Purification and characterization of phosphoenolpyruvate carboxylase of photomixotrophically cultured green tobacco [Nicotiana tabacum] cells

Phosphoenolpyruvate (PEP) carboxylase (PEPCase, EC 4.1.1.31) was purified to apparent electrophoretic homogeneity from photomixotrophically cultured tobacco cells by ammonium sulfate fractionation, DEAE-Sephacel-, hydroxylapatite-, Phenyl-Sepharose CL-4B-, and Sepharose CL-6B-chromatography, and fast protein liquid chromatography on Mono Q. The purified enzyme had a specific activity of 32 units per mg protein, and its purity was determined by denaturing polyacrylamide gel electrophoresis. The native enzyme, with a molecular weight of about 440,000, was a tetramer of four identical subunits and showed maximum activity at pH 8.5–9.0. Non-denaturing isoelectric focusing showed a single band at pl 5.4. Substrate-saturation kinetics of the purified enzyme for PEP, bicarbonate, and Mg2$ were typical Michaelis-Menten type, with Km-values of 60, 200, and 80μM, respectively. Most effectors which are known to influence the activity of C4- or bacterial PEPCase had only small effects on the activity of the purified enzyme at optimum pH, while some inhibitory effects by organic acids (malate, citrate and oxaloacetate) and.an activating effect by glucose-6-phosphate were observed at a suboptimal pH of 7.5.