PHOSPHOENOLPYRUVATE CARBOXYLASE IN ROOT-NODULES OF VICIA-FABA - PARTIAL-PURIFICATION AND PROPERTIES

Phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) was purified 56-fo ld from Vicia faba root nodules to a specific activity of 24.8 units m g(-1) protein. Native molecular mass was determined to be 443 kDa by g el permeation chromatography, whereas a molecular mass of 113 kDa was obtained for the subunit by means of SDS-PAGE, indicating that the enz yme is a homotetramer. One peak of activity was obtained by ion-exchan ge chromatography or gel filtration, and thus there was no evidence of isoenzymes. The effect of pH on PEPC activity was studied, the pH opt imum 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 mM. Inhibition b y malate or activation by glucose-6-phosphate was dependent on the pH of the reaction mixture. Malate behaved as a non-competitive mixed-typ e inhibitor with a K-i of 0.76 mM, a K-i(s) of 1.15 mM and a K-i(i) of 0.72 mM, at pH 7.0 while at pH 8.25 K-i was about 140 mM. Activation by glucose-6-P was 70% with 4 mM PEP at pH 7, whereas no effect was fo und at pH 8.25. Experiments with mixed effectors at pH 7 and 1 mM PEP, showed that glucose-6-P can reverse the inhibition caused by L-malate on the PEPC activity.