DICARBOXYLIC ACIDS IN RESPIRATION 227 



The formation of 4-carbon dicarboxylic acids has often been as- 

 cribed to the Thunberg condensation solely on the basis that resting 

 or growing cells given acetate form one or more of the dicarboxylic 

 acids. By itself, this observation is of no significance and cannot be 

 offered as evidence for the Thunberg reaction. 



The most effective evidence for some sort of Thunberg reaction in 

 fungi comes from studies on the formation of fumaric acid by Rhizo- 

 pns nigricans, and consists of the finding that the organism forms 

 fumarate from ethanol in yields so high as to make it unlikely that the 

 citric acid cycle is the pathway responsible (89). The reaction may be 

 written: 



2C*H 3 CH 2 OH + 2 ^ 2C*H 3 COOH 



-> HOOC— C*H=C*H— COOH (13) 



As indicated in the equation, the methyl carbons of ethanol appear as 

 the methine carbons of fumarate. However, in this instance the dis- 

 tribution of isotope is not of critical value, i.e., the labeling results do 

 not require us to postulate the Thunberg reaction (2, 283, 309); the 

 entrance of formic acid into the methyl group of pyruvate also com- 

 plicates the interpretation of the isotope data (151, 152). 



It has been suggested that an "acetic dehydrogenase" of Aspergillus 

 fumigatus carries out the Thunberg reaction (12), but the products of 

 the reaction have not been isolated. 



Studies on bacteria offer a possible model for the results on Rhizopus 

 nigricans. The enzyme malate synthetase of Escherichia coli carries 

 out the reaction (325): 



Acetyl phosphate + glyoxylate —> malate (14) 



Kornberg and Madsen (164) find the same enzyme in Pseudomonas sp. 

 and suggest the following sequence of reactions: 



Acetate + oxalacetate — » citrate O^a) 



Citrate —> isocitrate (15b) 



Isocitrate— > succinate + glyoxalate (15c) 



Acetate + coenzyme A—» acetyl-CoA (15d) 



Acetyl-CoA + glyoxylate-^ malate -f CoA (15e) 



Malate — > oxalacetate + 2[H] (15f) 



2 Acetate-^ succinate + 2[H] (15) 



This complex system requires condensing enzyme (15a), aconitase 

 (15b), isocitritase (15c), some mechanism generating acetyl-coenzyme A 

 (15d), malate synthetase (15e), and malic dehydrogenase (15f). Al- 



