Purification and Characterization of Phosphoenolpyruvate Carboxylase of Photomixotrophically Cultured Green Tobacco Cells

Phosphoenolpyruvate (PEP) carboxylase (PEPCase, EC 4.1.1.31 EC] )was purified to apparent electrophoretic homogeneity from photomixotrophicallycultured 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 purifiedenzyme had a specific activity of 32 units per mg protein, andits 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 maximumactivity at pH 8.5–9.0. Non-denaturing isoelectric focusingshowed a single band at pl 5.4. Substrate-saturation kineticsof the purified enzyme for PEP, bicarbonate, and Mg^2$ were typicalMichaelis-Menten type, with K_m-values of 60, 200, and 80µM,respectively. Most effectors which are known to influence theactivity of C₄- or bacterial PEPCase had only small effectson the activity of the purified enzyme at optimum pH, whilesome inhibitory effects by organic acids (malate, citrate andoxaloacetate) and.an activating effect by glucose-6-phosphatewere observed at a suboptimal pH of 7.5. (Received September 30, 1987; Accepted December 14, 1987)