THE CITRIC ACID CYCLE 225 



the role of an interrupted tricarboxylic acid cycle in the biogenesis of 

 citric acid (242). 



Extensive studies of the incorporation of isotopic carbon into the 

 citric acid molecule lend further and decisive support to the idea that 

 bulk citric acid synthesis in Aspergillus niger occurs via the citric acid 

 cycle. The labeling of citrate formed in the presence of C 14 2 is 

 consistent with fixation of carbon dioxide into oxalacetate and con- 

 densation of the oxalacetate with an acetyl fragment (191, 192, 198). 

 Lewis and Weinhouse (174) showed experimentally that the sym- 

 metrical citric acid molecule is asymmetrically synthesized, as first 

 suggested by Ogston (223). 



Experiments with the metabolism of labeled acetic acid (28, 49, 173) 

 similarly implicate the known initial reactions of the citric acid cycle 

 in the synthesis of citrate by A. niger. A study of the incorporation of 

 glucose-3,4-C 14 into citrate provides evidence that pyruvate, arising 

 from glucose, is converted to oxalacetate and an acetyl fragment, and 

 that these condense to yield citrate with almost theoretical incorpora- 

 tion of carbon dioxide and little or no randomization of the carboxyl 

 carbons of oxalacetate (58). The last observation is important in 

 showing that oxalacetate does not equilibrate with a symmetrical acid 

 like fumaric or succinic. 



Although it has been suggested (85) that a condensation of 2-carbon 

 fragments may yield the 4-carbon dicarboxylic acid skeleton for 

 citrate synthesis, work with isotopic acetate indicates that this pathway 

 is at best of minor significance (28, 173), and the studies of Cleland and 

 Johnson (58) with labeled glucose afford no evidence at all for such a 

 reaction. 



For Penicillium chrysogenum there is good evidence of the forma- 

 tion of citrate from acetyl phosphate and oxalacetate and for the 

 presence of enzyme systems oxidizing citrate, isocitrate, a-ketoglutarate, 

 succinate, and malate (51, 126, 229). A partially purified succinic 

 dehydrogenase is inhibited by malonate (110), and acetate oxidation 

 is depressed by the same poison (13). The incorporation of acetate 

 carbon into glutamic acid conforms to that expected if the citric acid 

 cycle is responsible for the metabolism of acetate (113). 



Extracts of Streptomyces coelicolor carry out the major reactions of 

 the cycle, including the formation of citrate, the oxidation of «- 

 ketoglutarate and other acids, and the decarboxylation of oxalacetate 

 (65). The incorporation of labeled carbon dioxide and acetate into 

 amino acids by S. griseus is also consistent with the operation of the 

 cycle (39, 104). 



