An Engineered Change in the L-Malate Sensitivity of a Site-Directed Mutant of Sorghum Phospho enolpyruvate Carboxylase: The Effect of Sequential Mutagenesis and S-Carboxymethylation at Position 8

Abstract

A recombinant, site-directed mutant form of sorghum phospho enolpyruvate 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 I 0.5 (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 phospho enolpyruvate (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 K m (total PEP) and V max values virtually identical to those of the unmodified control enzymes. Similar specific increases in the I 0.5 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 C 4-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 C 4-PEPC is caused by the introduction of negative charge into this N-termninal regulatory domain.