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Plant and Cell Physiology, 1997, Vol. 38, No. 12 1340-1345
© 1997

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

Long-Ying Dong, Yoshihisa Ueno, Shingo Hata and Katsura Izui1

Laboratory of Plant Physiology, Graduate School of Agriculture, Kyoto University Sakyo-ku, Kyoto, 606-01 Japan

Corresponding author: E-mail, izui{at}kais.kyoto-u.ac.jp; Fax, 75-753-6146.

Lys606, one of the two highly conserved lysine residues in maize C4-form phosphoenolpyruvate carboxylase (PEPC), was converted to Asn, GIu 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 (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(HCO–3) 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.

(Received June 12, 1997; Accepted October 1, 1997)
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