170 



III. OXIDATION AND METABOLISM 



COOH 

 HC— H 



COOH 

 Oxaloacetic 

 acid 



+ 



H 



H O 



COOH 



HCH 



O 



X 



Condensing 



enzyme 



,i. 



/■ 



» HOC— CH2C— X + H2O 



H 



Active 

 acetate 



k 



;ooH 



Citric acid 

 intermediate 



\ 



COOH 



I 

 HC— H 



HOC— COOH + HX 



HC— H 



COOH 

 Citric acid 



The Condensation of Oxaloacetate and of Activated Acetate to Form Citric Acid^^ 



The condensing enzyme is present in a number of animal tissues, includ- 

 ing skeletal muscle, liver, kidney, brain, heart, as well as in yeast and 

 several bacterial systems, Escherichia coli (Escherich's carbohydrate-fer- 

 menting intestinal bacillus) and Azotobacter agile (aerobic bacterium from 

 water and soil, which fixes free nitrogen). The enzyme which activates 

 acetate apparently has wide distribution in animal tissues, as well as in E. 

 coli and A . agile.}^'^ A comprehensive chart indicating how acetoacetate and 

 acetic acid enter the citric acid cycle is given in Fig. 2. 



By means of this oxidation of one acetic acid molecule, six molecules of 

 H2O are formed, and four molecules of H2O are used; two molecules of 

 CO2 are formed. The net result is given in the following equation: 



-> 2CO2 + 2H2O + 209,000 cal.^^* 



CH3COOH + 2O2 - 



Stern and Ochoa^^^ demonstrated that acetate or acetoacetate condenses 

 with oxaloacetate to give rise to citric acid rather than to czs-aconitic 

 acid or to isocitric acid. This proof was rendered possible by the prepara- 

 tion from pigeon liver of an enzyme system relatively free from the cis- 

 aconitase enzyme system. Novelli and Lipmann,^^^'^^'' as well as Stern 

 and Ochoa,^^^ demonstrated that ATP, Mg++ ions, and CoA were necessary 

 for the formation of citrate from acetate and oxaloacetate. Since, in this 



"0 E. S. West and VV. R. Todd, Textbook of Biochemistry, 2nd ed., Macmillan, New- 

 York, 1955. 



«i J. R. Stern and S. Ochoa, /. Biol. Chem., 179, 491-492 (1949). 



"2 G. D. Novelli and F. Lipmann, J. Biol. Chem., 171, 833-834 (1947). 



6" G. D. Novelli and F. Lipmann, /. Biol. Chem., 182, 213-228 (1950). 



