Studies on the mechanism of the citrate condensing enzyme reaction.

Studies with deuterium as a tracer have shown that wheat germ and pig heart malic dehydrogenase act on the keto form of oxaloacetic acid (1, 2). The keto form of this acid has also been shown to arise in the enzymatic carboxylation of phosphorylenolpyruvate by wheat germ phosphoryl-enolpyruvate carboxylase (3) and by avian liver phosphoryl-enolpyruvate carboxylase kinase (2). In an attempt to extend further our studies on the mechanism of the stereospecific reaction catalyzed by aconitase (4), it was deemed necessary to synthesize enzymatically monodeuterocitrate from stereospecifically labeled monodeuteromalate prepared in turn by the action of fumarase (5,6). To determine the feasibility of this experimental approach a study was undertaken to establish whether the keto or enol form of oxaloacetic acid is utilized by the condensing enzyme in the formation of citrate from acetyl coenzyme A. The results obtained in this study demonstrate that the keto form is the substrate of the citrate condensing enzyme.