Site-directed mutagenesis of the phosphorylatable serine (Ser8) in C4 phosphoenolpyruvate carboxylase from sorghum. The effect of negative charge at position 8.
The properties of the dephospho and in vitro phosphorylated forms of recombinant sorghum phosphoenolpyruvate carboxylase have been compared with those of the authentic dark (dephospho) and light (phospho) leaf enzyme forms and two mutant enzymes in which the phosphorylatable serine residue (Ser8) has been changed by site-directed mutagenesis to Cys (S8C) or Asp (S8D). Kinetic analysis of the purified recombinant, mutant, and leaf enzyme forms at pH 8.0 indicated virtually identical Vmax, apparent Km (phosphoenolpyruvate), and half-maximal activation (glucose 6-P) values of about 44 units/mg, 1.1 mM, and 0.23 mM, respectively. In contrast, the Ser8, S8C, and dark leaf enzymes were about 3-fold more sensitive to inhibition by L-malate at pH 7.3 than the Ser8-P, S8D, and light leaf enzyme forms. These comparative results indicate that: (i) Ser8 is an important determinant in the regulation of sorghum phosphoenolpyruvate carboxylase activity by negative (L-malate), but not positive (glucose 6-phosphate) metabolite effectors, (ii) phosphorylation of this target residue can be functionally mimicked by Asp, but not Cys, and (iii) negative charge contributes to the effect of regulatory phosphorylation on this C4-photosynthesis enzyme.
Wang
Y H
YH
Department of Biochemistry, University of Nebraska-Lincoln 68583-0718.
Duff
S M
SM
Lepiniec
L
L
Crétin
C
C
Sarath
G
G
Condon
S A
SA
Vidal
J
J
Gadal
P
P
Chollet
R
R
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003624:Darkness; D007700:Kinetics; D008027:Light; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010730:Phosphoenolpyruvate Carboxylase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010944:Plants / Q000201:enzymology*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012694:Serine
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D012694:Serine; D010730:Phosphoenolpyruvate Carboxylase