PARTIAL-PURIFICATION AND CHARACTERIZATION OF STOMATAL PHOSPHOENOLPYRUVATE CARBOXYLASE FROM VICIA-FABA

Stomatal phosphoenolpyruvate carboxylase (PEPCase EC 4.1.1.31), extracted from abaxial epidermal peels of Vicia faba L. cv. Fruhe Weisskeimige, was partially purified by ammoniumsulfate precipitation, and molecular sieve (Sepharose S-400) and ion exchange (DEAE-Sepharose) chromatography. The partially purified enzyme, essentially free of a PEPCase isoform existing in mesophyll and epidermal cells, had a specific activity of 300 nkat mg-1 protein at 25-degrees-C. Western immunoblot analysis revealed that the stomatal enzyme had two bands (M(r) of 110 000 and 112 000), crossreacting with PEPCase antibodies raised against PEPCase from Kalanchoe daigremontiana. The native molecular mass of the enzyme (467 000) points to a tetrameric subunit structure. The temperature optimum was found to be 35-degrees-C; cold treatments of PEPCase before assaying were accompanied by inactivation. The energy of activation was calculated to 51 kJ mol-1. The kinetic behaviour of the enzyme at fixed MgCl2 concentrations is characterized by a pH optimum between pH 8.0-8.2 with or without 1 mM malate or 5 mM glucose-6-phosphate (Glc-6-P), but a combination of both effectors resulted in a shift of the optimum to pH 7.6. The enzyme showed a pH sensitive inhibition by 1 mM malate and an activation by Glc-6-P. At low pH (6-7), Glc-6-P was able to compensate for the malate induced inhibition of the enzyme. Malate and Glc-6-P both affected K(m(PEP)) drastically and influenced V(max) at pH 7, but not at pH 8.3. The inhibition constant of malate was determined to be 1.2 mM at pH 7. From the Dixon plot, a competitive inhibition of malate was assumed under defined assay conditions.