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Phosphoenolpyruvate carboxylase in root nodules of Vicia faba : Partial purification and properties
Author(s) -
Ocaña Antonio,
Cordovilla María del Pilar,
Ligero Francisco,
Lluch Carmen
Publication year - 1996
Publication title -
physiologia plantarum
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.351
H-Index - 146
eISSN - 1399-3054
pISSN - 0031-9317
DOI - 10.1111/j.1399-3054.1996.tb00537.x
Subject(s) - phosphoenolpyruvate carboxylase , homotetramer , vicia faba , chemistry , size exclusion chromatography , phosphoenolpyruvate carboxykinase , molecular mass , chromatography , biochemistry , enzyme , enzyme assay , specific activity , protein subunit , biology , botany , gene
Phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) was purified 56‐fold from Vicia faba root nodules to a specific activity of 24.8 units mg ‐1 protein. Native molecular mass was determined to be 443 kDa by gel permeation chromatography, whereas a molecular mass of 113 kDa was obtained for the subunit by means of SDS‐PAGE, indicating that the enzyme is a homotetramer. One peak of activity was obtained by ion‐exchange chromatography or gel filtration, and thus there was no evidence of isoenzymes. The effect of pH on PEPC activity was studied, the pH optimum found at 8.25. The effect of substrate (phosphoenolpyruvate, PEP) on the enzyme activity was studied at five different pH values from 6.5 to 9.5. The K m (PEP) at pH 8.25 proved to be 0.064 m M. Inhibition by malate or activation by glucose‐6‐phosphate was dependent on the pH of the reaction mixture. Malate behaved as a non‐competitive mixed‐type inhibitor with a K i of 0.76 m M , a K i (s) of 1.15 m M and a K i (i) of 0.72 m M , at pH 7.0 while at pH 8.25 K i was about 140 m M. Activation by glucose‐6‐P was 70% with 4 m M PEP at pH 7, whereas no effect was found at pH 8.25. Experiments with mixed effectors at pH 7 and 1 m M PEP, showed that glucose‐6‐P can reverse the inhibition caused by L‐malate on the PEPC activity.