The dimeric form of phosphoenolpyruvate carboxylase isolated from maize: physical and kinetic properties

Phosphoenolpyruvate carboxylase purified from maize was a homodimer of molecular weight 200 kDa and was readily converted to a tetrameric form in the presence of Mg2+ plus PEP or Mg2+ alone. During the assay, the enzyme activity increased with time, reaching a steady state after a discernible lag, suggesting its hysteretic nature. The hystereses was not due to oligomerization of the enzyme as the lag time tau was independent of the enzyme concentration and the lag was not abolished on preincubation with 25 mM Mg2+, the condition under which the enzyme existed in tetrameric form. Nevertheless, the lag could be abolished on preincubating the enzyme with PEP plus Mg2+, indicating that the hystereses is due to a PEP plus Mg2(+)-induced slow transition of the enzyme to an activated state during the catalysis. During steady state, the enzyme showed cooperative kinetics for PEP and Mg2+ at pH 7. It had two binding sites with nearly 10-fold difference in affinities for PEP and Mg2+.