Purification and Properties of Phosphoenolpyruvate Carboxylase From Plants With Crassulacean Acid Metabolism

Abstract

Phosphoenolpyruvate (PEP) carboxylase was purified from three species of crassulacean acid metabolism (CAM) plants. There was no evidence for isoenzymes of PEP carboxylase in these plants and the purified protein was an active dimer of M_r 220 000-250 000 which dissociated to a monomer of M_r 110 000 after treatment with sodium dodecyl sulfate. Active, higher aggregates could be obtained on Sepharose 6B but the functional significance, if any, of these remains to be assessed. In the absence of effectors, normal Michaelis-Menten kinetics were obtamed with the substrates HCO₃^- and PEP. The purified enzyme shows a preference for HCO₃^-, rather than CO₂, at pH 6.1 and 8.1, with a K_m (HCO₃^-) of 10-20 µM. The V_max was relatively independent of pH between pH 5.5 and 8.5, but the K_m (PEP) (like most other kinetic properties) was pH dependent with a minimum of about 0.1 mM PEP at pH 6.8. Malate inhibition was more effective at pH 6.2 than at pH 8.2, and the inhibition evidently involved a slow binding of malate which increased the K_m (PEP) and resulted in non-hyperbolic kinetics. The K_m (PEP) was lowered about 5-10-fold by 1.0 mM glucose 6-phosphate which also overcame malate inhibition and restored hyperbolic kinetic relationships in the presence of malate. Possible roles for these properties in the regulation of CAM are discussed.