VOL. 12 (1953) 



PATHWAYS OF ACETATE OXIDATION 



245 



1800 



1500 



1200 



])ed when 40% of the added malonate was oxidized (Fig. 4). The R.Q. value of this 



oxidation was 1.47 an indication of complete oxidation (HOOCCHgCOOH + 2 Og -= 3 



CO2 + 2H20).The induction time, which may last as long as two hours when the 



amount of bacteria is small, was thought to 



be due to a slow decarboxylation of malonate 



preceding the oxidation (HOOCCH2COOH - 



CO2 + CH3COOH). There was, however, no COg 



production when the bacterial suspension was 



incubated in the presence of malonate with 



nitrogen as the gas phase. In spite of this 



oxidation, malonate inhibited effectively acetate 



oxidation (Fig. 5). 



Inhibition of acetate oxidation by malonate 

 maybe considered as evidence that the oxidation 

 proceeds through the dicarboxylic or the tricar- 

 boxylic acid pathway. There would be no inhi- 

 bition if the oxidation proceeded through another 

 pathway which had no succinate as an inter- 

 mediate oxidation product. Negative experi- 

 ments, however may mean only lack of penetra- 

 tion of malonate. Thus acetate oxidation by 

 moulds was not inhibited by malonate, although 

 there were found in the culture media all the 

 intermediates of the citric acid cycle. Malonate 

 inhibition experiments could be utilized as an 

 indication of the presence of the dicarboxylic 

 acid cycle by determination of citric and suc- 

 cinic acids after oxidation of acetate in the pre- 

 sence and in the absence of malonate. It is 

 known that baker's yeast produces citric and suc- 

 cinic acids on oxidation of acetic acid^^, and that 

 the pathway of this oxidation, mainly the tricar- 

 boxylic acid cycle, can also be the dicarboxylic 

 acid cycle^^'^'. In agreement with these views, on 



oxidation of acetate by yeast at pH 4.0 there were found 44 mm^ of citrate, and traces 

 of succinate, whereas in the presence of malonate citrate was four times as low and suc- 

 cinate increased considerably (Table VIII). In Corynebacterium creatinovorans, Barron 

 et al.^, found on oxidation of acetate in the presence of malonate an accumulation of 

 succinate with no citric acid formation. These experiments were repeated, using paper 

 chromatography for the detection of the intermediate acids formed. At pH 5.54, in the 

 presence of acetate, 2,030 mm^ of Og were used up. After ether extraction of the acidified 

 solution citric and a-ketoglutaric acids were detected, an indication that oxidation of 

 acetate proceeded via the citric acid cycle. In the presence of malonate, there was an 

 uptake of 200 mm^ and in the paper chromatograms only succinic acid was detected. 

 In Aerohacter aerogenes at pH 5.54 there was an O^ uptake of 2,280 mm^ in the presence 

 of acetate ; in the paper chromatograms there were detected citric, a-ketoglutaric, and 

 succinic acids. With acetate plus malonate, there was an Og uptake of 520 mm^; in the 

 References p. 24^. 



Fig. 5. Effect of Malonate on the Oxi- 

 dation of Acetate by A . aerogenes. 

 Phosphate buffer o,o3Af, pH 5.54. 

 Acetate, and malonate, o.oiAf. Abs- 

 cissa, time in minutes. Ordinate, Oj 

 uptake in /il. i. Acetate; 2. Acetate 

 + malonate; 3. Malonate (all figures 

 blank subtracted). Temp. 38°. 



