204 A SYMPOSIUM ON RESPIRATORY ENZYMES 



can also oxidize citric acid to alpha-ketoglutaric acid, the reaction 

 of Wood and Werkman involving the carboxylation of pyruvic 

 acid to yield oxalacetate (14) offers an attractive hypothesis for 

 alpha-ketoglutarate synthesis, i.e., oxalacetate + pyruvate -^ citrate 

 -> alpha-ketoglutarate. Slotin and I carried out experiments to test 

 this hypothesis, using carbon dioxide containing radioactive C" 

 and found that the succinic acid formed on the addition of pyruvate 

 to malonate-poisoned pigeon liver, presumably through the inter- 

 mediate formation of alpha-ketoglutaric acid, was devoid of radio- 

 activity. However, when we extended these experiments to the iso- 

 lation of alpha-ketoglutaric acid as the dinitrophenylhydrazone, we 

 could clearly observe the utilization of carbon dioxide in the forma- 

 tion of radioactive alpha-ketoglutarate (15). As the earlier succinate 

 experiments suggested, and as was confirmed later by direct exami- 

 nation of the isolated keto acid, the radioactivity is confined entirely 

 to the carboxyl group of this compound. 



The fact that carbon dioxide is used in the synthesis of alpha- 

 ketoglutarate from pyruvate in pigeon liver and that the assimilated 

 carbon dioxide is present entirely in the carboxyl group alpha to 

 the carbonyl oxygen, is now firmly established in view of the iden- 

 tical results of Wood, Werkman, Hemingway, and Nier (12), using 

 the stable C^^, and of Slotin and me, with the short-lived radioactive 

 C" (11, 15). These data cannot be reconciled with the intermedi- 

 ate formation of citric acid, since any symmetrical intermediate mole- 

 cule of this type would yield alpha-ketoglutarate with radio- 

 activity at both carboxyls. Therefore the original conception of 

 alpha-ketoglutarate synthesis occurring by way of pyruvate and 

 carbon dioxide condensation to oxalacetate, and the subsequent 

 reaction of this dicarboxylic acid with another mole of pyruvate 

 to yield citric acid, must be abandoned. Support for such a view 

 is also derived from experiments in which non-radioactive citrate 

 was added to liver suspension in radioactive bicarbonate medium 

 during the synthesis of alpha-ketoglutaric acid. Table 2 lists ex- 

 periments showing that the addition of 25 mg. of sodium citrate 

 affects neither the yield of alpha-ketoglutaric acid nor the ratio of 

 activity per mg. carbon of the alpha-ketoglutarate to that of the 

 medium. If citrate is an inteiTnediate in the formation of alpha- 

 ketoglutaric acid from pyruvate and carbon dioxide, the addition 

 of non-radioactive citrate to the synthesizing tissue should yield 

 alpha-ketoglutaric acid in which the radioactivity had been con- 

 siderably diluted. Failure to demonstrate such an effect may be 



