Evolution of the enzymatic characteristics of C-4 phosphoenolpyruvate carboxylase - A comparison of the orthologous PPCA phosphoenolpyruvate carboxylases of Flaveria trinervia (C-4) and Flaveria pringlei (C-3)

C-4 phosphoenolpyruvate (P-pyruvate) carboxylases have evolved from ancestral C-3 P-pyruvate carboxylases during the evolution of C-4 photosynthesis (Lepiniec et al., 1994). To meet the requirements of a new metabolic pathway, the C-4 enzymes have gained distinct kinetic and regulatory properties compared to C-3 enzymes. Our interest is to deduce the structure responsible for these C-4-specific properties. As a model system, the orthologous ppcA P-pyruvate carboxylases of Flaveria trinervia (C-4) and Flaveria pringlei (C-3) were investigated by expressing them in Escherichia coli using their cDNAs. The K-m (P-pyruvate) was about ten times higher for the C-4 enzyme (650 mu M) than for the C-3 enzyme (60 mu M). The activation by glucose 6-phosphate, which was shown by a decrease in the K-m (P-pyruvate), was about twice for the C-4 enzyme and three times for the C-3 enzyme. The C-3 enzyme showed a very high sensitivity to L-malate with an I-0.5 (50% inhibition) value of 80 mu M malate, whereas the C-4 enzyme was much less sensitive with a I-0.5 value of 1.2 mM malate. To locate the structural positions responsible for these differences in kinetic and regulatory properties, chimeras of these 95 % identical enzymes were made. In this study, the first 437 residues of the 966-amino-acid protein were interchanged. The results showed that the N-terminal part of the enzyme was responsible for a small but significant part of the kinetic difference observed between these two isoenzymes. Additionally, the results suggest that the N-terminus was the site for glucose 6-phosphate activation and was also responsible for the observed difference in activation by this sugar phosphate. The difference in inhibition by L-malate, however, is suggested to originate mainly from the C-terminal part of the enzyme.