Effects of Site-directed Mutagenesis of Conserved Lys606 Residue on Catalytic and Regulatory Functions of Maize C4-form Phosphoenolpyruvate Carboxylase

Lys606, one of the two highly conserved lysine residues in maizeC₄-form phosphoenolpyruvate carboxylase (PEPC), was convertedto Asn, GIu or Arg by site-directed mutagenesis. Resulted mutantenzymes expressed using pET system [Dong, L.-Y. et al (1997)Biosci. Biotech. Bio-chem. 61: 545] were purified by one stepprocedure through nickel-chelate affinity chromatograghy toa purity of about 95%. The replacement of Lys606 by Arg hadlittle effect on the kinetic and allosteric properties of theresulting mutant enzyme. In contrast, the maximum velocities(V_max were decreased to 22% and 2% of that of wild-type PEPCupon the substitution of Lys606 by Asn and Glu, respectively.The value of S_0.5(HCO^–₃) was increased 21—25 foldby the replacements, whereas the S_0.5(Mg²⁺) and S_0.5(PEP) valueswere increased only 5—8 fold. The extents of activationof mutant enzymes by glucose 6-phosphate and glycine were 2to 3-fold higher than those of wild-type enzyme. The mutantenzymes showed less sensitivity to malate inhibition, comparedwith the wild-type enzyme. The results suggested that the Lys606is not obligatory for the enzyme activity, but may be involvedin the bicarbonate-binding and contribute somehow to the allostericregulatory properties. (Received June 12, 1997; Accepted October 1, 1997)