title: Effects of site-directed mutagenesis of conserved Lys606 residue on catalytic and regulatory functions of maize C4-form phosphoenolpyruvate carboxylase.
abstract: Lys606, one of the two highly conserved lysine residues in maize C4-form phosphoenolpyruvate carboxylase (PEPC), was converted to Asn, Glu or Arg by site-directed mutagenesis. Resulted mutant enzymes expressed using pET system [Dong, L.-Y. et al. (1997) Biosci. Biotech, Biochem. 61:545] were purified by one step procedure through nickel-chelate affinity chromatography to a purity of about 95%. The replacement of Lys606 by Arg had little effect on the kinetic and allosteric properties of the resulting mutant enzyme. In contrast, the maximum velocities (Vmax) were decreased to 22% and 2% of that of wild-type PEPC upon the substitution of Lys606 by Asn and Glu, respectively. The value of S0.5(HCO3-) was increased 21-25 fold by the replacements, whereas the S0.5(Mg2+) and S0.5(PEP) values were increased only 5-8 fold. The extents of activation of mutant enzymes by glucose 6-phosphate and glycine were 2 to 3-fold higher than those of wild-type enzyme. The mutant enzymes showed less sensitivity to malate inhibition, compared with the wild-type enzyme. The results suggested that the Lys606 is not obligatory for the enzyme activity, but may be involved in the bicarbonate-binding and contribute somehow to the allosteric regulatory properties.
authors.affiliation: Laboratory of Plant Physiology, Graduate School of Agriculture, Kyoto University, Japan.
authors.forename: L Y
authors.initials: LY
authors.lastname: Dong
authors.forename: Y
authors.initials: Y
authors.lastname: Ueno
authors.forename: S
authors.initials: S
authors.lastname: Hata
authors.forename: K
authors.initials: K
authors.lastname: Izui
mesh_terms: D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D002384:Catalysis; D017124:Conserved Sequence; D005816:Genetic Complementation Test; D007700:Kinetics; D008239:Lysine / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010730:Phosphoenolpyruvate Carboxylase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003313:Zea mays / Q000201:enzymology / Q000235:genetics
chemical_list: D010730:Phosphoenolpyruvate Carboxylase; D008239:Lysine