Purification and molecular and kinetic properties of phosphoenolpyruvate carboxylase fromAmaranthus viridisL. leaves

摘要

Phosphoenolpyruvate carboxylase (EC 4.1.1.31) was purified 43-fold fromAmaranthus viridisleaves by using a combination of ammonium-sulphate fractionation, chromatography on O-(diethylaminoethyl)-cellulose and hydroxylapatite, and filtration through Sepharose 6B. The purified enzyme had a specific activity of 17.1 μmol·(mg protein)-1·min-1and migrated as a single band of relative molecular weight 100000 on sodium dodecyl sulphate-polyacrylamide gel electrophoresis. A homotetrameric structure was determined for the native enzyme. Phosphoenolpyruvate carboxylase fromZea maysL. andA. viridisshowed partial identity in Ouchterlony two-dimensional diffusion. Isoelectric focusing showed a band at pI 6.2. Kmvalues for phosphoenolpyruvate and bicarbonate were 0.29 and 0.17 mM, respectively, at pH 8.0. The activation constant (Ka) for Mg2+was 0.87 mM at the same pH. The carboxylase was activated by glucose-6-phosphate and inhibited by several organic acids of three to five carbon atoms. The kinetic and structural properties of phosphoenolpyruvate carboxylase fromA. viridisleaves are similar to those of the enzyme fromZea maysleav