Purification and characterization of two fructose diphosphate aldolases from Escherichia coli (Crookes' strain).
Two fructose diphosphate aldolases (EC 4.1.2.13) were detected in extracts of Escherichia coli (Crookes' strain) grown on pyruvate or lactate. The two enzymes can be resolved by chromatography on DEAE-cellulose at pH7.5, or by gel filtration on Sephadex G-200, and both have been obtained in a pure state. One is a typical bacterial aldolase (class II) in that it is strongly inhibited by metal-chelating agents and is reactivated by bivalent metal ions, e.g. Ca(2+), Zn(2+). It is a dimer with a molecular weight of approx. 70000, and the K(m) value for fructose diphosphate is about 0.85mm. The other aldolase is not dependent on metal ions for its activity, but is inhibited by reduction with NaBH(4) in the presence of substrate. The K(m) value for fructose diphosphate is about 20mum (although the Lineweaver-Burk plot is not linear) and the enzyme is probably a tetramer with molecular weight approx. 140000. It has been crystallized. On the basis of these properties it is tentatively assigned to class I. The appearance of a class I aldolase in bacteria was unexpected, and its synthesis in E. coli is apparently favoured by conditions of gluconeogenesis. Only aldolase of class II was found in E. coli that had been grown on glucose. The significance of these results for the evolution of fructose diphosphate aldolases is briefly discussed.
D000596:Amino Acids / Q000032:analysis; D000645:Ammonium Sulfate; D001322:Autoanalysis; D001894:Borohydrides; D002413:Cations, Divalent; D002474:Cell-Free System; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D004492:Edetic Acid; D004553:Electric Conductivity; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology*; D005033:Ethylmaleimide; D005634:Fructose-Bisphosphate Aldolase / Q000032:analysis / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D013053:Spectrophotometry; D013056:Spectrophotometry, Ultraviolet
D000596:Amino Acids; D001894:Borohydrides; D002413:Cations, Divalent; D046911:Macromolecular Substances; D004492:Edetic Acid; D005634:Fructose-Bisphosphate Aldolase; D005033:Ethylmaleimide; D000645:Ammonium Sulfate