Studies on citrate synthase.

I The mode of action of a number of inhibitors of citrate synthase has been studied. The results suggest that ?-oxoglutarate acts as an allosteric inhibitor of the enzyme from Gram negative facultatively anaerobic bacteria, but as an isosteric inhibitor of the enzyme from other sources. Similarly, NADH has been shown to be a powerful allosteric inhibitor of citrate synthases from Gram negative bacteria, but an isosteric inhibitor of the enzyme from Gram positive bacteria and eucaryotes. Other nucleotides have been shown to act only as isosteric inhibitors of citrate synthases from all sources examined. II Techniques have been developed which facilitate the rapid determination of the regulatory properties of a citrate synthase and its molecular size. The striking correlation between these properties and the Gram reaction of bacteria is discussed. It has been proposed that the rapid techniques described here could be of value in bacterial taxonomic studies and for bacteriological identification. III A citrate synthase deficient mutant of Escherichia coli has been isolated using a penicillin enrichment technique. A method has been developed which allows for the direct selection of citrate synthase deficient mutants by virtue of their intrinsic resistance to fluoro- acetate. Two citrate synthase deficient strains of Acinetobacter Iwoffi have been isolated using this method. IV A number of revertants, which have regained citrate synthase activity, were isolated from these citrate synthase deficient strains. A comparative study of the molecular, catalytic and regulatory properties of these enzymes has been carried out and possible structure-function relationships have been discussed. V Using revertant strains of E. coli which produce citrate synthases with regulatory properties different to those of the enzyme from the wild type organism, an attempt has been made to investigate the physiological significance of this altered regulatory behaviour of the enzyme. Revertants which have a citrate synthase which is not inhibited by ?-oxoglutarate (an allosteric inhibitor of the enzyme from E. coli wild type) appear to overproduce and excrete this (or a related) compound under certain growth conditions. Such a finding does suggest that the ?-oxoglutarate inhibition of citrate synthase has a physiological role in the regulation of this enzyme in this organism.