Citritase, the citrate-splitting enzyme from Escherichia coli. II. Reaction mechanisms.

Spectrophotometric Measurements of Enzymatic Activity-The enzymatic activities of aconitase and isocitric, Lu-ketoglutaric, malic, lactic, and glutamic dehydrogenases were followed in the Beckman DU spectrophotometer in the presence of either triphosphopyridine or diphosphopyridine nucleotide and the requisite substrates. Fumarase activity was detected by an increase in absorption at 240 rnp with malate as substrate (2). The color of the formazan derivative produced as a result of the reduction of 2,3,5triphenyltetrazolium chloride by succinic acid in the presence of enzyme was used as a qualitative index of succinic dehydrogenase activity (3). Acetateactivating enzyme and condensing enzyme activities were measured according to the methods of Novelli and Lipmann (4). Oxalacetate decarboxylase activity was determined manometrically and was based on the rate of decarboxylation of oxalacetic acid in the presence of enzyme, substrate, Mn ions, and buffer (5). Preparation of Radioactive Citric Acid-Radioactive citrate was prepared by incubating oxalacetate, 2-Cr4-acetate, ATP,l CoA, cysteine, Mg ions, and carrier substrate together with crude extracts obtained from acetate-grown