast and ventilation continued until practically all 

 the ether is removed. The preparation is then generally stored in a deep freeze. On thawing, the yeast 



* Fellow of the Atomic Energy Commission. 



** Present address: National Institute for Arthritic and Metabolic Diseases, National Institutes 

 of Health, Bethesda 14, Maryland. 



***The following abbreviations have been used: ATP, adenosine triphosphate; ADP, adenosine 

 diphosphate; AMP, adenosine monophosphate; CoA, coenzyme A; PP, inorganic pyrophosphate; 

 and Pj, orthophosphate. 



§ Cf. addendum at end of paper (p. 147). 



References p. i4g. 



