Effects of site-directed mutagenesis of conserved lys606 residue on catalytic and regulatory functions of maize [Zea mays] C4-form phosphoenolpyruvate carboxylase
1997
Dong, L.Y. (Kyoto Univ. (Japan)) | Ueno, Y. | Hata, S. | Izui, K.
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 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 (V(max)) 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 S(0.5)(HCO3(-)) 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 contribute somehow to the allosteric regulatory properties
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