Purification and regulatory properties of isocitrate lyase from Escherichia coli ML308.
Isocitrate lyase was purified to homogeneity from Escherichia coli ML308. Its subunit Mr and native Mr were 44,670 +/- 460 and 17,000-180,000 respectively. The kinetic mechanism of the enzyme was investigated by using product and dead-end inhibitors of the cleavage and condensation reactions. The data indicated a random-order equilibrium mechanism, with formation of a ternary enzyme-isocitrate-succinate complex. In an attempt to predict the properties of isocitrate lyase in intact cells, the effects of pH, inorganic anions and potential regulatory metabolites on the enzyme were studied. The Km of the enzyme for isocitrate was 63 microM at physiological pH and in the absence of competing anions. Chloride, phosphate and sulphate ions inhibited competitively with respect to isocitrate. Phosphoenolpyruvate inhibited non-competitively with respect to isocitrate, but the Ki value suggested that this effect was unlikely to be significant in intact cells. 3-Phosphoglycerate was a competitive inhibitor. At the concentration reported to occur in intact cells, this metabolite would have a significant effect on the activity of isocitrate lyase. The available data suggest that the Km of isocitrate lyase for isocitrate is similar to the concentration of isocitrate in E. coli cells growing on acetate, about one order of magnitude higher than the Km determined in vitro in the absence of competing anions.
MacKintosh
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Department of Biochemistry, University of Glasgow, Scotland, U.K.
Nimmo
H G
HG
D000838:Anions / Q000494:pharmacology; D002850:Chromatography, Gel; D004926:Escherichia coli / Q000201:enzymology*; D006016:Glycolates / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007522:Isocitrate Lyase / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D007652:Oxo-Acid-Lyases / Q000302:isolation & purification*; D010728:Phosphoenolpyruvate / Q000494:pharmacology; D011189:Potassium Chloride / Q000494:pharmacology; D013386:Succinates / Q000494:pharmacology; D019802:Succinic Acid
D000838:Anions; D006016:Glycolates; D013386:Succinates; C031149:glycolic acid; D011189:Potassium Chloride; D010728:Phosphoenolpyruvate; D019802:Succinic Acid; D007652:Oxo-Acid-Lyases; D007522:Isocitrate Lyase