5-Enolpyruvylshikimate-3-phosphate synthase of Klebsiella pneumoniae. 1. Purification and properties.
The shikimate pathway enzyme 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase (3-phosphoshikimate 1-carboxyvinyltransferase, EC 2.5.1.19) has been purified to apparent homogeneity from Aerobacter aerogenes, strain 62-1 (= Klebsiella pneumoniae ATCC 25306). A 3300-fold purification of the enzyme was achieved by ammonium sulfate fractionation, heat precipitation, chromatography on DEAE-cellulose, Sephadex G-75, and cellulose phosphate, and chromatofocusing as the final step. The recovery was 49%. An apparent relative molecular mass of 32400 was determined by calibrated gel filtration, while a single peptide chain of Mr = 42900 was found by sodium dodecyl sulfate/acrylamide gel electrophoresis. The isoelectric point was determined to be at pH 4.6. Two distinct pH optima (pH 5.4 and 6.8) were observed for the enzyme-catalyzed formation of EPSP from phosphoenolpyruvate (PEP) and shikimate 3-phosphate(S3P). For the reverse reaction, the pH optima were 5.6 and 7.6. No evidence for a metal cofactor was found. While the temperature optimum was at 60 degrees C, the activation energies were calculated to be 54.2 kJ/mol for the forward, and 64.1 kJ/mol for the reverse reaction. At low PEP and S3P concentrations, anions acted as activators of EPSP synthase at low concentrations, and as inhibitors at high concentrations. Non-linear Lineweaver-Burk plots were interpreted to result from the activation of EPSP synthase by its anionic substrates. The following dissociation constants were determined for the respective enzyme-substrate complexes: forward reaction: 43 microM (PEP) and 22 microM (S3P); reverse reaction: 1.3 microM (EPSP) and 2.6 mM (Pi). The kinetic patterns indicate a random sequential mechanism for the forward reaction.
D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase; D019883:Alkyl and Aryl Transferases; D000838:Anions / Q000494:pharmacology; D001665:Binding Sites; D002412:Cations / Q000494:pharmacology; D055598:Chemical Phenomena; D002621:Chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D007700:Kinetics; D007711:Klebsiella pneumoniae / Q000201:enzymology* / Q000254:growth & development; D008970:Molecular Weight; D013379:Substrate Specificity; D013696:Temperature; D014166:Transferases / Q000037:antagonists & inhibitors / Q000302:isolation & purification*
D000838:Anions; D002412:Cations; D014166:Transferases; D019883:Alkyl and Aryl Transferases; D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase