Effects of site-directed mutagenesis of conserved Lys606 residue on catalytic and regulatory functions of maize C4-form phosphoenolpyruvate carboxylase

被引:8
作者
Dong, LY [1 ]
Ueno, Y [1 ]
Hata, S [1 ]
Izui, K [1 ]
机构
[1] Kyoto Univ, Grad Sch Agr, Lab Plant Physiol, Sakyo Ku, Kyoto 60601, Japan
关键词
C-4; photosynthesis; enzymatic properties; phosphoenolpyruvate carboxylase (EC 4.1.1.31); site-directed mutagenesis; Zea mays;
D O I
10.1093/oxfordjournals.pcp.a029127
中图分类号
Q94 [植物学];
学科分类号
071001 ;
摘要
Lys606, one of the two highly conserved lysine residues in maize C-4-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 (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(Mg2+) 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.
引用
收藏
页码:1340 / 1345
页数:6
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