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.