3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase. Purification and molecular characterization of the phenylalanine-sensitive isoenzyme from Escherichia coli.
The phenylalanine-sensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (7-phospho-2-keto-3-deoxy-D-arabino-heptonate D-erythrose-4-phosphate lyase (pyruvate phosphorylating), EC 4.2.1.15) was purified to apparent homogeneity from extracts of Escherichia coli K12. The enzyme has a molecular weight of 140,000 as judged by gel filtration and sedimentation equilibrium analysis. The enzyme has a subunit molecular weight of 35,000 as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, suggesting that the native form of the enzyme is a tetramer. This was confirmed by cross-linking the enzyme with dimethylsuberimidate and by analyzing the cross-linked material by gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme shows a narrow pH optimum between pH 6.5 and 7.0. The enzyme is stable for several months when stored at -20 degrees C in buffers containing phosphoenolpyruvate. Removal of phosphoenolpyruvate causes an irreversible inactivation of the enzyme. The enzyme is strongly inhibited by L-phenylalanine and to a lesser degree by dihydrophenylalanine. Molecular parameters of the previously isolated tyrosine-sensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase from E. coli (Schoner, R., and Herrmann, K.M. (1976) J. Biol. Chem. 251, 5440-5447) are compared with those of the phenylalanine-sensitive isoenzyme from the same organism.
D010723:3-Deoxy-7-Phosphoheptulonate Synthase / Q000302:isolation & purification / Q000378:metabolism; D000446:Aldehyde-Lyases / Q000302:isolation & purification; D000596:Amino Acids / Q000032:analysis; D003035:Cobalt / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D010649:Phenylalanine / Q000494:pharmacology
D000596:Amino Acids; D007527:Isoenzymes; D046911:Macromolecular Substances; D003035:Cobalt; D010649:Phenylalanine; D010723:3-Deoxy-7-Phosphoheptulonate Synthase; D000446:Aldehyde-Lyases