Effects of Site-directed Mutagenesis of Conserved Lys606 Residue on Catalytic and Regulatory Functions of Maize C_4-form Phosphoenolpyruvate Carboxylase

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Lys606, one of the two highly conserved lysine residues in maize C_4-form phosphoenolpyruvate carboxylase (PEPOC), 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. Bio-chem. 61: 545] were purified by one step procedure through nickel-chelate affinity chromatograghy 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 (V_<max>) were de-creased to 22% and 2% of that of wild-type PEPC upon the substitution of Lys606 by Asn and Glu, respectively. The value of S_<0.5>(HCO^-_3) was increased 21-25 fold by the replacements, whereas the S_<0.5>(Mg^<2+>) and S_<0.5>(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 con-tribute somehow to the allosteric regulatory properties.
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