The mechanism of enzymatic carbon dioxide fixation into oxal-acetate.

Studies of the non-enzymatic decarboxylation of /3-keto acids have shown that it is the keto form of the 8-keto acid which loses CO2 to form an enol of the product. This was demonstrated for dimethylacetoacetic acid by Pedersen (1) and for dimethyloxalacetic acid by Steinberger and Westheimer (2). These latter authors studied the catalytic effect of metal ions on the decarboxylation reaction and suggested that the non-enzymatic reaction be taken as a model for similar enzymatic decarboxylations. Kornberg et al. (3) observed the formation of an enol band during the enzymatic decarboxylation of oxalacetic acid and of another P-keto acid, oxalsuccinic acid. They regarded their results as evidence that an enol form of the P-keto acid was decarboxylated, although their data do not appear to preclude the possibility that an enol appears as a product of the decarboxylation (4). Recent studies in this laboratory have shown that wheat germ extracts catalyze an irreversible carboxylation reaction which is essentially the reverse of a decarboxylation. This reaction consists of the addition of CO2 to phosphoenolpyruvate to form oxalacetate and inorganic phosphate (5).’ If the mechanism for the non-enzymatic decarboxylation should apply in reverse to this reaction, then the enol, PEP, should add CO2 to give the keto form of oxalacetate. Experiments with deuterium described in this paper have provided unequivocal evidence that the oxalacetate formed enzymatically in the carboxylation reaction is in fact the keto form and not an enol. These results have been described in a preliminary publication (6).