An engineered change in the L-malate sensitivity of a site-directed mutant of sorghum phosphoenolpyruvate carboxylase: the effect of sequential mutagenesis and S-carboxymethylation at position 8.
A recombinant, site-directed mutant form of sorghum phosphoenolpyruvate carboxylase (PEPC), in which the phosphorylatable serine residue (Ser-8) was changed to cysteine (S8C), was chemically modified by iodoacetic acid and iodoacetamide for the purpose of testing the effect of introducing a negative charge at position 8. S-Carboxymethylation of the Cys-8 enzyme by iodoacetic acid decreased its sensitivity to L-malate from an I0.5 (50% inhibition) value of 0.12 to 0.35 mM at pH 7.3 when the active-site domain was protected during modification by the substrate phosphoenolpyruvate (PEP). In contrast, neither S-carboxymethylation of the wild-type enzyme nor modification of the mutant enzyme by iodoacetamide caused any change in the enzyme's sensitivity to L-malate. The modified, substrate-protected forms of the Ser-8 and S8C PEPCs had Km(total PEP) and Vmax values virtually identical to those of the unmodified control enzymes. Similar specific increases in the I0.5 value of L-malate have been reported previously for in vitro phosphorylated leaf and recombinant Ser-8 PEPCs, the site-directed mutant Asp-8 enzyme, and C4-leaf PEPC purified from light-adapted sorghum or maize (in vivo phospho-form). Therefore, these data from different but complementary experimental approaches provide convincing evidence that the effect of phosphorylation of Ser-8 on the L-malate sensitivity of sorghum C4-PEPC is caused by the introduction of negative charge into this N-terminal regulatory domain.
Duff
S M
SM
Department of Biochemistry, University of Nebraska at Lincoln 68583-0718.
Lepiniec
L
L
Crétin
C
C
Andreo
C S
CS
Condon
S A
SA
Sarath
G
G
Vidal
J
J
Gadal
P
P
Chollet
R
R
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D007460:Iodoacetamide / Q000494:pharmacology; D007461:Iodoacetates / Q000494:pharmacology; D019807:Iodoacetic Acid; D007700:Kinetics; D008293:Malates / Q000494:pharmacology*; D016297:Mutagenesis, Site-Directed; D010730:Phosphoenolpyruvate Carboxylase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006109:Poaceae / Q000201:enzymology*; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012694:Serine
D007461:Iodoacetates; D008293:Malates; D011994:Recombinant Proteins; D012694:Serine; D010730:Phosphoenolpyruvate Carboxylase; D019807:Iodoacetic Acid; D007460:Iodoacetamide