PMID:9249011
| Citation |
Yano, M and Izui, K (1997) The replacement of Lys620 by serine desensitizes Escherichia coli phosphoenolpyruvate carboxylase to the effects of the feedback inhibitors L-aspartate and L-malate. Eur. J. Biochem. 247:74-81 |
|---|---|
| Abstract |
Chemical modification of Escherichia coli phosphoenolpyruvate carboxylase (P-pyruvate carboxylase) by 2,4,6-trinitrobenzene sulfonate, a specific reagent for amino groups, causes desensitization to allosteric inhibitors, L-aspartate and L-malate, as well as inactivation. When L-malate is included in the modification mixture, P-pyruvate carboxylase was markedly protected from both desensitization and inactivation [Naide, A., Izui, K., Yoshinaga, T. & Katsuki, H. (1979) J. Biochem. (Tokyo) 85, 423-432]. To determine the lysine residue(s) involved in allosteric inhibition, the lysine residues that were protected from modification by L-malate were investigated by analyzing trinitrophenylated peptides liberated by digestion with glutamyl endopeptidase (V8-protease). The identified residues were Lys491, Lys620, Lys650, and Lys773. Each of these residues was individually replaced with an alanine or serine residue by site-directed mutagenesis to produce mutant enzymes. The mutant enzyme whose lysine residue was replaced with serine ([Ser620]P-pyruvate carboxylase) showed a marked desensitization to L-aspartate and L-malate, while retaining almost the same maximal catalytic activity as the wild-type P-pyruvate carboxylase. Essentially no changes in enzymatic properties were observed for the [Ala491]- and [Ala650]P-pyruvate carboxylases, while for the [Ala620]- and [Ala773]P-pyruvate carboxylases the polypeptides of the expected size were not significantly accumulated in the transformed E. coli cells, presumably due to intracellular degradation. |
| Links | |
| Keywords |
Amino Acid Sequence; Aspartic Acid/pharmacology; Enzyme Inhibitors/pharmacology; Escherichia coli/enzymology; Feedback; Kinetics; Malates/pharmacology; Molecular Sequence Data; Mutagenesis, Site-Directed; Phosphoenolpyruvate Carboxylase/antagonists & inhibitors; Phosphoenolpyruvate Carboxylase/chemistry; Structure-Activity Relationship |
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