\-0L. 12 (1953) PATHWAYS OF ACETATE OXIDATION 247 



oxidize acetate only by the dicarboxylic acid pathway but in other cells as well. The 

 enzyme has been found in the mitochondria of rat liver, of pigeon breast muscle, and in 

 rat skeletal muscle. It has not yet been possible to isolate the protein component, and 

 efforts are now being directed towards this goal. 



DISCUSSION 



The oxidation of acetate by the tricarboxylic acid cycle requires the presence of 

 citrogenase, Ochoa's condensing enzyme, and of isocitric acid dehydrogenase. Once 

 these two enzymes have been found in a particular cell or tissue, it may be concluded 

 that acetate metabolism can proceed via the citric acid cycle. Search for these two 

 enzymes in microorganisms thought to oxidize acetate through alternative pathways 

 has demonstrated their presence. Both enzymes were found in Aerobacter aerogenes, 

 Corynebaderium creatinovorans and Rhyzopus nigricans, previously considered to oxidize 

 acetate via the dicarboxylic acid cycle alone^~^. They were also found in the moulds 

 M. niveus and M. tremellostts, which had been thought to oxidize acetate via glycolic 

 acid^". Moreover, the culture fluid of these moulds contained citric, a-ketoglutaric, suc- 

 cinic, and malic acids - all intermediates of the citric acid cycle. The presence of glycolic 

 acid in the culture fluid of these moulds (as found by us) and in Aspergillus (as found by 

 Weinhouse^^) must be due to hydrolysis of malic acid by the enzyme malic hydrolase, 

 which was found in plants by Link et al.'^. The protozoa Tetrahymena geleii was also found 

 to oxidize acetate via the citric acid cycle. 



Although the citric acid cycle is thus extended to cover the mechanism of acetate 

 oxidation in cells hitherto considered to utilize alternative pathways, the origin of the 

 oxalacetic acid necessary for condensation with acetylcoenzyme A needs to be explain- 

 ed. Oxidation of acetate proceeds in these cells after an induction time of variable 

 length. The accumulation of succinic acid on oxidation of acetate in the presence of 

 malonate ivithout citric acid or glycolic acid formation is proof that acetic acid does 

 oxidize to succinic acid. In cell-free extracts of bacteria and moulds, as well as in mito- 

 chondria of liver and muscle, there was found an enzyme system which oxidized acetate 

 anaerobicalty as shown by the reduction of methylene blue. Furthermore, it was found 

 that after dialysis oxidation of acetate required the addition of coenzyme A, DPN, FAD 

 and Mg+2. From these experiments the anaerobic oxidation of acetate may be postul- 

 ated to proceed as follows : 



a) 2 Acetyl-S-CoA +DPN +2H20^HOOC-CH2CH2COOH +DPNH +H+ +2 HSCoA 



b) DPNH + H+ -f FAD ^ DPN -f FADH^ 



c) FADH2 + MB ^ FAD -f MBH2. 



The first reaction proceeds at a slow rate because of the low oxidation-reduction 

 potential of DPN as compared with that of acetate ^ succinate. It was found in fact 

 that reaction stopped when only 0.02 °o of the DPN was reduced. Once succinate is 

 formed, the production of oxalacetic acid is easily seen through its stepwise oxidation 

 to fumarate, fumarate - malate, and malate - oxalacetate. It is our belief that this 

 enzyme is universally present in all cells and that it is the source of the oxalacetate 

 required to initiate the oxidation of acetate via the citric acid cycle. It is quite possible 

 that impairment of this enzyme is responsible for the large aerobic glycolysis found by 

 References p. 24g. 



