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Partial purification and characterization of stomatal phosphoenolpyruvate carboxylase from Vicia faba
Author(s) -
Denecke Martin,
Schulz Margot,
Fischer Christoph,
Schnabl Heide
Publication year - 1993
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.1399-3054.1993.tb08796.x
Subject(s) - phosphoenolpyruvate carboxylase , vicia faba , biology , enzyme , biochemistry , phosphoenolpyruvate carboxykinase , pyruvate carboxylase , enzyme assay , molecular mass , malate dehydrogenase , microbiology and biotechnology , botany
Stomatal phosphoenolpyruvate carboxylase (PEPCase EC 4.1.1.31), extracted from abaxial epidermal peels of Vicia faba L. cv. Frühe Weiβkeimige, was partially purified by ammoniumsulfate precipitation, and molecular sieve (Sepharosc 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°C. Western immunoblot analysis revealed that the stomatal enzyme had two bands (M : of 110000 and 112000), crossreacting with PEPCase antibodies raised against PEPCase from Ka‐lanchoe daigremontiana . The native molecular mass of the enzyme (467000) points to a tetrameric subunit structure. The temperature optimum was found to be 35°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 MgCl 2 concentrations is characterized by a pH optimum between pH 8.0–8.2 with or without 1 m M malate or 5 m M 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 m M 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 m M at pH 7. From the Dixon plot, a competitive inhibition of malate was assumed under defined assay conditions.