title: Evolution of the enzymatic characteristics of C4 phosphoenolpyruvate carboxylase--a comparison of the orthologous PPCA phosphoenolpyruvate carboxylases of Flaveria trinervia (C4) and Flaveria pringlei (C3).
abstract: C4 phosphoenolpyruvate (P-pyruvate) carboxylases have evolved from ancestral C3 P-pyruvate carboxylases during the evolution of C4 photosynthesis (Lepiniec et al., 1994). To meet the requirements of a new metabolic pathway, the C4 enzymes have gained distinct kinetic and regulatory properties compared to C3 enzymes. Our interest is to deduce the structure responsible for these C4-specific properties. As a model system, the orthologous ppcA P-pyruvate carboxylases of Flaveria trinervia (C4) and Flaveria pringlei (C3) were investigated by expressing them in Escherichia coli using their cDNAs. The K(m) (P-pyruvate) was about ten times higher for the C4 enzyme (650 microM) than for the C3 enzyme (60 microM). The activation by glucose 6-phosphate, which was shown by a decrease in the K(m) (P-pyruvate), was about twice for the C4 enzyme and three times for the C3 enzyme. The C3 enzyme showed a very high sensitivity to L-malate with an I(0.5) (50% inhibition) value of 80 microM malate, whereas the C4 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.
authors.affiliation: Department of Biochemistry, Stockholm University, Sweden. per.svensson@biokemi.su.se
authors.forename: P
authors.initials: P
authors.lastname: Svensson
authors.forename: O E
authors.initials: OE
authors.lastname: Bläsing
authors.forename: P
authors.initials: P
authors.lastname: Westhoff
mesh_terms: D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D019143:Evolution, Molecular; D007700:Kinetics; D008969:Molecular Sequence Data; D010730:Phosphoenolpyruvate Carboxylase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010944:Plants / Q000201:enzymology*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
chemical_list: D018076:DNA, Complementary; D011994:Recombinant Proteins; D010730:Phosphoenolpyruvate Carboxylase