Enzymatic synthesis of citric acid. I. Synthesis with soluble enzymes.

The synthesis of citrate from acetate, ATP,’ and oxalacetate by soluble enzyme preparations from pigeon liver was briefly reported in 1949 (1). CoA was found to be required for activity. Similar results were later reported by Novelli and Lipmann (2) with preparations from yeast and Escherichiu coli, and by Elliott and Kalnitsky (3) with kidney acetone powder extracts. It was further shown (1) that citrate rather than cisaconitate or isocitrate was the product of the “acetate”-oxalacetate condensation. Acetoacetate (1) and other P-keto acids (4) could also serve as acetyl donors for citrate synthesis in pigeon liver preparations. It was further reported (4) that the above synthesis of citrate proceeds through at least two steps: (Q) acetate + ATP + “active acetate,” and (b) “active acetate” + oxalacetate -+ citrate. While the condensing enzyme catalyzing step (b) was found to be readily soluble and could be extracted from a variety of animal tissues, yeast, and bacteria, the enzyme or enzymes catalyzing step (a) was present in or could be extracted from only a few sources. These included pigeon liver, lyophilized yeast, E. coli, and Axotobacter agile. These preparations also contained condensing enzyme, but some preparations were obtained from E. coli which, while very active in catalyzing step (a), contained little or no condensing enzyme. Citrate was readily synthesized from acetate, ATP, and oxalacetate when these E. coli preparations were combined with preparations from a number of tissues (4). An account of the above work is given in the present paper. This work led to the isolation and crystallization of the condensing enzyme from pig heart, and this, in turn, facilitated an understanding of the