Analysis of the metal requirement of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Escherichia coli.
The three isozymes of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Escherichia coli were overproduced, purified, and characterized with respect to their requirement for metal cofactor. The isolated isozymes contained 0.2-0.3 mol of iron/mol of enzyme monomer, variable amounts of zinc, and traces of copper. Enzymatic activity of the native enzymes was stimulated 3-4-fold by the addition of Fe2+ ions to the reaction mixture and was eliminated by treatment of the enzymes with EDTA. The chelated enzymes were reactivated by a variety of divalent metal ions, including Ca2+, Cd2+, Co2+, Cu2+, Fe2+, Mn2+, Ni2+, and Zn2+. The specific activities of the reactivated enzymes varied widely with the different metals as follows: Mn2+ greater than Cd2+, Fe2+ greater than Co2+ greater than Ni2+, Cu2+, Zn2+ much greater than Ca2+. Steady state kinetic analysis of the Mn2+, Fe2+, Co2+, and Zn2+ forms of the phenylalanine-sensitive isozyme (DAHPS(Phe)) revealed that metal variation significantly affected the apparent affinity for the substrate, erythrose 4-phosphate, but not for the second substrate, phosphoenolpyruvate, or for the feedback inhibitor, L-phenylalanine. The tetrameric DAHPS(Phe) exhibited positive homotropic cooperativity with respect to erythrose 4-phosphate, phophoenolpyruvate, and phenylalanine in the presence of all metals tested.
Department of Biology, University of Virginia, Charlottesville 22901.
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Stephens
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Bauerle
D010723:3-Deoxy-7-Phosphoheptulonate Synthase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D001667:Binding, Competitive; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology*; D007527:Isoenzymes / Q000378:metabolism; D007700:Kinetics; D008670:Metals / Q000378:metabolism*; D010649:Phenylalanine / Q000494:pharmacology; D011994:Recombinant Proteins / Q000378:metabolism; D013057:Spectrum Analysis; D013403:Sugar Phosphates / Q000378:metabolism
D007527:Isoenzymes; D008670:Metals; D011994:Recombinant Proteins; D013403:Sugar Phosphates; D010649:Phenylalanine; C026959:erythrose 4-phosphate; D010723:3-Deoxy-7-Phosphoheptulonate Synthase