During growth of Escherichia coli ML308 on pyruvate in a continuous culture (turbidostat) or batch culture, flux of carbon into the cells exceeds the amphibolic capacity of the central pathways. This is balanced by diversion of carbon flux to acetate excretion which in turn diminishes the efficiency of carbon conversion to biomass [g] dry wt (mol substrate)-1]. However, restriction of carbon supply in a chemostat diminishes flux to acetate excretion and at a dilution rate (D = mu) of 0.35 h-1 or less, no flux to acetate excretion was sustained thus permitting perfect balance between carbon input on the one hand, and the output to biosynthesis and energy generation on the other. This, in turn, improves the efficiency of carbon conversion to biomass. Inclusion of 3-bromopyruvate (an inhibitor of pyruvate dehydrogenase) at a concentration which diminishes growth rate (mu) to 0.35 h-1 or less also prevented flux to acetate excretion. Furthermore, in a family of fluoroacetate-resistant strains, excessive flux of pyruvate was balanced by diversion of carbon flux to lactate excretion rather than acetate and a higher growth rate (mu = 0.63 h-1) was sustained.
SUMMARYEscherichia coZi grown aerobically in glucose + salts medium excretes acetate. On glucose exhaustion, the cells synthesize the enzymes of the glyoxylate bypass which permits growth on acetate as sole source of carbon and energy. Concurrently, the activity of isocitrate dehydrogenase falls to 20 % and, when the acetate is exhausted, is restored to 75 % of the original level. Similar results are obtained after growth on substrates (e.g. glycerol) which do not promote excretion of acetate, provided acetate is added to the medium at the end of growth. This control of isocitrate dehydrogenase activity is apparently a mechanism which restricts the flow of carbon round the tricarboxylic acid cycle and favours operation of the glyoxylate bypass.
1. In Escherichia coli ML308 isocitrate dehydrogenase is partially inactivated during growth on acetate [Bennett, P. M. and Holms, W. H. (1975) f. Gee. Microhiol. 87,[37][38][39][40][41][42][43][44][45][46][47][48][49][50][51].2. The active form of isocitrate dehydrogenase was purified to homogeneity from cells grown on glycerol. The key step in the procedure was chromatography on procion-red-Sepharose, from which the enzyme was specifically eluted with NADP'.3. Two forms of isocitrate dehydrogenase were purified to homogeneity from cells grown on acetate. One form did not bind to procion-red-Sepharose and was essentially inactive; this form could be resolved from the active form by non-denaturing gel electrophoresis. The other form was specifically eluted from procion-red-Sepharose and was partially active; analysis of this form by non-denaturing gel electrophoresis suggested that it was a mixture of the active and inactive forms.4. The three forms comigrated on denaturing gel electrophoresls and were identical by the criterion of onedimensional peptide mapping.5. Analysis of the active and inactive forms by sedimentation equilibrium centrifugation and non-denaturing gel electrophoresis showed that they differed in charge but not in size. Amino acid analysis and two-dimensional peptide mapping showed that both forms were dimers of identical subunits. 6.The active form of the enzyme contained no detectable alkali-labile phosphate, the inactive form contained 0.8 molecule/subunit and the partially active form contained an intermediate amount.7. The data suggest that the active and inactive forms of isocitrate dehydrogenase differ only in the presence of one phosphate group per subunit in the latter form; this is consistent with our results from phosphorylation of isocitrate dehydrogenase in vitro (Following paper in this journal).8. The nature of the partially active form of isocitrate dehydrogenase and the significance of the results are discussed.When Escherichia coli grows on acetate as sole source of carbon and energy the enzymes of the glyoxylate bypass, isocitrate lyase and malate synthase, are induced in order to provide the necessary precursors for biosynthesis [l] [9,10]. The phosphorylation of ICDH is of particular interest because this system is the first example of metabolic control of a bacterial enzyme by this mechanism. We are engaged in a study of the molecular details and the metabolic significance of the phosphorylation of ICDH in E. coli ML308. We report here the isolation of homogeneous preparations of ICDH from cells grown o n glycerol or acetate and we demonstrate the existence of a totally inactive phosphorylated form of the enzyme. In the accompanying papers we discuss the puritication, characterisation and regulatory properties of ICDH kinasc and ICDH phosphatase [ l l , 121.
1. Isocitrate dehydrogenase kinase and isocitrate dehydrogenase phosphatase were purified over 1 000-fold from Escherichiu coli ML308 by a procedure involving fractionation with (NHJ2S04 and chromatography on DEAEcellulose, blue-dextran-Sepharose and Sephadex G 1 50. 4. Analysis of a partial acid hydrolysate of phosphorylated isocitrate dehydrogenase showed that the only phosphoamino acid present was phosphoserine. 2.5. Isocitrate dehydrogenase phosphatase catalysed the release of 32P from 32P-phosphorylated isocitrate dehydrogenase; it required either ADP or ATP for activity. In the presence of ADP, or ATP plus an inhibitor of the kinase, the phosphatase cdtalysed full reactivation of isocitrate dehydrogenase and there was a good correlation between reactivation and the release of phosphate. In the presence of ATP alone the phosphatase catalysed the release of 32P from phosphorylated isocitrate dehydrogenase but the activity of the dehydrogenase remained low, indicating that the kinase and phosphatase were active simultaneously in these conditions.6. The active and inactive forms of isocitrate dehydrogenase can be resolved by non-denaturing gel electrophoresis ; the two forms of the enzyme were interconverted by phosphorylation and dephosphorylation in uitro. The extent of the interconversion correlated well with the changes in isocitrate dehydrogenase activity. This system is of considerable interest because it is the first example of metabolic regulation involving reversible phosphorylation of an enzyme to be discovered in bacteria. Recently Laporte and Koshland [6] showed that the enzymic activities responsible for the phosphorylation and dephosphorylation of ICDH, namely ICDH kinase and TCDH phosphatase, copurify and seem to comprise a bifunctional enzyme. However, the reactions cdtalysed by the kinase and the phosphatase were not fully characterised and the effects of phosphorylation and dephosphorylation on the activity of ICDH were not studied.
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