EFFECTS OF SITE-DIRECTED MUTAGENESIS OF CONSERVED LYS606 RESIDUE ON CATALYTIC AND REGULATORY FUNCTIONS OF MAIZE C-4-FORM PHOSPHOENOLPYRUVATE CARBOXYLASE
Ly. Dong et al., EFFECTS OF SITE-DIRECTED MUTAGENESIS OF CONSERVED LYS606 RESIDUE ON CATALYTIC AND REGULATORY FUNCTIONS OF MAIZE C-4-FORM PHOSPHOENOLPYRUVATE CARBOXYLASE, Plant and Cell Physiology, 38(12), 1997, pp. 1340-1345
Lys606, one of the two highly conserved lysine residues in maize C-4-f
orm 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 a
ffinity chromatography to a purity of about 95%. The replacement of Ly
s606 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 exte
nts 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 en
zymes showed less sensitivity to malate inhibition, compared with the
wild-type enzyme, The results suggested that the Lys606 is not obligat
ory for the enzyme activity, but may be: involved in the bicarbonate-b
inding and contribute somehow to the allosteric regulatory properties.