Studies on the 5-enolpyruvylshikimate 3-phosphate synthase of Escherichia coli

1. A method for the purification of EPSP synthase of E. coli K12 has been developed. The purification procedure consisted of ammonium sulphate fractionation, ion-exchange chromatography and hydrophobic chromatography. The final step involved substrate elution from a phosphocellulose column. EPSP synthase was purified 843-fold and in 22% yield over the (NH4)2SO4 fraction. 2. E. coli EPSP synthase has been shown to be a monomeric enzyme. The subunit Mr was estimated to be 49,000 by SDS PAGE, and native Mr values of 42,000 and 55,000 were determined by gel filtration. Kinetic parameters for E. coli EPSP synthase are reported. The enzyme was inhibited by the herbicide glyphosate, inhibition was competitive with respect to phosphoenolpyruvate. 3. EPSP synthase has also been purified from an overproducing strain, E. coli AB2829/pKD501. The overproduced enzyme was purified 50-fold and in 30% yield over the crude extract fraction. EPSP synthase can be purified in milligram quantities from the overproducing strain. 4. The overproduced enzyme has been shown to be identical in its physical and kinetic properties to EPSP synthase purified from E. coli K12. The amino acid composition and N-terminal amino acid sequence of E. coli EPSP synthase are reported. 5. Chemical modification of EPSP synthase by 3-bromopyruvate has been examined. Although substrate protection against inactivation was observed, bromopyruvate did not appear to be an/ an active-site-directed reagent for E. coli EPSP synthase. 6. Phosphoserine aminotransferase has been purified from the overproducing strain, E. coli AB2829/pKD501. The purification procedure was similar to that developed for EPSP synthase; (NH4)2SO4 fractionation, ion-exchange chromatography and hydrophobic chromatography. The final step was ion-exchange chromatography on a mono-Q column. PSAT was purified approximately 7-fold over the crude extract fraction. 7. The subunit Mr of E. coli PSAT has been shown to be 39,000 and this enzyme appeared to be dimeric. The amino acid composition and N-terminal amino acid sequence of PSAT are reported. Some kinetic properties of this enzyme are also described