C, phosphoenolpyruvate (P-pyruvate) carboxylases have evolved from ancestral C, P-pyruvate carboxylases during the evolution of C, photosynthesis (Lepiniec et al., 1994). To meet the requirements of a new metabolic pathway, the C, enzymes have gained distinct kinetic and regulatory properties compared to C, enzymes. Our interest is to deduce the structure responsible for these C,-specific properties. As a model system, the orthologous ppcA P-pyruvate carboxylases of Flaveria trinervia (C,) and Flaveria pringlei (C,) were investigated by expressing them in Escherichia coli using their cDNAs. The K,,, (Ppyruvate) was about ten times higher for the C, enzyme (650 pM) than for the C, enzyme (60 pM). The activation by glucose 6-phosphate, which was shown by a decrease in the K, (P-pyruvate), was about twice for the C, enzyme and three times for the C, enzyme. The C, enzyme showed a very high sensitivity to L-malate with an (50% inhibition) value of 80 pM malate, whereas the C, enzyme was much less sensitive with a 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.