256 Charles Heidelberger and Van R. Potter 



has helped to remove the objections to the inclusion of citric 

 acid in the Krebs cycle, has given further information on the 

 stereochemistry of substrate-enzyme interaction, and may 

 be used as a tool for the investigation of new phenomena in 

 intermediary metabolism both in vitro and in vivo. 



REFERENCES 



Buchanan, J. M., Sakami, W., Gurin, S., and Wilson, D. W. (1945). 



J. biol. Chem.y 159, 695. 

 Evans, E. A., Jun., and Slotin, L. (1941). J. biol. Chem., 141, 439. 

 IsHERWOOD, F. A. (1946). Biochem. J., 32, 108. 

 LoRBER, v.. Utter, M. F., Rudney, H., and Cook, M. (1950). J. biol. 



Chem., 185, 689. 

 Martius, C, and Schorre, G. (1950). Liebig's Ann., 570, 140, 143. 

 Ogston, a. G. (1948). Nature, London, 162, 963, 

 Potter, V. R., and Heidelberger, C. (1949). Nature, London, 164, 



180. 

 Potter, V. R., and Heidelberger, C. (1951). Unpublished. 

 Potter, V. R., Heidelberger, C, and Busch, H. (1951). Unpublished. 

 Stern, J. R., and Ochoa, S. (1949). J. biol. Chem., 179, 491. 

 Weinhouse, S., Medes, G., Floyd, N. F., and Noda, L. (1945). J. 



biol. Chem., 161, 745. 

 Wilcox, P. E., Heidelberger, C, and Potter, V. R. (1950). J. 



Amer. Chem. Soc., 72, 5019. 

 Wood, H. G., Werkman, C. H., Hemingway, A., and Nier, A. O. 



(1942). J. biol. Chem., 142, 31. 



DISCUSSION 



Arnstein: In the synthesis of citric acid (Fig. 7) some of the radio- 

 activity was incorporated into the tertiary carboxyl group. Have you 

 any idea of the mechanism of this reaction? 



Heidelberger: We added HCN to ethyl y-chloroacetoacetate and 

 got this compound 

 ^ OH 



CI-CH2-C-CH2COOH 



CN 



The cyanide group was then hydrolysed to a carboxyl group but we 

 could not recrystallize the acids. There is some possibility that there 

 may have been some nitrile left that was not completely hydrolysed 

 under these conditions; if there were, this might have exchanged with 

 the labelled cyanide we added in the next step, and given us the result 

 that was obtained. This is a possible explanation, but fortunately 

 it did not affect the results of the a-keto story. 



